CA2304823A1 - Regulation of gene expression - Google Patents

Abstract

The application provides nucleic acid isolates which have an SCL gene regulatory element; the isolate being free of SCL coding sequence and/or promoter for said SCL gene wherein the regulatory element can alter transcription from an SCL gene if the gene is operably linked thereto.

Description

REGULATION OF GENE EXPRESSION
The present invention relates to the identification of regulatory elements involved in the transcriptional control of the SCL gene and to the use of such regulatory elements to control gene expression. In particular, the present invention concerns materials and methods relating to regulatory elements which are involved in the transcriptional control of the SCL gene. For example, the present invention further relates to uses of nucleic acid encoding SCL regulatory elements, in particular for controlling expression of non-SCL genes and SCL genes in cell types in which the regulatory el'ments are able to function. The regulatory elements as provided hereby may be employed to regulate gene expression for experimental or therapeutic purposes.
The mechanisms whereby a pluripotent stem cell generates multiple distinct differentiated cell types remain almost completely obscure. Haematopoiesis, the formation of blood cells, provides a powerful paradigm for probing this process. Several lines of evidence demonstrate a central role for transcription factors. The present inventors have investigated the transcriptional regulation of the SCL gene (also known as TAL-1) which encodes a basic helix-loop-helix transcription factor, is expressed in pluripotent haematopoietic stem cells and functions as a pivotal regulator of haematopoiesis.
The transcription factor encoded by the SCL gene is essential for the development of all haematopoietic lineages. Ectcpic expression results in T cell leukemia.
S CL
is expressed ~: normal pluripotent haematopoietic stem cells. Its expression is maintained d~~ring differentiation along erythroi, mast and megakaryocytic lineages, but SUBSTITUTE SHEET (RULE 26) 2 . PCT/GB98/02914 extinguished following commitment to other cell types.
SCL was originally discovered as a result of its activation by chromosome rearrangements in T-cell acute lymphoblastic leukaemia (T-All) (Begley, C.G, et al. PNAS
USA 86: 10128, 1989; Finger, L.R, et al. PNAS USA 86:
5039, 1989 and Chen, Q et al. EMBO J 9: 415,1990). SCL -expression is activated in these tumours by several mechanisms including chromosome translocation and, more r commonly, interstitial deletions which place the SCL gene under the control of the SIL gene promoter (Aplan, P.D, et al. Mol Cell Biol 10: 6426, 1990; Bernard, O, et al.
Genes, Chromosomes and Cancer 1: 194, 1990; Brown, L, et al. EMBO J 9: 3343, 1990). The SCL gene is also expressed ~15 in a substantial proportion of T-ALL cells in the absence of any overt rearrangement, and evidence for both cis and transacting mechanisms has been presented (Bash, R.O, et al. Blood 86: 566, 1995). The SCL protein is phosphorylated on serine, contains an N-terminal transactivation domain (Goldfarb, A.N, et al. Blood 58:
537, 1992; Cheng, J.T, et al. Mol Cell Biol 13: 801, 1993; Sanchez-Garcia, I et al. PNAS USA 91: 7869, 1994) and binds to a specific E-box as a heterodimer with class A bHLH proteins (Hsu, H.L, et al. Molecular and Cellular Biology 14: 1256, 1994). More recently, SCL has also been shown to bind to LMO-2, a LIM domain protein which does not appear to bind DNA itself (Waximan, I, et al. EMBO J
13: 4831, 1994; Valge-Archer, et al. PNAS USA 91: 8617, 1994). The LMO-2 and SCL null phenotypes are similar and LMO-2 is also a T cell oncogene (Boerm, T, et a1. PNAS USA 88: 4367, 1991;
Royer-Pokora, B, et a1. Oncogene 6: 1887, 1991; Warren, A. J, et al. Cell 78: 45, 1994).
Mice lacking SCL protein die by embryonic day 10, and exhibit a complete absence of all haematopoietic cells SUBSTITUTE SHEET (RULE 26~

WO 99116782 PCTIGB98l02914 (Robb, L, et al. PNAS USA 92: 7075, 1995; Shivdasani, R.A, et al. Nature 373: 432, 1995; Procher, C, et al.
Cell 86: 47, 1996; Robb, L. Et al. EMBO J 15: 4123, 1996). Target genes for SCL have not yet been identified, but previous antisense experiments have suggested that SCL may perform distinct functions in different haematopoietic cell types. Introduction of anthozoans -constructs into a multipotent cell line resulted in reduced proliferation and self-renewal (Green, A.R. et ;..
al. EMBO J 10: 4153, 1991) whereas analogous experiments inhibited differentiation of a committed erythroid cell line (Aplan, P.D, et al EMBO J 11: 4073, 1992). In addition, SCL has been implicated in the prevention of apoptosis in leukaemic T cells (Leroy-Viand, K, et al.
~15 EMBO J 14: 2341, 1995).
SCL is expressed in haematopoietic cells, endothelial cells and in brain (Visvader, J, et al. Oncogene 6: 195, 1991; Green, A.R. et al. Oncogene 7: 553, 1992: Hwang, L-Y, et al. Oncogene 8: 3043, 1993: Kallianpur, A.R., et al. Blood 83: 1200, 1994; Pulford, K, et al. Blood 85:
675, 1995). Within the haematopoietic system SCL is expressed in committed erythroid, mast and megakaryocytic cells as well as in IL-3 dependent cell lines (Green, A.R, et al. Supra; Kallianpur, A.R, et al. Supra;
Pulford, K et al. Supra; Green, A.R, et al. Oncogene 6:
475, 1991; Visvader, J, et al. TIES 16: 330, 1991; Mouthon, M.A, et al. Blood 81: 647, 1993). Growth factor induced erythroid differentiation of a multipotent progenitor cell line was accompanied by up-regulation of SCL mRNA, whereas induced granulocyte/monocyte differentiation resulted in extinction of SCL expression (Cross, M.A, et al. Oncogene 9: 3013, 1994). Indeed, down-regulation of SCL
expression not only accompanies, but may actually be required for normal myeloid differentiation, since over-expression of exogenous SCL impaired macrophage SUBSTITUTE SHEET (RULE 2B) WO 99/1b782 PCT/GB98/02914 differentiation of M1 cells (Tanigawa, T, et al. PNAS USA
90: 7864, 1993) .
Given the pattern of SCL expression it is not known whether the same regulatory elements are responsible for expression in stem cells and in committed erythroid, mast and megakaryocytic cells. GATA-1 regulates the SCL
promoter in erythroid cells (Aplan P.D. et al. EMBO J 11:
4073, 1992: Lecointe, N, et al. Oncogene 9: 2623, 1994;
Bockamp, E, et al. Blood 86: 1502, 1995) and is also expressed at high levels in mast and megakaryocytic cells. GATA-2 recognises a subset of GATA-1 binding sites and is expressed at high levels in multipotent progenitors. The lineage-restricted pattern of SCL
expression could therefore reflect the presence of one or more regulatory elements responsive to both GATA-1 and GATA-2. Further, it is not known what drives SCL
expression in haematopoietic stem cells. SCL appears to be downstream of GATA genes in committed haematopoietic lineages yet SCL null mice exhibit a more complete absence of haematopoiesis than is found in mice null for GATA-I or GATA-2 (Robb, L, et al. PNAS USA 92: 7075, 1995; Shivdasani, R.A, et al. Nature 373: 432, 1995;
Peony, L et al. Natura 349: 257, 1991; Tsai, F, et al. Nature 371: 221, 1994;
Fujiwara, Y, et al. PNAS 93: 12355, 1996). Also the basis for the down-regulation of SCL expression following commitment to non-expressing lineages is not known. It might be regulated by absence of GATA-1 and/or GATA-2, or SCL may be subject to silencing. Since ectopic SCL
expression in T cells is tumorigenic, SCL down-regulation during haematopoiesis is likely to be subject to stringent regulation.
The murine SCL gene has been previously isolated (Begley, C.G, et al. Gene 138: 93, 1994). Promoter la and promoter SUBSTITUTE SHEET (RULE 26) lb for the SCL gene exhibit lineage-restricted activity in transient reporter assays (Bockamp, ~, et al. Blood 86: 1502, 1995) 5 Broadly, this application concerns the identification of regulatory elements which modulate transcription of the SCL gene. The regulatory elements can be used to control expression of the SCL gene or non-SCL genes in appropriate cell types.
;...
The regulatory elements identified herein which modulate the transcription of the SCL gene have uses in several areas of biotechnology. Such regulatory elements enable further elucidation of the molecular pathway leading to cell lineage commitment, a process which may be applicable to cell types other than those of the haemopoietic lineage. In the medical field the regulatory elements provide a means of regulating expression of genes including non-SCL genes in cell ~ypes (e. g.
haematopoietic cells), where the regulatory elements are able to function.
Thus the present inventors provide a plurality of nucleic acid fragments comprising regulatory elements from the vicinity of an SCL gene. There are provided nucleic acid fragments comprising regulatory elements which function to alter transcription of an SCL,gene and also non-SCL
genes operably linked thereto. There are disclosed nucleic acid fragments which can serve to enhance transcription of e.g. an SCL gene and nucleic acid fragments comprising regulatory elements which can serve to silence transcription of e.g. an SCL gene. Nucleic acid fragments comprising such regulatory elements are effective in relation to modulating activities of genes not normally associated with (i.e. heterologous to) such regulatory elements of an SCL gene.
SUBSTITUTE SHEET (RULE 26j The use of nucleic acid fragments as provided is in relation to controlling expression of a gene of interest in cell types in which the regulatory elements are able to function. This may be for experimental or therapeutic S purposes. Another major use of the nucleic acid fragments provided is in screening for substances able to modulate their activity. It is well known that pharmaceutical research leading to the identification of a new drug generally involves the screening of very large numbers of ~S
candidate substances, both before and even after a lead compound has been found. This is one factor which makes pharmaceutical research expensive and time-consuming. A
method or means assisting in the screening process will have considerable commercial importance and utility.
1.5 Substances identified as regulators of gene activity provide basis for design and investigation of therapeutics for in vivo use. In this case the ectopic expression of the SCL gene results in T cell leukaemia.
Thus the nucleic acid fragments comprising regulatory elements hereby provided, give basis for design and investigation of therapeutics for use in relation to T
cell leukaemia.
According to a first aspect, the present invention provides a nucleic acid isolate which comprises a regulatory element which is able to alter transcription from an SCL gene. The alteration.,.Fnay be such that transcription is enhanced. Alternatively, the alterations may be such that transcription is silenced.
The nucleic acid isolate may be such that a said regulatory element is mostly free of substantially the greater portion of nucleic acid which flanks the regulatory element in the native situation. Thus the nucleic acid isolate may be such that it comprises no more than about l.5kb of the nucleic acid which flanks the regulatory element in the native situation. The SUBSTITUTE SHEET (RULE 26) nucleic acid isolate may comprise substantially less than l.5kb of the nucleic acid which flanks the regulatory element in the native situation. The nucleic acid isolate may comprise less than lkb, less than SOObp, less than 600bp, less than 400bp, less than 200bp, less than 100bp, less than 50bp of nucleic acid which flanks the regulatory element in the native situation. _ The present disclosure provides a plurality of such nucleic acid isolates.
Figures 8 and 9 disclose nucleotide sequences for murine and human hypersensitive sites identified herein as comprising regulatory elements which serve to alter 5.5 transcription of an SCL gene. Other species will have genes orthologous to the murine and human SCL genes. The present disclosure allows those skilled in the art to obtain for themselves nucleic acid isolates similar to those described herein which comprise regulatory elements which serve to alter transcription of the orthologous SCL
gene in such other species by using the methodologies disclosed herein or by using the disclosure of significant seauence homology between. the murine and human hypersensitive sites to identify by use of standard methodologies substantially homologous hypersensitive site sequences in nucleic acid material deriving from other species. The sites can be bested as described herein for the presence of regulatory elements which may be employed as suggested. Nucleic acid isolates as described herein may be obtained from non-mammalian species, e.g. from fish or birds. Genes and associated regulatory sequences from fish (e. g. Zebra fish and Fugu) and birds (e. g. chicken) are compact and therefore easy to manipulate ex vivo.
The present invention thus covers both human, non-human and non-mammalian nucleic acid isolates which comprise a SUBSTITUTE SHEET (RULE 26) regulatory element which is able to alter transcription from an SCL gene. In particular, the present invention provides a nucleic acid isolate which comprises a regulatory element which is able to alter transcription from an SCL gene which nucleic acid isolate comprises (i) a sequence as shown in Figure 8 or 9 or a sequence complementary to a sequence as shown in Figure 8 or 9: or-(ii) a sequence with sufficient homology to either a sequence as shown in Figure 8 or 9 or a complementary r, sequence to a Figure 8 or Figure 9 sequence, to allow annealing thereto under stringent hybridization conditions. With reference to Fig.8 the -3/4.5HS sites are important for endothelial cell expression, the +1/+3HS sites in combination with the SCL promoters ~15 direct expression to mid-brain. If the la promoter is absent one loses expression in brain but retains expression in spinal neurones. The +i7/+18HS sites direct expression to foetal liver and endothelium. Thus the present invention provides the use of nucleic acid isolates as hereby disclosed for regulating expression of a gene in a cell type as mentioned above or a cell type mentioned elsewhere herein. The skilled person will be able to readily determine for himself the precise cell types a given regulatory element is effective in using both his common general knowledge and the disclosures herein.
The present invention also comprises a nucleic acid isolate which comprises two or more regulatory elements which either individually or in concert are able to alter transcription from an SCL gene. Depending upon the gene to be regulated and the transcription system utilized, the two or more regulatory elements may in concert serve to enhance transcription. Alternatively they may in concert serve to silence transcription. The isolate may comprise greater than two regulatory elements. The isolate may comprise 3, 4 or greater than 4 regulatory SUBSTITUTE SHEET (RULE 26) elements.
A regulatory element in a nucleic acid isolate as hereby provided may be chromatin-dependent.
Where the nucleic acid isolate comprises two or more regulatory elements, a regulatory element of the isolate may be chromatin-dependent.
Also provided are nucleic acid isolates which comprise an antisense version of a regulatory element has herein disclosed. Such isolates comprising antisense sequences may be obtained in accordance with standard procedures using the disclosures herein and employed to regulate gene expression.
The present invention also provides for the use of a nucleic acid isolate as hereby provided which comprises a regulatory element which is able to modulate transcription from a human or non-human SCL gene for controlling expression of an SCL gene or non-SCL gene in a cell. It has been found for example that SCL regulatory elements can control expression of heterologous genes in haematopoietic cells (e. g. cells of the erythroid, mast or megakaryocyte lineages) endothelial cells, neuronal cells, yolk sac cells.
The present invention also provides for the use of a nucleic acid isolate as hereby provided for the preparation of a reagent or a medicament for use in controlling expression of an SCL gene or non-SCL gene in a cell.
Also provided are oligonucleotides which comprise fragments of a sequence (i) as shown in Figure 8 or 9 or a sequence complementary to a sequence shown in Figure 8 or 9; or (ii) which has sufficient length and homology to SUBSTITUTE SHEET (RULE 26) either a sequence as shown in Figure 8 or 9 or a complementary sequence to a Figure 8 or 9 sequence, to allow annealing thereto under stringent hybridization conditions.

Such oligonucleotides may be specific to regulatory elements which serve to control transcription of an SCL _ gene (human or non-human). The oligonucleotides may themselves comprise a nucleotide sequence which serves to 10 act as a regulatory element by controlling transcription of an SCL gene (human or non-human).
Preferably such fragments are the minimal sequence required to modulate promoter (e. g. SCL promoter) activity, and may be at least about l0 nucleotides in length, more preferably at least 20, 40, 60, 80, 100, 120, 140, 160, 180 or 200. The minimal sequence required for modulation of transcription from a promoter (e.g. an SCL promoter) can be easily identified by one skilled in the art by restriction enzyme digests followed by an appropriate assay, for example as described herein using luciferase constructs, until the smallest fragment which retains regulatory activity is obtained. In one embodiment of the present invention the minimal sequence may be one or more GATA motifs. Such fragments themselves individually represent aspects of the present invention.
Such fragments may be used inter..;alia as expression regulators, as primers or probes to identify further regulatory elements having homology to those disclosed above, or may be used in the isolation of the regulatory elements disclosed herein by PCR techniques, or may be used in methods concerned with determining the presence in a test sample of a sequence indicative of cancer susceptibility.
The term "locus" when used means the SCL gene, both the coding sequence (exons) and intervening sequences SUBSTITUTE SHEET (RULE 26) (introns), and its regulatory elements for controlling transcription and/or translation. The SCL locus covers allelic variations within the locus.
The terms "SCL gene" or "SCL allele" are used for normal alleles of the SCL gene, and also alleles carrying one or more variations that are linked to a predisposition to cancers such as T cell leukemia. Alleles including such mutations are also known in the art as susceptibility alleles.
The term "regulatory element" is used to denote a sequence of nucleotides which modulate, either by enhancing or silencing, the activity of the promoter or ~5 further regulatory sequences to which they are operably linked e.g. downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA), thus effecting the degree of transcription from the promoter or further regulatory elements.
"Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA
operably linked to a regulatory element is "under transcriptional regulation" of the element.
"SCL promoter modulating activity" means the ability of a regulatory sequence to control the transcription of a gene by regulation of the SCL promoter, to which the regulatory sequence is operably linked. The control may have the effect of enhancing, reducing or conferring specificity of the SCL promoter. However the regulatory elements hereby provided may be able to function operably linked to other promoters.
"Chromatin-dependent" means that the activity of the regulatory element or promoter which controls SUBSTITUTE SHEET (RULE 26) transcription of a gene is dependent upon integration into chromatin in order to mediate its full effects.
The present invention extends to a regulatory element having SCL promoter modulating activity which comprises having a nucleotide sequence which is an allele, mutant, variant or derivative, by way of nucleotide addition, -insertion, substitution or deletion of a regulatory element sequence provided herein. The allele, mutant, ;., variant or derivative may have at least 50% homology with any one of the Figure 8 or 9 sequences provided herein or complementary to the Figure 8 sequences, preferably at least about 60% homology with any one of the sequences, preferably at least about 70o homology, more preferably at least about 80% homology, more preferably at least about 90% homology, more preferably at least about 95%
homology.
Nucleic acids having the appropriate level of homology with a given sequence may be identified by using hybridisation and washing conditions of appropriate stringency. For example, hybridisations may be performed, according to the method of Sambrook et al., using a hybridisation solution comprising: 5X SSC, SX
Denhardt's reagent, 0.5-loo SDS, 100 ~,g/ml denatured, fragmented salmon sperm DNA, 0.050 sodium pyrophosphate and up to 50% formamide. Hybridisation is carried out at 37-42°C for at least six hours. Following hybridisation, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS: (3) 30 minutes-1 hour at 37°C in 1X SSC and 1% SDS; (4) 2 hours at ~2-65°C in 1X SSC and to SDS, changing the solution every 30 minutes.
One common formula for calculating the stringency conditions required to achieve hybridisation between SUBSTITUTE SHEET (RULE 26) nucleic acid molecules of a specified sequence homology is (Sambrook et al., 1989):
Tm = 81.5°C + 16.6Log [Na+] + 0.41{% G+C) - 0.63 (% formamide) - 600/#bp in duplex As an illustration of the above formula, using [Na+] -[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T" is 57°C. The Tm of a DNA duplex decreases by 1 - 1.5°C with every to decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridisation temperature of 42°C.
r .
It follows from the above that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
"Stringent hybridisation conditions" or "high stringency conditions", as used herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M
sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridisation a denaturing agent, such as formamide, for example, 50% (v/v) formamide with O.lo bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50mM sodium phosphate buffer at pH
6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA {50 ~.g/ml), 0.1% SDS, and 10%
dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC
SUBSTITUTE SHEET (RULE 2fi) (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C.
S The sequer_ce in accordance with an embodiment of the invention may hybridise with any one of the shown sequences, or the complementary sequences (since DNA is .
generally double-stranded). The sequence may have the ability to modulate the activity of the SCL promoter (i.e. have "regulatory element activity") in haematopoietic cells le.g. cells of the erythroid, mast or megakaryocyte lineages) endothelial cells, neuronal cells, yolk sac cells.
Systematic or random metagenesis of nucleic acid to make an alteration to the nucleotide sequence may be performed using any technique known to those skilled in the art.
Alternations to a regulatory element as provided herein may ssrve to increase or decrease modulating activity, or increase or decrease the magnitude of the effect of a substance able to modulate the regulatory elements activity.
"Regulatory element activity" is used herein to refer to ability of the regulatory elements to modulate the activity of a promoter or further regulatory element to which they are ooerably linked, thus effecting the ability of the promoter or regulatory element to initiate transcription. The level of regulatory element activity is quantifiable for instance by assessment of the amount of mRNA produced by transcription from the promoter or regulatory element to which they are operably linked, or by assessment of the amount of polypeptide product produced by translation of m RNA produced by transcription from the promoter or regulatory element.
The amount of a specific mRNA present in an expression system may be determined for example using specific oligonucleotides which are able to hybridise with the SUBSTITUTE SHEET (RULE 26) mRNA and which are labelled or may be used in a specific amplification reaction such as the polymerase chain reaction. Use of a reporter gene as discussed further below facilitates determination of promoter activity by S reference to protein production.
Further provided by the present invention is a nucleic acid construct comprising a nucleic acid isolate as described above, operably linked to all or part of a :, promoter. The promoter may be that of an SCL gene, or may be a heterologous promoter. The nucleic acid isolate may be operably linked to either or both of the SCL la and lb promoters. A regulatory element of the nucleic acid isolate may be operably linked to the whole promoter, or to a part of the promoter. If a part of the promoter is used, it is preferably a part able to initiate transcription from a gene. Generally, a regulatory element of the nucleic acid isolate is operably linked, either upstream or downstream, to the promoter sequence to form a construct which may then be used to regulate the transcription of a gene. Examples of promoters which may be used include the SV40 early prcmoter.
Further provided by the present invention is a nucleic acid construct comprising a nucleic acid isolate or a construct comprising a regulatory element operably linked to a promoter sequence, each as c~.escribed above, operably linked to a heterologous gene, e.g. a coding sequence. By "heterologous" is meant a gene other than SCL. Generally, the gene may be transcribed into mRNA which may be translated into a peptide or polypeptide product which may be detected and preferably quantified following expression.
3S A gene whose encoded product may be assayed following expression is termed a "reporter gene", i.e. a gene which "reports" on promoter activity. The reporter gene may SUBSTITUTE SHEET (RULE 26) encode an enzyme which catalyses a reaction which produces a detectable signal, preferably a visually detectable signal, such as a coloured product. Many examples are known, including f3-galactosidase and luciferase. f3-galactosidase activity may be assayed by production of blue colour on substrate, the assay being by eye or by use of a spectrophotometer to measure _ absorbance. Luminescence, for example that produced as a result or luci~erase activity, may be quantitated using a luminometer. Radioactive assays may be used, for instance using chloramphenicol acetyltransferase, which may also be used in non-radioactive assays. The presence and/or amount of gene ;.=oducc resulting from expression from the reporter gene may be determined using a molecule able to ~5 bind the produc=, such as an antibody or fragment thereof. The bi::ding molecule may be labelled directly or indirectly using any standard technique. Those skilled in the art are well aware of a multitude of possible reporter genes and assay techniques which may be used to determine gene activity. Any suitable reporter/assay may be used and it should be appreciated that no particular choice is essential to or a limitation of the present invention.
Expression of a heterologous gene, e.g. a gene, the polypeptide produce of which is desirable for a therapeutic ef~ect or a reporter.,.gene from or. or more regulatory elements as provided may be in an in vitro expression system or may be intracellular (in vivo).
Expression generally requires the presence, in addition to the one or more regulatory elements, a promoter which initiates transcription, a translational initiation region and transcriptional and translational termination regions. One o. more introns may be present in the gene, along with mRNA processing signals (e. g. splice sites).
Systems for clcr:ing and expression of a polypeptide in a SUBSTITUTE SHEET (RULE 26) variety of different host cells are well known. Suitable host cells generally include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cells are preferred for use in the present invention. Mammalian cell lines available in the art for exaression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, human glioma cells, and may others, including other human cell lines. A commor_, preferred bacterial host is E.coli.
The present invention also provides a nucleic acid vector comprising a nucleic acid isolate as disclosed herein.
Such a vector may comprise a suitably positioned restriction enzyme site or other means for insertion into the construct of a sequence heterologous to a regulatory element therein. In the present invention, such a vector may be any vector suitable for the insertion of the one or more regulatory elements therein including phage, phagemids, plasmids, Yeast Artificial Chromosomes or Human Artificial Chromosomes. Suitable vectors can be chosen or constructed, containing appropriate further regulatory seauences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring-:Harbor Laboratory Press. Procedures for introducing DNA into cells depend on the host used, but are well known.
Thus, a further aspect of the present invention provides a host cell containing a nucleic acid vector comprising one or more regulatory elements as disclosed herein, operably linked to the whole or part o' a promoter sequence, or a heterologous gene, or both. A still further aspect crovides a method comprising introducing such a vector =nto a host cell. The introduction may SU8ST1TUTE SHEET (RULE 26) employ any available technique, including for eukaryotic cells, calcium phosphate transfection, DEAF-Dextran transfection, electroporation, liposome-mediated transfection and transduction using retrovirus. The introduction may be followed by causing or allowing expression of the heterologous gene under the control of the regulatory sequence or plurality o~ regulatory .
elements e.g. by culturing host cells under conditions for expression of the gene.
In a preferred embodiment of the present invention the vector comprising one or more regulatory elements as provided and one or both of promctsr sequence and gene, is integrated into the genome (e.g. c~Yomosome) of the host cell. Preferably, the inserted sequence adopts a chromatin confirmation analogous ~o that of native chromatin. Integration may be promoted by inclusion in the vector of sequences which promote recombination with the gene, in accordance with standard techniques.
A host cell according to the present invention, may be comprised (e.g. in the soma) wit~~in an organism which is an animal, particularly a mammal, whic':z may be human or non-human, such as rabbit, guinea pig, rat, mouse or other rodent, cat, dog, pig, sheep, goat, cattle or horse, or which is a bird, such as a chicken. Genetically modified or transgenic animals, ~a.rds or fishes (e. g.
Zebra fish or Fugu) comprising such a cell are also provided as further aspects of to present invention.
This may have a therapeutic aim. (Gene therapy is discussed below). The presence o. one or more additional regulatory elements) or mutant, allele or variant of such elements, within cells of an organism, particularly when in place of a homologous er_dogencus sequence, may allow the organism to be used as a model in testing and/or studying the role of the regulatory element, or the movie of control of the gene, cr substances which SUBSTITUTE SHEET (RULE 26) modulate activity of the one or more regulatory elements.
Instead of or as well as being used for the production of a polypeptide encoded by a gene under the control of a regulatory element or plurality of regulatory elements as provided herein, host cells may be used as a nucleic acid factory to replicate the nucleic acid of interest in _ order to generate large amounts of it. Multiple copies of nucleic acid of interest may be made within a cell when coupled to an ampli~iable gene such as DHFR. Host cells transformed with nucleic acid of interest, or which are descended from host cells into which nucleic acid was introduced, may be cultured under suitable conditions, e.g. in a fermenter, taken from the culture and subjected to processing to purify the nucleic acid. Following purification, the nucleic acid or one or more fragments thereof may be used as desired, for instance in a diagnostic or prognostic assay as discussed elsewhere herein.
As a further alternative, the nucleic acid isolate provided herein could be used in a method of gene therapy, comprising one or more regulatory elements to treat a patient who requires a particular active polypeptide, nucleic acid or other macromolecule. Vectors such as viral victors have been used in the prior art to introduce genes into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transfection can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide. The transfected nucleic acid may be permanently incorporated into the genome of each of the targeted tumour cells providing long lasting effect, or alternatively ~he treatment may have to be repeated periodically.
SUBSTITUTE SHEET (RULE 26) WO 991167$2 PCT/GB98/02914 A variety of vectors, both viral vectors and plasmid vectors, are known in the art, see US Patent No.
5,252,479 and WO 93/07282. In particular, a number of viruses have been used as gene transfer vectors, 5 including papovaviruses, such as SV40, vaccinia virus, herpesviruses, including HSV and EBV, and retroviruses.
Many gene therapy protocols in the prior art have been -used disabled murir_e retroviruses.
10 As an alternative to the use of viral vectors other known methods of introducing nucleic acid into cells includes electroporation, calcium phosphate co-precipitation, mechanical tecnr~icru~s such as microinjection, transfer mediated by liposomes and direct DNA uptake and receptor-15 mediated DNA transfer.
As mentioned above, the aim of gene therapy using nucleic acid isolates encoding a regulatory elements) as disclosed herein, or an active portion thereof, is to 20 alter (e.g.increase) the amount of the expression of the protein under tre control of these elements in cells in which the level of production in the wild-type is undesirable. Such treatment may be therapeutic in the treatment of cells which are already diseased or prophylactic in the treatment of individuals known through screening to have a genetic defect and therefore predisposed to certain medical conditions.
Gene transfer techniques which selectively target the regulatory element or regulatory elements to cell types in which the regulatory elements are able to function, e.g. to the blood stream, bone marrow, spleen, liver, cells of the haematopoietic lineage, endothelial cells or neuronal are preferred. Examples or this included receptor-mediated gene transfer, in which the nucleic acid is linked to a protein ligand via polylysine, with the ligand being specific for a receptor present on the SUBSTITUTE SHEET (RULE 26) surface of the target cells.
Many known techniques and protocols fcr manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA
into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Hiology, Second Edition, Ausubel et al. Eds., John Wiley & Sons, 1992, the disclosure of which is incorporated herein by reference.' Nucleic acid isolates, molecules, constructs and vectors according to the present invention may be provided isolated and/or purified (i.e. from their natural environment) in substantially pure or homogeneous form, free or substantially free of a SCL promoter or coding sequence, or free or substantially free of nucleic acid or genes of the species of interest or origin other than the regulatory element/elements. Nucleic acid according to the present invention may be wholly or partially synthetic. The term "isolate" encompasses all these possibilities.
Nucleic acid constructs comprising a regulatory element or plurality of regulatory elements (as disclosed herein) or regulatory element operably linked to whole or part of a promoter and/or heterologous gene (reporter), may be employed in screening for a substance able to modulate activity of a regulatory element or plurality of regulatory elements. For therapeutic purposes, e.g. for treatment of T-cell leukemia, a substance able to down-regulate expression of the SCL gene may be sought. A
method for identification of a substar_ce which binds to a regulatory element or regulatory elements may involve standard techniques such a band shift assays, site directed mutagenesis of the regulator~,r elements) to determine the exact binding site and transactivation SUBSTITUTE SHEET (RULE 26) WO 99/16782 PC"T/GB98/02914 experiments. A method of screening for ability of further substances to modulate activity of a regulatory elements) may comprise contacting an expression system, such as a host cell, containing a nucleic acid construct as herein disclosed with a test or candidate substance and determining expression of the heterologous gene.
The level of expression in the presence of the test substance may be compared with the level of expression in the absence of the test substance. A difference in expression in the presence of the test substance indicates ability of the substance to modulate gene expression. An i~crease er decrease in expression of the heterologous gene compared with expression of another gene not linked to a regulatory element cr regulatory elements as disclosed herein indicates specificity of the substance for modulation of the regulatory element(s).
A construct comprising one or more regulatory elements may be transfected into a cell line using any technique previously described or available to the skilled person to produce a stable cell line containing the reporter construct integrated into the genome. The cells may be grown and incubated with test compounds for varying times. The cells may be grown in 96 well plates to facilitate the analysis of large numbers of compounds.
The cells may then be washed and.-.the reporter gene expression analysed. For some reporters, such as luciferase, the cells will be lysed then analysed.
Constructs comprising one or more regulatory elements may be used to screen far a substance able to modulate the corresponding aspect of the regulatory element e.g. cell or lineage, or state specific e:cpression, or promoter specificity.
Following identizication of a substance which modulates SUBSTITUTE SHEET (RULE 26) or affects the activity of at least one regulatory element, the substance may be investigated further.
Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals. -Thus, the present invention extends in various aspects not only to nucleic acid isolates comprising regulatory elements and substances identified as modulators of a regulatory element or plurality of regulatory elements as provided by the present disclosure, but also to pharmaceutical compositions, medicaments, drugs.or other compositions comprising such substances; methods comprising administration of such compositions to a patient, e.g. for regulating expressions of a gene (for decreasing SCL expression, for instance in treatment of T
cell leukemia and other cancers associated with SCL), use of such substances in manufacture of a composition for administration, (e.g. for use in treatment of cancer?, and a method of making a pharmaceutical composition comprising admixing such substances with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
Administration will preferably ber:in a "therapeutically effective mount" this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated.
Prescription of treatment, e.g. decisions on dosage, etc, is within the responsibility of general practitioners and other medical doctors. A composition may be administered alone or in combination with of the treatments, either simultaneously or sequentially dependent upon the SUBSTITUTE SHEET (RULE 26) condition to be treated.
Pharmaceutical compositions according Lo the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well know-to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the r;ute of administration, which may be oral, or 'ay injection, e.g.
cutaneous, subcutaneous or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liqui~ form. A tablet may comprise a sold carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
For intravenous, cutaneous or subcutaneous injection, or injection at t:~:e site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicals such as Sodium Chloride Injection, Ringer's Injection. Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Instead of a substance identified using a regulatory element or plurality of regulatory elements or substance SUBSTITUTE SHEET {RULE 26) shown to bind to these sequences as disclosed herein, a mimetic or mimic of the substance may be designed for pharmaceutical use. The designing of a mimetic to a known pharmaceutically active compound is a known approach to 5 the developmen~ of pharmaceuticals based on a "lead"
compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g._ peptides are unsuitable active 10 agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design synthesis and testing may be used to avoid randor:ly screening urge numbers of molecules for a target property.
There are several steps commonly taken in the design of a mimet=c from a compound having a given target property.
Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or =esidues constituting the active region of the compound are known as its "pharmacophore".
Once the pharrnacophore has been found, its structure is modelled to according its physical properties, e.g.
stereochemistry, bonding, size and/or charge, using data 30' from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which modes the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process. In a variant of this approach, the three-dimensional s~ructure of the ligand and its binding partner are modelled. This can be especially useful where SUBSTITUTE SHEET (RULE 26) the ligand and/or binding partner change conformation on binding, allowing the model to take account of this for the design of the mimetic.
A template molecule is then selected o~~to which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optim_sation or modification can then be carried out tc arrive at one or more final mimetics for in vivo or clinical testing.
Mimetics of substances identified as having ability to modulate SCL promoter activity using a screening method as disclosed herein are included with_n the scope of the present invention.
Nucleic acid isolates cornprisir~g a regulatory element or plurality of regulatory elements according to the present invention, such as a full regulatory Q_ement sequence or fragment thereof (e. g. oligonucleotides as discussed earlier? may be provided as part of a kit, e.g. in a suitable container such as a vial~in which the contents are protected from the external environment. The kit may include instructions for use of the nucleic acid isolate, e.g. in PCR, screening for substances which bind to a regulatory element, gene therapy, or i3entification of homologous sequences. A kit wherein t::e nucleic acid isolate is intended for use in PCR may include one or more other reagents required for the r'action, such as polymerase, nucleosides, buffer solut'_~n etc. The nucleic acid may be labelled. A kit =or use y.. determining the presence or absence of a regulatory e'_'ment in a test SUBSTITUTE SHEET (RULE 26) sample may also include one or more articles and/or reagents for performance of the method, such as means for providing the test sample itself, e.g, a swab for removing cells prom the buccal cavity or a syringe for removing a blood sample (such components generally being sterile). In a further aspect, the present invention provides an apparatus comprising storage means including-the nucleic acid isolate comprising regulatory element or plurality of regulatory elements according to the present invention, the stored nucleic acid being used to compare the ability of the test sample to modulate SCL promoter activity, or to compare the sequence of the test nucleic acid to determi::e the presence of mutations.
The present application also discloses a new mapping technique. The method comprises using a frequent cutter restriction enzyme to identify in known sequences, sites analogous to HS sites (those highly sensitive to DNaseI) and comparing the deduced sites at which the enzymes could cleave the sequence (i.e. the correct recognition sites for the enzyme) with those actually cleaved. The latter, like HS sites, are likely to correspond to regions of DNA which are exposed, i.e. not "wrapped up"
in chromatin, and therefore indicate possible locations of regulatory elements.
Aspects of the present invention~:and experimental work relating there~o will now be further described with reference to the accompanying drawings, by way of example and not limitation. Further aspects of the invention will be apparent to those of ordinary skill in the art.
Generally, Figure 1 relates to the analysis of DNase2 hypersensitive sites associated with the murine SCL gene.
Hypersensitive mapping was performed.
Figure lA shows a partial restriction map of the murine SUBSTITUTE SHEET (RULE 26) WO 99116'782 PCT/GB98/029i4 SCL locus illustrating the probes (pl-4) used for DNaseI
hypersensitive analysis. The exons of the SCL gene are indicated with boxes, filled boxes representing coding exons. The restrictions sites are indicated using letter A, K, R where A is, ApaI; K is KpnI; R is Eco RI.
Figure 1B shows 5' hypersensitive sites detected by probe pl on ApaI restricted DNaseI - treated DNA in erythroid (F4N), primitive myeloid (M1) and T cell BW5147 (BW) cell lines. In the Figure, filled arrowheads represent germline fragments; open arrowheads represent fragments generated by DNaseI cleavage.
Figure 1C shows 3' hypersensitive sites detected by probe p2 on KpnI-restricted DNaseI-treated DNA.
Figure 1D shows hypersensitive sites within the SCL gene detected by probe p4 on Kpnl-restricted DNaseI-treated F4N DNA.
Figure lE shows hypersensitive site within the SCL gene detected by probe p3 on EcoRI-restricted DNaseI-treated M1 DNA.
Figure 2 shows the DNaseI hypersensitive sites associated with the murine SCL gene. The upper part shows hypersensitive sites identified i:~n erythroid (F4N), primitive myeloid (M1) and T cell BW5147 (BW) cell lines shown as arrows, where restricted sites are K, KpnI; B, BamHi; A, ApaI. The lower part of figure 2 illustrates the strategy used to study the function of the hypersensitive sites. Fragments encompassing the -lOHS, +7HS and +17/18 HS were inserted downstream of the luciferase (luc) gene in -0.2E3. The function of the -3/4.5 HS was studied by testing the activity of -7.OE3.
SUBSTITUTE SHEET (RULE 26) -0.2E3 refers to a fragment extending prom 0.2 kb upstream of exon la as far as exon 3. Similar terminology is used for other fragment positive numbers referring to sites downstream of example la.
Figure 3 shows the results of transient transfection assays of SCL promoter constructs. Transient transfections were performed using F4N, M1 and BW5147 cells as indicated. SCL promoter constructs are depicted to the left. Luciferase values are the mean (~SD) of at least four independent electroporations using two different DNA preparations.
Figure 4 shows the results of tranS~.2::~ transfection analysis of hypersensitive site function. Transient transfections were performed using F4N, M1 and BW5147 cells as indicated. The construes and their contained hypersensitive sites (HS) are indicated to the left. Fold activation values represent luci~erase activity (normalised against B-gal values) relative to background luciferase activity obtained using pGL-2b basic.
Figure 5 shows the results of stable transfection analysis of hypersensitive site '~..;:.ction. Stable transfections were performed using F4N, M1 and BW5147 cells as indicated. The constructs and their contained hypersensitive sites are indicated to the left.
Figure 6 shows the promoter speci_icity of the SCL 3' enhancer. Transient (A) and stable (B) transfection analysis in F4N cells of the +17/18HS in conjunction with SCL promoter la (-0.2E1a) SCL promoter lb (-0.2E1B) and SV40 early promoter (SV40).
Generally, Figure 7 relates tc ,.=e mapping of t~e +17 and +18 HS sites.
SUBSTITUTE SHEET (RULE 26) Figure 7A shows the relative positions, sizes and orientations of fragments inserted downstream of a -0.2E3/luc construct. Restriction sites are A, ApaI; B, BamHI; Bg, BgiII; H, HindIII K, Kpr.I; S, Sau3AI.

Figure 7B shows the results of transient transfection analysis of the constructs illustrated in 7A. Transient transfections were performed as before, using F4N cells.
Fold activation vales represent luciferase activity 10 (normalised against a-gal values) relative to the luciferase activity obtained using 0.2E3.
Figure R shows sequence alignments of mouse and human SCL
DNA. Black boxes indicate regions of complete 15 conservation; g=ey boxes indicate positions at which one sequence has a:: adenine nucleotide and the other a guanine. Figures 8A to 8Ei show regions of high homology correspc:.ding t~ the -10, -4.5, -, +l, ~3, +7, +17 and +i8 S sites respectively.
Figures 3A and ~3 show PCR amplification fragments contai~~i~:g similar :nurine DNA sequences to those shown in the sequence alignments of Figures 8G and Sri, and corresponding t-~ the +17 HS and +18 HS sites respectively. ?rimers used are ~.etailed in Example 6.
Figure i0 shows the results of a novel endonuclease accessibility assay. Solid arrow indicates probe hybri~'ising with o '.~cb germline Sari fragment. Hollow arrow i::dicates p=obe hybridising with a 2.5 kb fragment 5' to t~:e Saci fragment, with increasing concentration of 2.5 kb _=agmer_t °~rith increasir:g HaeIII concentration.
Dashes on the exploded 6 kb fragment indicated deduced HaeIII =estriction sites. Upward arrow indicates site at which c_eavage occurs to produce 2.5 kb fragment, which SUBSTITUTE SHEET (RULE 26) WO 99/16782 PC'T/GB98/02914 corresponds to the +18 HS.
Figure 11 shows the expression in different mouse embryo tissues of LacZ reporter constructs cc:~taining different promoters and with or absent a murine SCL fragment carrying the +17 and +18 regulatory regions. The abbreviation AGM refers to embryonic aorta, gonad and mesonephros tissue.
Figure 12 shows the e:~pression in different mouse embryo tissues (Figure 12B) ef LacZ reporter constructs containing a series o. SCL inserts con=aining different promoters and different regulatory regions. The structure of these constructs is s.~ow~ =n Figure 12A.
Figure 13 shows deletion analysis of ~n~ 5' regulatory regions of SCL. (A) Diagram of the SC~ gene as described in Fig.l3A, with deletion constr~,:cts s:~own below.
Endothelial, midbrain and hindbrain/s~.=nal cord enhancers are indicated. (B) to (H) Whole mour.~ X-gal stained 12.5 dpc transgenic embryos snowing r~pres~-:~ative staining Tor the indicat'd ccr.structs. iv, _nte=somitic vessels;
rne, mesencephaion; sp, spinal cord.
Figure 14 illustrates a functional analysis of the SCL
elements between -7kb and +2.8kb. (A) Diagram of the murine SCL locus with DNase I hypersensitive sites indicated by arrows with site locations indicated in kb from the transcriptional start sits a~ exon la. Open and closed boxes represent untranslated antranslated exons of SCL respectively, with exon numbers below. Construct -7E3/lacZ is sown to scale. (B) 'nlho~.e mount 12.5 dpc embryo from a -7F.,3/lacZ transgenic li~e stained for lacZ
expression.. =acZ staining can be see~ in the mesencephalon line), blood vessels (v) and spinal cord SU8ST1TUTE SHEET (RULE 26) (sp) with ectopic expression in the ear placode. No staining is visible in fetal liver (fl). (C) Saggital section (100um) of the embryo shown in B, demonstrates staining in diencephalon (d), rnesencephalon (me), heart (h), spinal cord (so) and blood vesse~s (v). mt, metencephalon; my, myelencephalon; t, telencephalon. (D) Autoradiograph of a wild type 1~.5 dpc embryo hybridised with an SCL specific oligonucleotide. (E) Nissl counterstain of the=sectien in D.
la Figure 15 illustrates vascular and haematopoietic expression o= t:~:a -%F3/lac.Z tra::saer.~. (A) Lateral view of a 7.5 dpc embr,~o shows IacZ e_~aression in extraembryonic mesoderm (xm). ~;B) Ssction through embryo from Fig.l5A demonstrating lacZ e:~oression in extraembryonic mesoderm (xm). ec, ectcplacental cavity, ex, exocoel; ps, primitive streak. ',~) Rostro-ventral and (D), caudo-ventral view of 8.0 d~c embryo shows staining in paired dorsal aortae 'daj, endocardium of the heart primordium (e), allantois (al), and presumptive angicblasts ~:~rYowheads) para'lel 'c t:~e aortae. ys, yolk sac; ~., headfcl::s. (E) 5aggit~~ section of embryo from Fig.lSC shows lacZ expression in the endocardium (e), dorsal aorta (daj, angioblasts (arrowheads) and forming vessels (a) in the cephalic mese;ychyme (cm). ne, neuroectoderm. (F) Section of embry_~, from Fig.ISC
showing clusters of presumptive angiobiasts (arrowheads) at the base of the ailantois (al). ne, neuroectoderm, en, endoderm; md, :~esoderm. (G) Exoressv~on cf IacZ in the developing 8.5 dpc vasculature showing staining in the looping embryonic heart !:), dorsal aortae (da) and fused allantoic stalk (al). (H) Ganeral vascular staining in a 9.5 dpc embryo ~rith prom~.nent staff.~.i.~.g in the interscmitic -.essels (arrowheads'. ,dote absent staining in ;yolk sac _sj. (Ij Transverse section of 18.5 dpc SUBSTITUTE SHEET (RULE 26) fetal tho=ax, shows strong staining in the endothelium of the aorta ;ao) ar_d pulmonary artery (pa) emanating from the hear. (~), w'_th considerably Sneaker staining in the endotheli::m of t:~e superior vena cava (svc) and pulmonary vein (pv). Weak expression is also seen in the endocardwu:~ (e). (J) Blood island cf the embryo in Fig.lSC s:~ows bot~ haematopoietic (hc) and endothelial (ve) stai::i ng. ~C) Clusters of stained cells attached to 10.5 dpc lateral endbthelium (ve) of the dorsal aorta.
(L) Mult~.:~ucleare cell with megakaryocytic morphology and (M), small haema~opoietic-like cells positive for lace expressi~:~ in a =2.5 dpc fatal li~;~r.
Figure 1~ illust=arcs neural expressicn of the -7E3/lacZ
transgene. (A) ~ateral view of ti-:e head of a 10.5 dpc transgenvc embry: showing the firs stage at which neural expression: is observed. LacZ expression is shown within the posterior co~-;~~nissure (pc) and in rostro-ventral mesencephalon (me). t, telencephalc:~; ~?, diencephalon;
mt, mete..~.cephalc~; my, myelencepha~_or,; ey, eye. (B) Saggital secticr. of the 1I.5 dpc ~:~bryc in Fig.l6C with arrows _ _ indica ~e ~r2r.tral me tencewna'~an (mt ) , myelencec~alon (:'.iy) and spinal con (sp) staining. (C) Lateral -view of 11.5 dpc brain showing strong lacZ
staining in the bosterior commiss~u=a with rostral extensio.~. into the diencephalon (arrowhead) and caudal extensio:: of expression in the ve~tral mesencephalon.
Note the intertectal axons (a) projecting towards the dorsal m=d-line. ~~Ihite bars show the locations of the coronal sections shown in Figs.loD, E, F. (D), (E) and (F) Coro::al sections through the posterior commissure and mesencep::alon. c, cell bodies; a, axons; v, ventricle.
(G) Dorsal view of a 12.5 dpc heat, snowing la::Z
expressi~r: in .a posterior ccmmissure (pc) and ventro-lateral ~~ssencev:~alon (rne). Tnter=octal axons are SUBSTITUTE SHEET (RULE 26) crossing the dorsal midline anteriorly but n-ot posteriorly. (H) Coronal section through 12.5 dpc mesencephalon showing axons crossing tie dorsal mid-line of the tectum (tc). (I) Transverse section through 12.5 dpc spinal cord shows lacZ expression in the mantle layer (ml) and in developing vessels within surrounding rnesenchyme (m). drg, dorsal root ganglion; el, ependymal layer. (J) Whole mount 18.5 dpc brain shows continued lacZ expression within the mesencephalor. (me), spinal cord (sp) and caaillary plexus surrouncing the brain (ep). cb, Ce=ebellum; t, telancenhalon.
EXAMPLES
Example 1 Identification of DNase I Hypersensitive Sites To identify regulatory elemer_~s important for SCL
expression the inventors mapped DNase _ hypersensitive sites in a region of 45kb er_compassing the marine SCL
gene. They used two different types of SCL expressing cell; marine erythroleukemia cells (F4V) which correspond to committed erythroid precursors between the CFU-E and BFU-E stages of differentiation (Dube, S,K et al. PNAS
USA 72: 1863, 1975) and primitive. myeloid cells (M1) positive for the stem cell antigen CD34 (May, G et al.
EMBO J 14: 564, 1995). Ml cells can be induced to differentiate into monocytes and so the inventors realised that their analysis may provide clues not only to the regulation of SCL in primitive CD34 positive cells, but perhaps also to the mechanisms responsible for SCL down-regulation following commitment to the monocyte lineage. In addition the inventors analysed a T cell line (BW5147) which dces not express SCL.
SUBSTITUTE SHEET (RULE 26) The murine erythroid cell lines F4N and J2E, the murine T-cell line BW5147 and the murine CD34 positive cell line M1 have been described previously (Bockamp, E, et al.
Supra 1995; Ichikawa Y. J Cell Physiol 74: 223, 1969).
5 BW5147 and M1 were cultured in RPM1 1640 plus 10% foetal calf serum and F4N in DNEM plus l0% foetal calf serum.
Approximately 3x10a cells were rinsed in PBS and resuspended in 2om1 of homogenisation buffer (lOmM Tris 10 pH 7.4, lSmM NaCl, 0..15mM spermine, 0.5 mM spermidine, 1 mM EDTA, 0.1 mM EGTA, 0.2% NP-40, 5% sucrose). After Dounce homogenisation, the homogenate was spun through a 10o sucrose lays= and resuspended in buffer (lOmM Tris pH
7.4, 15 mM NaCI, 60 mM KC1, 0.15 mM spermine, 0.5 mM
15 spermidine). Alter adding DNaseI (Sigma) (0.8 - 16 ug), samples were incubated at 37°C in the presence of 50 mM
MgCl2 and 50 mNl CaCl~ for 10 minutes. Reactions were stopped by adding 12.5 mM EDTA and 1~ SDS and the DNA was isolated following proteinase K digestion/phenol 20 extraction and ethanol precipitation. After digestion with restriction enzymes, the DNA was analysed by Southern blotting. Probes used were as follows: pl - a 220 by Sau3AI/Acal fragment 50 by upstream cf exon Ia; p2 - a 596 by XmnI/KpnI fragment from exon VI; p3 - a 1200 25 by HindIII fragment 260 by upstream of the EcoRI site in exon VI; p4 - a 810 by KpnI/SacI fragment extending 3' of SCL exon VI.
The strategy tc map the DNase I hypersensitive site is 30 shown in Figure lA. Figure 1B shows DNase I
hypersensitive sites upstream of the SCL gene. Probe pl (Figure lA) was hybridised to a Southern blot of DNase I
treated DNA digested with ApaI. In addition to the germline fragment of approximately 23 kb (closed 35 arrowhead), ore additional band was cbserved in F4N cells (-lOHS) and 3 additional bands in M1 cells 1-lOHS, -4.5HS, -3HS), w zereas no DNase I hypersensitive sites SUBSTITUTE SHEET (RULE 26) were detected in the T cell line BW5147. DNase I
hypersensitive site nomenclature refers to the position of the hypersensitive site relative tc the beginning of exon la.
DNase I hypersensitive sites downstream of the SCL gene are shown in Figure 1C. DNase 1 treated DNA was digested.
with KpnI and hybridised with probe p2 (Figure 1A). In addition to tre l0 kb germline band (closed arrowhead), two bands of approximately 3 and 4 kb were evident in F4N
cell (denoted +17HS and +18HS). No hypersensitive sites downstream of the SCL gene were detected in Ml or in BW514 7 .
To study DNase ~ hypersensitive sites within the body of the SCL gene, DNase I treated DNA was digested with either KpnI (Fig 1D) or EcoRI (Fig lE). The pattern of hypersensitive sites in F4N cells was best demonstrated using a KpnI digest followed by hybridisation with probe p3 (Figure lE and data not shown).
In addition to the germline band of 20 kb, bands of approximately '_2, 10 and 6 kb were observed (Figure 1D).
The 12 kb band corresponds to the promoter region, the 10 kb band corresponds to a hypersensitive site upstream of exon 3 (+3HS) and the 6 kb band corresponds to a hypersensitive site at the beginrr~,.ing of exon 4 (+7HS). In analogous Southern blots of M1 and BW5147 DNA, the +7HS
but not the +3HS was evident in M1 ce p s and no hypersensitive sites were detected in BW5147 cells (data not shown). The +7HS could also be detected in M1 and F4N
cells using an EcoRI digest and hybridisation with probe p3.
The experiments therefore showed hypersensitive sites (+3HS, +17HS, -18HS) to be present in erythroid cells, but not in CD34 positive myeloid cells or in T cells. To SUBSTITUTE SHEET (RULE 26) confirm the generality of these results a second erythroid cell line (JE2) generated in a completely different way was studied. The F4N cell line used above was derived from a marine erythroleukaemia generated by neonatal infection with the friend marine leukaemia virus complex (Dube, S.K., et al. 1975 Supra). The J2E cell line was derived by in vitro infection of marine foetal .
liver cells with a replication incompetent retrovirus containing myc and raf (Klinten S.P, et al. PNA USA 85:
8506, 1988). J2E cells are growth factor independent and undergo terminal erythroid differentiation in response to erythropoietin. The pattern cf DNase T_ hypersensitive sites associated with the SCL gene was identical in F4N
and J2E cells (data not shown).
The DNase I hypersensitive sites associated with the SCL
gene are summarised in the tcp part of Figure 2.
Erythroid and CD34 positive primitive myeloid cells have shared and unique hypersensitive sites, whereas no hypersensitive sites were found in T cells. These data indicate that distinct combinatior_s of regulatory elements control SCL expression in different haematopoietic cell types.
Example 2 Preparation of Reporter Constructs Luciferase reporter constructs were generated in pGL-2 basic (Promega). SCL genomic DNA contained in the -0.2E3, -7.OE3 and +?HS inserts was subcloned from a phage Balb/c genomic library (Begley, D.G, et al. 1994 Supra). The insert in -0.2E3 extended from an EspI site 187 by upstream of exon Ia to a XcmI site 91 by into exon III, the -7.0E3 insert from a KpnI site 7 kb upstream of exon Ia to the same Xcml site in exor_ III and the +7HS insert SUBSTITUTE SHEET (RULE 26) from a Sau3AI site 1 kb upstream of exon IV to an NdeI
site 75 by into exon V. The +17/18HS insert was subcloned from a 129a fix genomic library (Stratagene) and extended from a BglII site 670 by upstream to a Sau3AI site 4.5kb downstream of the poly A site. The 3' insert in -0.2E3 extended from the same BglII site to a BamHI site 2.6kb 3' of the polyA site. The 3' insert in -0.2E3/d extended.
from the same 3' BamHI site to the Sau3AI site 4.6 kb downstream of the polyA site and the 3' insert in -0.2E3/e from the same BamHI site to a HindIII site 4.2 kb downstream of the polyA site. The 3' inserts in -0.2E3/a and -0.2E3/b contained a 2.5 kb HindIII fragment extending from 1.7 to 4.2 kb downstream of the polyA site in opposite orientations. The -lOHS insert was subcloned from a P1 clone isolated from a C57/B16 genomic library (Zehetner, G et al Naure 367: 489, 1994) and extended from a BgIIII sits at -10 kb to a KpnI site at -7 kb.
Example 3 Transient transfection assays The SCL regulatory elements were assessed using transient luciferase reporter assays. Additional genomic DNA
fragments containing the -lOHS, +17HS and +18HS were isolated since these were not contained in the phage contig previously described (BegJ~ey, C.G et al, 1994 Supra). Luciferase reporter constructs generated in pGL-2 basic as described above, were introduced into F4N and BWS147 cells.
Transient transfections were carried out as described (Bockamp, E-O, et al. 1995 Supra) with the exception that only 6~,g of the pEF-BO~ lacZ vector was used for normalisation of each pulse. Also, in order to adjust for the large size differences between tre reporter constructs used _n this study, the molar amount of SUBSTITUTE SHEET (RULE 26) reporter construct added to each pulse was kept constant using 10 ~.g of pGL-2 basic and proportionally more of the larger constructs. The total amount of DNA added to each pulse was kept constant by adding the required amount of the plasmid pGEMIIZF+ (Promega). Luciferase and f~-galactosidase assays were carried out as described (Bockamp E-O, et al. 1995 Supra.) The relative light units presented are the mean of at least four independent experiments and the results obtained were confirmed using a second DNA preparation for each construct.
In F4N cells the core promoter lb (+0.2E1b) was silent whereas the core promoter la (-0.2E1a) was modestly active (Figure 3). A construct containing both promoters with the luciferase gene fused into exon 3 (-0.2E3), and which therefore contained the +3HS, was approximately 4 fold more active. A very different pattern was obtained in M1 cells. Promoter la was inactive, promoter lb was strongly active and the inclusion of sequences between axon lb and axon 3, which contained the +3HS, had no effect. In T cells all three constructs procured low levels of luciferase activity relative to F4N or M1 cells. These results are consistent with the +3HS acting as an enhancer in erythroid but not in primitive myeloid cells. The data could also reflect a lineage specific effect on RNA processing and/or stability resulting form the inclusion of introns in the -.Ø2E3 construct. This seems unlikely, particularly because the parental pGL-2 basic vector already includes an intron. Additional regulatory elements might act on promoter la or promoter lb or both. Both promoters were therefore included in subsequent constructs. The complex alternative splicing pattern associated with the 5' untranslated axons of the murine SCL gene results in axon 3 being the first axon that is shared between transcripts originating at promoters la and lb. The effect of the other hypersensitive sites on the activity of -0.2E3 was also SUBSTITUTE SHEET (RULE 26) studied since this construct contains both SCL promoters together with a physiological splice acceptor at the beginning of exon 3. The strategy that was adapted is summarised in the lower part of Figure 2. DNA fragments S containing the -lOHS, +7HS and +17/18HS were subcloned downstream of the iuciferase gene in -0.2E3. The -3HS and -4.5HS were tested by including 7kb of sequence upstream-of exon la (-7.03E). The effect of all these hypersensitive sites on the activity of -0.2E3 in 10 transient reporter assays is shown in Figure 4. In F4N
cells the -lOHS, -3/4.5HS and +7HS all resulted in approximately two fold enhancements. T~:e most pronounced effect was seen with the +17/18HS whic~ resulted in a six fold increase over the already considerable activity of -15 0.2E3 and 240 fold increase relative to pGL-2 basic. In M1 cells a very different pattern was obtained. Inclusion of the -3/4.SHS (construct -7.OE3) produced a two fold increase in activity relative to -0.2E3 alone, whereas the -lOHS, +7HS and +17/18HS had no detectable effect. In 20 BW5147 cells, which do not express endogenous SCL mRNA, all of the constructs produced only low levels of activity. Taken together these data show that the -lOHS, +7HS and +17/18HS functioned as enhancers in F4N but not M1 cells, whereas the -3/4.5HS functioned as an enhancer 25 in both F4N and M1 cells.
Example 4 Stable transfection assays Chromatin structure places important constraints on the functional activity of transcriptional regulatory elements (Felsenfeld, G. Nature 355: 219, 1992; Lewis, B.
Cell 79: 379, 1994; Ernst, P et a1. Immunity 2:
311,1995). Therefore the activity of the various SCL
regulatory elements following integration into chromatin was assessed. The same series of reporter constructs used SUBSTITUTE SHEET (RULE 26) for the transient transfection assays were introduced into F4N, M1 and BW5147 cells and pools of stably transfected cells were derived. Analysis of pools rather than individual clones was performed to minimise position effects. Pools of cells carrying pGL-2 basic were used as negative controls, and pools carrying pA3RSVluc (in which luciferase is driven by the RSV LTR) were used as positive controls.
3 x 10' F4N, BW5147~or M1 cells were electroporated (960~,Fd, 250 V) with 3 ~.g of linearised PGK purpoPa vector (an alternative vector which could be employed is pMCI available from 'Stratagene' with subsequent neomycin selection) and a tenfold molar excess of the linearised Z5 luciferase plasmid. After 24 hours, electroporated cells were transferred into selective medium containing 2~.g/ml (F4N and BW5147) or 10 ~.g/ml (M1) puromycin (Sigma).
Cells electroporated with SCL reporter constructs were maintained as 3 independent pools.
Puromycin resistant pools were derived 2-4 weeks following electroporation and luciferase assays were performed using extracts derived from 3 x 10' cells for each assay point as described above. For each experiment, assays were performed in duplicate on each pool and a positive control (a pool of cells.:transfected with pA3PSVluc) and a negative control (a pool of cells transfected with pGL-2 basic) were included in each experiment. This experiment was repeated on 3 separate occasions for each SCL construct. Results were expressed as fold elevation over the negative control. In addition, this relative luciferase activity was normalised for luciferase DNA content by Southern blot analysis of BamHl and HindIII digested DNA. Filters were probed with a non-repetitive sequence from the mouse vav gene locus (A.
Elefanty et al, unpublished results) to normalise DNA
SUBSTITUTE SHEET (RULE 2S) loading and with the HindIII/BamHl fragment of the luciferase gene from pGL-2 basic to normalise the luciferase DNA content of test pools to the control pool carrying pGL-2 basic.
Both core promoter la and core promoter lb display minimal activity following stable transfection into F4N
cells (data now shown). By contrast, -0.2E3 which contained both promoters and the +3HS give rise to a 88 fold increase in luciferase activity relative to pGL-2 basic (Figure 5). Incorporating the -lOHS did not significantly alter the activity of -0.2E3. However, the +7HS and -_7/18HS Beth further increased the relative luciferase activity of -0.2E3 to 225 and 250 respectively, values which were well in excess of those obtained using the positive control plasmid, pA3RSVluc (data not shown). The -7.OE3 construct (containing the -3/4.5HS) displayed reduced activity relative to -0.2E3.
These results raise the possibility of a negative regulatory element in the vicinity of the -3/4.5HS in erythroid cells, active after chromatin integration.
In M1 cells the two core promoters were again effectively silent following integration in chromatin, whereas the -0.2E3 construct was strongly active and produced a 79 fold increase in luciferase activity over background (Figure 5l. This indicates that a<positive element in the vicinity of the +3HS was functional in M1 cells as well as in F4N cells. Further experiments will be required to determine whether this element is the same as the one active in F4N cells. The -10HS had no effect on the function of -0.2E3, but inclusion of the +17/18HS
resulted in a 20 fold fall in activity. Even more striking were the effects of the -3/4.5HS and the +7HS
both of which effectively abolished expression from -0.2E3.
SUBSTITUTE SHEET (RULE 28) WO 99!16782 PCT/GB98102914 Much lower levels of luciferase activity were obtained in BW5147 cells. The -0.2E3 construct produced only background activity. The maximum activity obtained was 35 fold over background, and this was achieved using -7.OE3.
Taken together with the transient transfection data, these results identify the key components of a complex interacting network of regulatory elements that control the pattern of SCL expression during haematopoiesis. Four elements are of particular significance. The +17/18HS
elements acted as a potent erythroid enhancer in transient and stable assays, yet had chromatin-dependent silencer activi~y in primitive myeloid cells. The +7HS
functioned as a powerful chromatin-dependent silencer in primitive myeloid cells, but as an enhancer in erythroid cells. The -3/4.5HS displayed chromatin-dependent silencer activity in M1 and F4N cells. The DNA fragment containing the -3/4.5HS also exhibited modest enhancer activity in transient assays in both cell types. This may reflect the presence of two functionally distinct elements within the DNA fragment. The +3HS functioned as a chromatin-dependent enhancer in M1 cells. In F4N cells, inclusion of tre +3HS produced a modest (4 fold) enhancement relative to promoter la (Figure 3). However in stable assays inclusion of the +3HS produced a 100 fold increase in activity relative to promoter la (Figures 5 and 5). These data suggest that integration in chromatin is required for full activity of the +3HS in both M1 and F4N cells. It is interesting to note that the DNA fragments containing the +3HS and +17/18HS functioned as potent regulatory elements in M1 and F4N cells and yet the hypersensitive sites themselves were only detected in F4N. Perhaps the endogenous +3HS and +17/18HS elements are functionally active in M1 cells but have adopted a chromatin structure that is not hypersensitive to DNaseI.
Alternatively, it is possible that the endogenous +7HS
exerts a strong bidirectional silencing activity in M1 SUBSTrTUTE SHEET (RULE 26) cells which "shuts down" the flanking +3HS and +17/18HS.
Exa~le 5 The 3' enhances displayed promoter specificity The -0.2E3 construct contained both promoter la and promoter lb and so the 3' enhances may have been interacting with one or both SCL promoters. To address the promoter specificity of the 3' enhances, a further series of constructs were therefore generated containing promoter la, promoter lb, or the SV40 early promoter together with the +17/18HS element. As can be seen from Figure 5A, in transient assays using F4N cells the +17/lBFiS produced a two fold increase in the activity of the SV40 promoter but had little or no effect on SCL
promoter la or 1b. There is the possibility that the 3' enhances might reauire integration in chromatin for some aspects of its activity. The same constructs were therefore used in a series of stable transfection experiments in F4N cells (Figure 6B). These revealed a marked enhancing effect of the +17/18HS on the SV40 promoter and on SCL promoter 1a but only a minimal effect on promoter lb. The results show that the +17/18HS
element exhibits promoter specificity and can distinguish between the two SCL promoters.
Discussion of Examples 1 - 5 The inventors have studied the regulation of the murine SCL gene. They have characterised distant regulatory elements in cell lines representing three haematopoietic cell types: committed erythroid cells which express SCL;
CD34 positive primitive myeloid cells which also express SCL; and T cells which do not express SCL. The results demonstrate that the two SCL expressing cell types exhibited overlapping patterns of DNase I hypersensitive SUBSTrfUTE SHEET (RULE 26) sites, with each cell type displaying both unique and shared sites. Furthermore the DNase I hypersensitive sites flagged the positions of both positive and negative regulatory elements that functioned in a cell type-s specific manner. Four of these elements are of particular interest.
The +17/18HS element functions as a potent erythroid 10 enhancer. Sequence analysis has demonstrated the presence of several GATA motifs in the region of the +18HS (data not shown) and so this 3' enhancer is likely to be GATA-1 responsive in erythroid cells. M1 cells express GATA-2 (but no GATA-1) yet lack the +17/18HS, and so GATA-2 15 appears insufficient for either the formation or the enhancer activity of the +17/18HS. The 3' enhancer was found to up-regulate SCL promoter la but not SCL promoter lb.
Promoter la, but not promoter lb, contains functionally 20 important GATA-1 sites (Aplan, P.D et al. 1992, Supra;
Lecointe, N et al. Oncogene 9: 2623, 1994; Bockamp, E-O, et al. 1995 Supra). This may be significant since it has been suggested that interactions between a promoter and its cognate enhancer can be mediated by GATA-1 self-25 association (Crossley, M et al. Moi Cell Biol 15: 2448, 1995). As well as acting as an erythroid enhancer the 5kb fragment containing the +17/18HS~~also functioned as a silencer in M1 cells. The +17/18HS enhancer may function as two discrete elements or a single bifunctional element 30 capable of responding differently to the transcriptional environment present in the two cell types.
The characteristics of the +3HS element were very different from the 3' enhancer. In transient assays the 35 +3HS was modestly active in F4N cells but was inactive in M1 cells. By contrast it was very active following stable transfection experiments in both of these cell types.
SUBS"f1~'I~~'E SHEET (RULE 26) Full activity of the +3HS was therefore chromatin dependent and did not require the presence of GATA-1. The sequence immediately upstream of exon III is highly conserved between the human and murine loci, suggesting the presence of an important regulatory element.
The +7HS and -3/4.5HS marked the sites of potent negative regulatory elements which completely abolished the enhancing effect of the +3HS on the SCL promoter region in M1 cells. In addition the +7HS functioned as an enhancer in F4N cells. As with the +17/18HS, it is not clear whether this reflects the presence of two separate elements or a single bifunctional element. The presence of silencers functioning in M1 cells but not in F4N cells is consistent with the observation that SCL mRNA levels in M1 cells were several fold lower than those in erythroid cells (Green, A.R et al. Oncogene 6: 574, 1991) and suggests that down regulation of SCL following commitment to non-expressing lineages is not merely a passive consequence of GATA-1 extinction. The fact that the endogenous SCL gene was transcriptionally active in M1 cells implies that the effect of the silencers is countered by one or more positive elements which remain to be identified.
Chromatin structure appears to play a pivotal role in regulating gene transcription (F'~lsenfeld, G et al. 1992, supra; Lewin, B et al. 1994 supra; Ernst, P, et al. 1995 supra). Several cis-acting regulating elements including locus control regions (LCRs) and silencers appear to function by influencing chromatin structure within a region and are frequently dependent upon integration in chromatin for their activity. As a result, two broad classes of enhancer are now recognised. The first of these are the classical enhancers which do not require to be integrated in chromatin for their activity and which therefore function well in transient reporter assays.

Activators binding these elements interact directly or indirectly with components of the general transcriptional machinery at the promoter (Kinstong, R.E, et al. Curr Biol 4: 325, 1994; Zawel, L et al. A Rev Biochem 64: 533, 1995). The second category consists of chromatin-dependent enhancers which exhibit little or no activity in transient assays but function well in stable _ transfection or transgenic experiments. Examples include the f3-globin HS3 and 4 (Tuan D.H, et al. PNAS 86:
2554,1989; Eraser, Pet al. Nucleic Acids Res 18: 3503, 1990; Pruzina, S, et al. Nucleic Acids Res 19: 1413, 1991) the 5kb MyoD enhancer (Tanscott, S.J, et al.
Molecular and Cellular Biology I2: 4994, 1992) and the 5' enhancer of the CD34 gene (May, ~, et al. 1995 Supra). At least some of these elements have been shown to influence chromatin structure. However, the functional distinction between chromatin-dependent and chromatin-independent enhancers is not straightforward. Thus, the f3-globin HS2 and metallothionein enhancer can both function well in transient assays but recent evidence also suggests that they suppress the development c~ repressive chromatin structures (Waiters, M.C, et al. Genes & Development 10:
185, 1996). Perhaps in these cases the effect on chromatin structure is indirec~ and secondary to altered transcriptional activity of the locus, whereas chromatin-dependent enhancers directly modulate chromatin structure.
The data demonstrates the existence of at least three SCL
enhancers with contrasting degrees of chromatin dependence. The SCL +3HS displayed little or no activity in transient assays in F4N or ~!i cells respectively, but was strongly active following _:~tegration in chromatin in both cell lines.
The SCL locus also contained three potent lineage-specific arid chromatin-dependent silencers in the SUBSTITUTE SHEET (RULE 26) vicinity of the -3/4.5HS, +7HS and +17/18HS. Silencing was first described in yeast (Brand, A.H et al. Cell 41:
41, 1985) but has subsequently been extensively analysed in flies (Faro, R. Genetics 6: 416, 1990; Bienz, M et al.
Bioessays 17: 775,1995). In Drosophila, evide__~_ce has been accumulating for two classes of cis-acting negative regulatory elements which respectively initiate or .
maintain repression. The members of the first category initiate repression early in development, function transier_tly, act at~short range to repress closely linked enhancers or promoters, and in cell specific proteins such as hunchback, Kruppel, even skipped and engrailed (Bienz, '~, et al. 1989 Supra; Biggin, M.D, et al. Cell 58: 433, 1989; Jaynes, J.B et al. EMBO J 10: x.427, 1991).
~15 The second category is more similar to yeast silencers in that its members confer long-term silencing and function over large distances. Yeast silencers were originally defined as being position-independent, orientation-independent and capable of repressing heterologous promoters (Brand, A.H, et al 1985 Supra).
However, it is now apparent that silencers are functionally heterogeneous and that some may be position-dependent (Vacher, J et al. Science 250: 1732, 1990) orientation dependent (Zink, D et al. EMBO J 14: 5660, 1995) or may exhibit promoter-specificity (Busturia, A, et al. EMBO J 12: 1415, 1993; MuZ~ler, J, et al. EMBO J
14: 1209, 1995). The SCL -3/4.5HS, +7HS and +17/18HS
resemble yeast silencers and the second category of Drosophila negative regulatory elements since they operate over several kilobases and are chromatin-dependent. These results suggest that they are likely to function by directly influencing chromatin structure as has been invoked for the regulation of matir_g type in S.cerevisiae (T~aurenson, P, et al. Microbfiol. Rev 56:
543, 1992) and homeotic genes in Drisophila (faro, R.
1990 Supra; Bienz, M, et al. 1995 Supra).
SUBSTITUTE SHEET (RULE 26) Example 6 Mapping of the +17/+18 hypersensitive sites The marine SCL gene was sequenced and the location of further restriction enzyme sites within the gene were deduced. The location of the +17/+18 HS sites within the HgIII/Sau3AI f=agment of Example 2 was further investigated by cloning smaller restriction fragments into the -0.2E31uc reporter construct and assaying luciferase activity in the transient transfection assay.
The position, orientation and size of the restriction ~ragmer.ts and the results of the trans=ection assay are shown at a to a i.~, Figure 7A and 7B respectively. The insert in -0.2E3/e is a BamHI/HindIII =ragment of l.5kb.
'nlithin this fragment is an Apal restriction site defining a BamHI/ApaI sequence of 1.23kb.
0.2E3/a, 0.2E3/b and 0.2E3/c all contain a HindIII/BamHI
sequence comprising +17HS which is substantially lkb.
-0.2E3/f contains a 514bp insert ~~om '7.01 to 17.53kb 3' of -3I GATA site. This fragment was c~~ned after ?CR
amplification (template: -0.2E3/a primers: forward 5'AACAGCTGCAGAGTGCTGGATTACAGG 3' reverse 5' AAAGGATCCAGTGGGAGGAGAAGGGCTG 3'. .,, -0.2E3g contains a 434bp insert from 13.08 to 18.52kb 3' of -37 GATA site. This fragment corresponds to a genomic NcoI/AscI fragment contained within t:~.~ downstream insert of -0.2E3/a.
Regions of interest or the human SCL ge.~.e, generally corresponding to those regions of interest found in the marine gene, were sequenced. Comparison of the marine and human nucleic acid sequences showed a high degree of ° SUBSTITUTE SHEET (RULE 26j homology in, among others (see Example 8), two regions located at approximately +l7kb and +l8kb (numbers refer to position downstream of the murine exon la). Sequence alignments are shown in Figures 8G and 8H. A 164 by 5 double stranded fragment of mouse DNA incorporating the entire length shown in the +1? HS sequence alignment was amplified by PCR using -0.2E3/a (Fig.7) as a template and using the following primers:
(forward) J'-GAGAGGATCCCAGATGTTGAATTTTGGTTTA-3'; and 10 (reverse) 5'-AGAGCTGCAGCATACCTGGGGTCCCATG-3'.
Underlined letters (capital letters in Fig. 9) identify nucleotides r.o~ present in the endogenous muri:~e SCL
locus, the modvf~.catiens having been ~_.troduced for cloning purposes. Similarly a 149 by fragment 15 incorporating :r.~s~ of the length shown. in the +18 HS
sequence alignme::~ was amplified using the following fragments:
(forward) ~'-GAGAGGATCCACTATCATTCTGAGGTTTGGTTTC-3';
and 20 (reverse) ~,'-AGAGCTGCAGCTCTTTCTATAGAATCATTTTTTAATG.
The seauences o:: :.he amplified fragments are shown in Figures 9A and 9B. The sequence betw'en n~~zc~~eotides 59 and 149 of Fig. 9A matches that between nucleotides 1 and 91 of Fig. 8G and the sequence between. nucleotides 7 and 25 145 of Fig. 9B matches that between nucleotides and 4 and 142 of Fig. 8H.
The two amplified fragments were cloned into the -0.2E3 luc reeorter construct (Figure ?A, at h and j) and their 30 activities in the transient transfection assay determined (Figure 7B).
Thus t!~.e sea-uences identified as potential regulatory sites cy virtue of a high level of homology between the 35 murine and human sequences we=a shown to greatly enhance SUBSTITUTE SHEET (RULE 26) fold activation of the -0.2E3 luc repcrter construct in the transient transfection assay relative to the construct lacking any SCL sequence outside -0.2E3. The absence of a reduction in activity between the larger restriction fragments and the smaller amplified fragments (e.g. between t'.~.~ BglII/Sau3AI fragmen~ and d, e, g, and j and between c, f and h) indicates that the core regulatory sequences have been localised to the amplified fragments.
Example Fine mapping of. chromatin structure by endonuclease accessibility A nove_ °ndonuclease accessibility assay, analogous to the DNase I hycersensitivity assay, was used to fine map the position cf the +18 ~iS site. 'T'he endonuclease accessiwility assay is based on t::~e observation that consens~:s sites for restriction e::donuclease present in defined JNaseI :S sites are accessible when purified nuclei c~ chromatin are incubated with the respective restriction. e..~.zvme.
Nuclei are digested with a frequen_ly cutting restriction enzyme (i.e. one having a 4 base<pair recognition site, suitable frequent cutters include HaeIII HhaI, ScrFI, Sau96I, AvaII and HinfI), which digests DNA only at exposed sites, corresponding to regions of open chromatin. After digestion, DNA is isolated, cut with a rare c~.:tter a-:u analysed by Southern blotting with an end probe correspc.~.ding to one end cf a known rare cutter fragment. Meas~~:rement of the size of bands appearing allows accurate location of regulatory elements, partic~~~-arly ~= the sequence ~f ..~a a::alysed region is SUBSTITUTE SHEET (RULE 2fi) known, since actual and potential cleavage sites may be compared.
2 x l0a F4N or 6- cells (6- is a murine mast cell line S grown in RPMI TlOo ECS were harvested, washed twice with PBS, resuspended in 8 ml lysis buffer (50 mM KC1, 10 mM
MgSO" 3 mM DTT, 5 mM HEPES (pH 7.4), 0.050 NP40, 1 mM
PMSF) and incubated =or 1 hour at room temperature with mixing. Nuclei were spun down at 100 x g for 5 min and the pellet washed i~ 50 ml RSB (10 mM NaCI, 10 mM Tris (pH 7.4), 3 mM MgCl_;. Nuclei ~.~ere resuspended in 9.5 ml RSB and 400 u'_ ~ M ~3aCi, 70 u' ~ M MgCl_ and 100 ~1 0.1 M
DTT were added. Restriction digests ~.aere set up using 400 ul nuclei suspension and 0 - 400 units HaeIII (a frequent cutter) and incubated rcr 60 min at 37°C. 400 ul proteinase :~ digestion buffer (50 rru'~ Tris (pH 7.9), 100 mM EDTA, 200 mM NaCl, l~ SOS, 250 ug/ml proteinase K) was added to each digest. Digests were incubated at 55°C
for 4 hours to overnight and extracted once with phenol, once with phenol/chlcroform aid once with chloroform. 2 volumes ethane' wera added and mixed and the DNA
precipitated at -20'C for 20 m~in. C?~1A was spun down (microfuge, 10 min), the pellet washed in 70% ethanol, dried and resuspended in 50 ul water. DNA was digested with Sacl and Southern blotting e:arried out with a labelled end probe (comprising 407bp SacI/ScaI fragment 15.69kb 3' of -37 GATA site).
As shown in Fig. 10, increasing concentration of the frequent cutter yielded a band at 2.5 kb, indicating a cleavage site 2.5 kb from the end probe. Analysis of the possible cleavage sites (shown as dashes on the SacI
fragmer_t of Fig. 10' indicated a cleavage site corresponding tc the location cT the +18 HS as determined by sequence cc~~arison, DNaseI '.:;ypersensiti-rity and in SUBSTITUTE SHEET (RULE 26) vitro transfection assays. The HaeIII site at which cutting occurs is 18.19kb 3' of -37 G.TA site. There is no HaeIII site in the +17HS core region.
Example 8 Mapping of other HS sites Fine mapping of other previously iden=ified HS sites by the endonucleas~ accessibility assay ;as described in L0 Example 7) was carried out to further localise the core regulatory elements. An additional e:iposed region (corresponding to a hypersensiti-re region in t:~e DNaseI
hypersensitivit-_; assay) was identified at +1 kb and was named +1 HS. "''~e +1 HS site lies between exons lb and I5 2b; its positio:: is shown in Figure 12.
Fine mapping was followed by sequence analysis, carried out as in Example 6. Sequence alignments for the -10, 4.5, -3, +1, +3 and +7, +17 and '~3 ciS sites are shown in 20 Figures 8A to 8c. In all cases, a region of high homology between marine and huma:~ sequence was found in the region ider.~ified by DNase _ lpe=sensityTri~y and/or endonuclease accessibility as potentially encoding a regulatory element. The following table shows the 25 positions of the homologous regions i:: both marine and human SCL genes. Positive and negati-re numbers indicate the number of bases down- or upstream of the -37 GATA
site, respecti~rely.
30 HS site Position i:~ mur'_ne Position in human gene gene -10 -8689 to -9236 -9357 to -8842 -4.5 -4216 tc -3705 -3542 to -2942 -3 -2825 to -2379 -2101 to -1353 SU9STITUTE SKEET (RULE 26) +1 +583 to +1105 +721 to +1221 +3 +2424 to +2884 +2652 to +3068 +7 +6424 to +f884 +6473 to +6933 +17 +17272 to +17352 +18329 to +18419 +18 +18266 to +18426 +19552 to +19700 The various studies carried out to localise the +17 and +18 HS sites (namely: DNaseI hypersensitivity;
endonuclease accessibility; homology between mouse and human sequences in the identified regions of interest;
and tre transien~ ~ransfecticn assay using constructs containing cere regulatory sequences as identified by sequence analysis? showed highly consistent results. In view o~ this, y~ is taught that the other sequences shown in Figure 8 !:~~hica were identified by homology and either DNaseI hypersensitivity or endonuclease accessibility) represen~ or i:~clude the core regulatory sequences.
Exampla 9 Regulation of expression of a I~ac2 reporter gene by +17/1$ HS in vivo Constructs and transaenic mice ..>.:
A 10.5 kb SCL cassette, +6E5/LacZ/+17/18, was constructed by cloning 2.7kb +6E5 Sau3AI/iVdel fragment tin a XholHindIII cassette) into the Xno/H.indIII pGl2-LacZ
plasmid site. The 5.2kb BglII/Sall fragment containing enhances +17/18 was cloned downstream into the SaII/BamHI
cloning site of plasmid pGL2+6E5/LacZ. Both the 5.7kb +6E5/LacZ fragment and 10.5 kb +6E5/LacZ/+17/18 fragment were obtained by digestion with XnolSalI. A -0.9E3/LacZ/+'7/18 cassette was similarly constr~.:cted from a 3.8kd BamHI/Xcmi fragment. A SV40/LacZ/+17/18 cassette SUBSTITUTE SHEET (RULE 26) was constructed by cloning the 8glII/SalI fragment into a standard SV40 expression vector.
Fertilized (CBAxC57Bl/6)F1 oocytes were microinjected 5 with the DNA constructs. Constructs were purified to remove all vector sequences. PCR analysis of tail DNA
was used to identify transgenic mice within a litter.
(Cell 51: 975-985, 1987).
10 f3-qalactosidase assays and antibody staining For analysis of 3-galactosidase ac~i~Jity in viable transgenic thymus, spleen, bone :~arrow and lymph node cells, 10'-10 single cells were =esuspsnded in 20~z1 of 15 PBS 5°s FCS and incubated 10 minu~~s at 3?°C prior to loading with 20 ul of 2 mM fluorescein di-(f~-D-galactopyranoside) ( FDG) in dH~O a:~d fell owed by incubation at 37°C for 50-70 seco.~.ds. The FDG (Sigma) uptake was stopped by addition c. 400 ul of ice-cold PBS
20 5o FCS and the reaction was alle~.a~d to proceed for 1-3 hours on ice in the dark. Cc-sza~..~.irg =~rith PE or biotin-conjugated antibodies against cei surface anr.igens was performed during FDG incubation ~:~ identify cells of haematopoietic lineage. Before .low cytometric analysis, 25 propidium iodide (Sigma) was added to a final concentration of 0.5 ul/ml to allow the exclusion of dead cells. The fluorescence generated by ~3-galactosidase was detected on the FACSsort (Becton-7ic'.~cinson) on a EITC
analysis channel. Whole amount ambrycs were isolated 30 into ice-cold PBS and fixed ~i~ X-gal fix (0.2~
glutaraldehyde) at 4°C for 20-60 :~~~.nutes, depending upon size. Embryos were stained ove=::yght at room temperature in lmg/ml X-gal (Sigma).
35 Results of the LacZ assay in di=;=~er.t tissues of an 11 SUBSTITUTE SHEET (RULE 26) day post-coiturn embryo are shown in Figure li. Cells were identified as being of haematopoietic cell lineage by morphology and cell surface antigen expression. A
comparison of LacZ expression using the +6E5/LacZ/+I7/IS
and +6E5/LacZ constructs indicates that the 5.lkb +17/18HS fragmer.~ is able in the absence of other regulatory regions to drive expression from t::e exon 4 promoter in defined haematopoietic tissues (in this case liver, yolk sac and=AGM), and vessel endothel~~um and endocardium of t~e li day embryo. A comparison between +6E5/LacZ/+I7/~8 and SV40/LacZ/+17/18 indicates that regulation of exoressicn is not dependent on t:ne exon 4 promoter being used. Differential expression between +6E5/LacZ/+17/'~~ and -0.9E3/LacZil7/18 may be due either to the influence o~ other regulatory regions !e.g. +1 HS, +3 HS) present in the -0.9E3 fragment, or to the presence of different promoters (promoters la and lb).
Similar results (not shown) indicate that bct:~
+6E5/LacZ/+17/'8 and (to a lesser extent) -0.9E3/LacZ/+17/i8 constructs drive expression in the thymus and sp'~e°:~ of newborn mice. LacZ is co-expressed with CD2, CD4,CD8 cell surface antigens, but r,ot with Ter119. In adult mice, LacZ expression driven by the +6E5/LacZ/+17/18 construct is detected in a small proportion of thymus and bone marrow cells. ~apression of this construct is also observed in CD34 positive cells from 11 d.p.c. foetal liver.
Exam~ie 10 Regulation of expression of a LacZ reporter gene by different regulatory regions in vivo Preparation of rransaenic reeorter ccnstrucrs and transaenic mice SUBSTITUTE SHEET (RULE 26) WO 99/1b782 PCT/GB98/02914 In order to construct SCL-LacZ reporter transgenes, 5' regions from the mouse SCL gene (8egley et al supra 1994) were cloned upstream of the bacterial LacZ gene. The transgenes ccntained the following: in -10E3/LacZ, a 13.2 kb region from -i0kb (BgIIII site) to exon III (XcmI
site) ; in -?.OE3/LacZ, a 10.1 kb region. from -7 kb (Kpnl .
site) to exon III; in -0.9E3/LacZ, a 3.3 kb region from -0.9 kb (BamHI site) to exon III; in +0.02E3/LacZ; a 2.9 kb fragment frcm exon Ia (BssHII site) to exon III; -0.9E1a/lacZ, a 943 by from the -0.9 kb (BamHI site) to exon la (BssHII site); and in +0.02E28/LacZ, a 1.1 l:b fragment from eyon Ia (BssHII) to X~~oI site, engineered by PCR, 32 by into exon IIb. Construct SV/lacZ was constructed by replacing the luci~~rase gene in pGL2 promoter-vector (Promega) with the lacZ gene, and -7-0.9/SV/lacZ contained the region from -? kb (Kpn I site) to -0.9 kb (BamHI site) cloned upstream of SV/lacZ.
Constructs were made using 8luescript KS (Stratagene), pGL-2 basic (Promega) or pGEM-il (~romega) as the plasmid backbones.
A schematic diagram showing reporter transgene construction is shown in Figure 12~.. Downward arrows refer to HS regions and to promoters la and lb.
Fragments for microinjection were prepared by digesting 50 ug of plasmid DNA with restriction enzymes to remove the plasmid backbones. Fragments were separated by gel electrophoresis in LMP agarose (Bioresolve) for large fragments or normal agarose (~ibco 8RL) for small fragments (<7 kb). Fragments were extracted by f3-agarase (NEB) from LMP agarose or Qiaex I~ (Qiagen) from normal agarose. Fragments were diluted to 3-4 ~g/ml in a buffer containing lOmM Tris (pH 7.4) and 0.25mM EDTA (pH 7.4).
Pronuclear injections were perfor:~ed into CBA x C57/B1 6 SUBSTITUTE SHEET (RULE 26) fertilised mouse oocytes which were allowed to divide to two cells prior to implantation into t~~e oviducts of pseudopregnant CD1 female mice (Hogan et al in "Manipulating the mouse embryo", 2nd eu, Cold Spring Harbor Press, 1994). In some cases, _regnancy was terminated after 12.5 days of pregnanc.,i and embryos were stained with X-gal (Melford Laborator=es Ltd) for f~-galactosidase activity (Miles et a1 Development 124: 537-547, 1997). Lines were established fcr some transgenes.
In these instances CBA x C57/B1 6 F1 females were mated with transgenic males and embryos were analysed after 12.5 days post-coitum. I:~ a'.~i cases, DNA from yolk sac was prepared aa.d tested for the presence of the LacZ
transgene by PCR with an internal myocenin control (Miles et a1. supra).
The results are shown in Figure 12B a~d Figure 13. The various constructs have progresSiV~lyr :ewer of the identified regulatory regions. Comparison o~ the -7E3/LacZ and -0.9E3/LacZ constructs _::dicates that the -4.5 HS site and/or the -3 HS si~2 =s capable of driving expression in 1 fiver, AGM, -ressel a~:dc~~~elium and endocardium in the 12.5 day embryo (Fig.l3D). Expression of lacZ in hindbrain was unafected (~ig.l3D and Table 13I). Comparison of the -0.9E3/hcacZ and +0.02E3/LacZ
constructs indicates that a regulatoryr region exists between 0.9 kb upstream and +0.02 kb downstream of promoter la, which drives expression in mid brain. Loss of expression may be due to loss of ..::nction of promoter la, or to loss of a -0.2 ::S site, four:d by Leroy-Viard et al. (supra), but not in the cell lines studied by the present inventors. From a total of 1? PCR positive embryos, 6 exhibited expression of lacZ in hindbrain (Fig.l3G; Table i3I). Prcgressi-re less of expression in vessel endothelium and spine ~:ay re'lect regulation by SUBSTITUTE SHEET (RULE 28) more than. one regulatory region.
To determine whether the region from -0.9 kb to -7 kb was also sufficient for endothelial and haematopoietic expressi:~n, it was linked to an SV40 minimal promoter (-7-0.9/SV/lacZ, ~'ig.l3A). Strong endothelial staining was observed in transgenics generated with this construct (Fig.l3L) together with weaker staining in a minority of fetal li-rer cells (riot shown). No vascular endothelial or haernatopoietic staining was observed with a construct containi.~.g the SV40 minimal promoter alone (SV/lacZ, Fig. i A and P , '='abl a 13J) . ThereTore, a o. 1 kb region upstream of the SCL promoters contains one or more enhanc es whic:-~ were both necessary and sufficient for directing lacZ expression to vascular endothelium and a population of fetal liver haematopoietic cells.
To deter:aine whether the 940 by regicn from -0.9 kbp to +0.02 k'cp was also sufficient for mesencephalic expression, the fragment was linked tc lacZ (-0.9E1a/lacZ, Pig.l3A), and founder tra::sgenics were analysed. Frcm a total of 14 PCR posv~=iv2 embryos, 6 expressed lacZ in the mesencephaion in a pattern identical to that previously observed (Fig.l3H; Table 13J). No hindbrain or spinal cord staining was observed (Fig.l3H; Table 13J). These data demonstrate that promoter 1b was not needed for midbrain expression and that t:ze 940 by region containing promoter la was both necessary and sufficient for directing lacZ expression to the developing midbrain.
To furt::er define elements responsible for directing expression to ~ze hindbrain and spinal cord, a region containing DNaseI hypersensitive site i3 was deleted from construct +0.02E3/lacZ to generate +O.G2E2b/lacZ
SUBSTITUTE SHEET (RULE 26) (Fig.l3A). Using the latter construct, 2/10 founder transgenics expressed lacZ in the hindbrain. Expression was also seen i-:~the spinal cord. However, expression in both tissues was weak suggesting t::at an enhancer within 5 intron 3 was necessary for full hindbrain and spinal cord expression.
Example ~1 SCI 5' sequences direct lacZ expression to developing 10 brain, spinal cord and endothelium In order to derermine further t~_e _n vitro function of the SCL 5' elements, a construct containing the region from -7kb (relative L: exon la transcriptional start) to +2.8 15 kb (exon 3), was cloned upstream of a ':acZ reporter gene (-7E3/lacZ, Fig.l4A) and expression was analysed in transgenic mice generated in accordance with example 10.
In total, six transgenic lines and one rounder embryo were analysed for lacZ express_cn by X-gal staining at 20 12.5 dpc. One c. the transgenic lines was omitted from further analysis because of a ccmpiet2 absence of lacZ
express~.on.
Analysis of who'~.e mount transgenic embryos at 12.5 dpc 25 demonstrated transgene expressiocr at high levels in the mesencephalon (midbrain me, Eig.143) in accordance with the above results, with weaker expression observed in blood vessels ( ~J) and expression i:: the spinal cord ( sp, Fig.l4B). Saggital sections revealed transgene 30 expression in t:!e diencephalon (d) and heart (h) (Fig.l4C). In addition, stain;~g was observed in the ventral metencephaion (mt) and ;~yelencephalon (my), but this s~aining was very weak in comparison with the intensi~y of lacZ expression seen in t~:e mesencephalon.
SUBSTITUTE SHEET (RULE 26) Discussion of Examples 9, 10 and 11 These examples provide clear evidence chat the regulatory sequences discovered and investigated :~.erein are capable of directing expression of transgenes prom different promoters in discrete sets of tissues 'n vivo.
Furthermore, i~: combination with Example 5 (and Figure 6), these results suggest that the +17/18 HS site enhances expr~ssion~'from the exon 4, exon la and SV40 promoters, but not from the exon lb promoter.
Exampl° 12 SCL 5' elements differentially target distinct endothelial cell populations during development Expression of the -7E3/lacZ transgene was also observed within endothelial cells. Staining was first noted within the extraembryonic mesoderm at 7.5 dpc (Figs.l5A, B) which differentiates into haematopoietic and endothelial cells. In 8.0 dpc embryos, the endothelium of the yolk sac blood islands in the yolk sac expressed lacZ (Fig.lSC, D, and J). LacZ was also expressed within the dorsal aorLae (da, Fig.lSC and D), allantoic bud (al, Fig.lSD), endocardium of the heart primordia (e, Fig.l5C
and E), developing vessels (v) and clusters of presumptive angioblasts in the cephalic mesenchyme (arrowheads in Fig.lSE). Clusters of presumptive angioblasts adjacent to the aortae (arrowheads in Fig.l5C
and D) and within the mesoderm at the base of the allantois (arrowheads in Fig.lSF) also expressed lacZ.
In 9.5 dpc embryos, the transgene was expressed in sprouting intersomitic vessels arising from the dorsal aorta (arrowheads in Fig.lSH), in the truncus arteriosus, aortic arches, and a small region on the floor of the left atrium (data not shown). Developing capillary networks over the brain and extending ~hrougi-iout the SUBSTITUTE SHEET (RULE 26) embryo were also stained (Fig.l5H). However, in marked contrast to the extensive endothelial expression of the transgene within the embryo, transgene expression within the yolk sac was only detected in rare yolk sac endothelial cells (Fig.lSH and data not shown). This differential endothelial expression was observed in 4/4 transgenic lines and suggests that SCL regulatory elements can detect functional differences between distinct endothelial populations.
At 18.5 dpc lacZ expression was markedly stronger in arteries than veins, as shown by the more intense staini~g in the pulmonary artery and aorta relative to the pulmonary vein and superior vena cava (Fig.lSI).
Weak lacZ expression was also observed in 18.5 dpc endocardium (Fig.lSI). LacZ expression within the embryonic vasculature remained evident at birth, but was not detectable by 6 months (data not shown).
Our data therefore demonstrate that the transgene contained one or more enhancers that directed expression to endothelial cells in vessels formed by vasculogenesis (eg. dorsal aortae and cephalic capillary network) and angiogenesis (eg. intersomitic vessels) (Risau, 1997).
Moreover, the endothelial enhancer could detect functional differences between spatially and temporally distinct endothelial cell populat~.ons.
Example ~3 Transgene expression within the developing nervous system In order to determine the time-course of transgene expression during neural, vascular and haematopoietic development embryos from at least two -7E3/lacZ
transgenic lines were analysed at multiple time points from 7.5 dpc to 18.5 dpc. Expression of lacZ was first detected in the nervous system at 10.5 dpc (Fig.l6A).
SUBSTITUTE SHEET (RULE 26) Strong staining was observed in the rostro-ventral region of the mesencephalon (me) and in dorsally projecting posterior commissural axons at the boundary between the diencephalon and mesencephalon. Expression of the transgene in the hindbrain and spinal cord also commenced at this time point (data not shown).
At 11.5 dpc (Fig.l6B), lacZ expression was also observed within the ventral region of the metencephalon (mt) or presumptive pons and extended caudally in the myelencephalon (my) and spinal cord (sp) as indicated by arrowheads. Staining in the posterior commissural axons and cell bodies was intense (Fig.l6C,D and E).
Expression extended caudally it lateral cell bodies in the ventro-lateral mesencephalon, and was followed by a wave of ventral-to-dorsal axorai growth towards the dorsal midline (Fig.l6C, E and F). Also at this time, two stripes of expression in both cell bodies and axons, extended anteriorly into the diencephalon (arrowheads in Fig.l6C and D).
At 12.5 dpc lacZ positive axons within the anterior 1/3 of the mesencephalon crossed the dorsal midline (Fig.l6G
and H) in the intertectal commissure i~lilson et al., 1990). In the caudal mesencephalon the ventral-to-dorsal axonal growth had not yet reached the dorsal mid-line (Fig.l6G), but by 15.5 dpc lacZ a,~ons covered the whole tectum except for the most caudal region adjacent to the midbrain/hindbrain junction (data not shown). LacZ was also expressed in large cells in the spinal cord mantle layer at 12.5 dpc (Fig.l6I). '~'he size and location of these cells suggest they represent motor neurones.
Strong transgene expression continued to 18.5 dpc within the diencephalon, midbrain, hindbrain and spinal cord (Fig.l6J and data not shown) but by 5 months of age, lacZ
expression persisted only in to adult mesencephalon SUBSTITUTE SHEET (RULE 26) (superior and inferior colliculi - data not shown).
Endogenous SCL transcripts were observed in a similar pattern in 18.5 dpc midbrain and hindbrain (data not shown)- Taken together our data demonstrate that a 10.1 kb region of the SCL locus contained regulatory elements sufficient for appropriate spatial and temporal reporter gene expression during neural development.
Discussion of Example 13 Our data demonstrate that the SCL midbrain element was initially switc'_'_~.ed on at 10.5 dpc in more rostral neurones withi:: the ventro-lateral region of the developing midb=ain and then in progressively more caudal neurones. Activation of this element was accompanied by a synchronous wave of dorsal axonal growth, demonstrating a close temporal link between SCL activation and axonal extension. The rostrol SCL-positive neurones appear to correspond to posterior commissure neurones identified recently using,lipophilic tracer dyes (Mastick and Easter, 1996). The function of the SCL-positive neurones is not clear. However the corresponding regicn of the adult brain contains the occulomotor and Edinger-Westphal nuclei, responsible for occulomotor and pupillary control (reviewed in Mason and Kandel, 1991), as well as several other nuclei of uncertain function including the intermediate nucleus of Cajal and:the nucleus of Darkschewitsch (williams, 1995).
This region of the developing midbrain and the posterior commissure perform at least two major tasks: co-ordinate control of eye position in response to visual, acoustic, cortical and proprioceptive inputs; and reflex pupillary and accommodation adjustments required for accurate perception (Mason and Kandel, 1991). These functions are clearly ancient phylogenetic features and this is consistent with our demonstration that the SCL midbrain SUBSTITUTE SHEET (RULE 26) element is highly conserved, both at the level of sequence and also at a functional level. Moreover the pattern of SCL expression within the rnidbrain/diencephalon was strikingly similar in mouse, 5 chicken and zebrafish. Our data therefore suggest that SCL-positive neurones in this region may be important for establishing the neural circuitry within occulomotor, pupillary and/or retinotectal pathways. This would also be consistent with expression of the -7E3/lacZ transgene 10 in the superior and, inferior colliculi. The midbrain element described here provides a specific and powerful tool to test this speculation.
SUBSTITUTE SHEET (RULE 26) REFERENCES

Begley, C.G., et al.Proc Natl Acad Sci USA 86: 10128, Finger,L.R., et al.Proc Natl Acad Sci USA 86: 5039, 1989 Chen, Q-, et al. EMBO
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415, P.D., et al.Mol Cell Biol 10: 5426, 1990 Aplan , Bernard, O., et al.Genes, Chromosomes and Cancer 1: 194, Brown, L., et EMBO J 9: 3343, 1990 al.

Bash, R.O., et Blood 86: 666, 1995 al.

Goldfarb, A. N., et al. Blood 58: 537, 1992 Cheng, J.T., et al.Mol Cell Biol 13: 801, 1993 Sanchez-Garcia, I.,et al. Proc Natl Acad Sci USA 91:

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8617, 1994 Boehm, T., et al. Proc Natl Acad Sci USA 88: 4367, 1991 Royer-Pokora, B., et al. Oncogene 5: 1887, 1991 SUBSTITUTE SHEET (RULE 26) Warren, A.J., et al. Cell 78: 45, 1994 Robb, L., et al. Proc Natl Adad Sci USA 92: 7075, 1995 Shivdasani, R .A., et al. Nature 373: 432, 1995 Porcher, C., et al. Cell 86: 47, 1996 Robb, L., et al. EMBO J 15: 4123, 1996 Green, A.R., et al: EMBO J 10: 4153, 1991 Aplan, P.D., et al. EMBO J 11: 4073, 1992 Leroy-Viard, K., et al. EMBO J 14: 2341, 1995 Visvader, J., et al. Oncogene 6: 195, 1991 Green, A.R., et al. Oncogene 7: 653, 1992 Hwang, L.Y., et al. Oncogene 8: 3043, 1993 Kallian~ur, A.R., et al. Blood 83: 1200, 1994 Pulford, K., et al. Blood 85: 675, 1995 Green, A.R., et al. Oncogene 6: 4'75, 1991 Visvader, J. , et al. TIBS 16: 330, 1991 Mouthon, M.A ., et al. Blood 81: 647, 1993 Cross, M.A., et al. Oncogene 9: 3013, 1994 Tanigawa, T. , 't al. Proc Natl Acad Sci USA 90: 7864, SUBSTITUTE SHEET (RULE 26) WO 99116782 PCTlGB98/02914 Lecointe, N., et al. Oncogene 9: 2623, 1994 Bockamp, E.O., et al. Blood 86: 1502, 1995 Pevny, L., et al. Nature 349: 257, 1991 Tsai, F.Y., et al. Nature 371: 221, 1994 Fujiwara, Y., et al. PNAS 93: 12355, 1996 Begley, C.G., et al. Gene 138: 93, 1994 Bockamp, E.O., et al. J Biol Chem in press Dube, S.K., et al. Proc Natl Acad Sci USA 72: 1863, 1975 May G, et al. Embo J 14: 564, 1995 Klinken, S.P., et al. Proc Natl Acad Sci USA 85: 8506, Felsenfeld, G., et al. Nature 355: 219, 1992 Lewin, B., et al. Cell 79: 397, 1994 Ernst P., et al. Immunity 2: 311, 1995 Crossley, M., et al. Mol Cell Biol 15: 2448, 1995 Tapscott, S.J., et al. Molecular and Cellular Biology 12:
4994, 1992 Waiters, M.C., et al. Genes & Development 10: 185, 1996 Kingston, R.E., et al. Curr Biol 4: 325, 1994 Zawel, L., et al. A Rev Biocnem 54: 533, 1995 SUBSTITUTE SHEET (RULE 26) Tuan, D.H., et al. Proc Natl Acad Sci 86: 2554, 1989 Fraser, P., et al. Nucleic Acids Res 18: 3503, 1990 Brand, A.H., et. al. Cell 41: 41, 1985 Paro, R., et al. Trends in Genetics 6: 416, 1990 Bienz, M., et al. BioEssays 17: 775, 1995 Biggin, M.D., et al. Cell 58: 433, 1989 Vacher, J., et al. Science 250: 1732, 1990 Zink, D., et al. EMBO J 14: 5660, 1995 Busturia, A., et al. EMBO J 12: 1415, 1993 Mizller, J., et al. EMBO J 14: 1209, 1995 Laurenson, P., et al. Microbiol Rev 56: 543, 1992 Mason, C. & Kandel, E.R. Principles o~ Neural Science, (1991), pp420-439. Elsevier, New York Mastick, G.S. & Easter, S.S. Dev. Biol. 173, 79-94., (1996) Williams, P. L. Gray's anatomy: The anatomical basis of medicine and surgery. Churchill Livingstone, New York, (1995) Wilson, S.W., Ross, L.S., Parrett, T., & Easter, S.S.
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S
SUBSTITUTE SHEET (RULE 26)

Claims

Claims 1. An isolated nucleic acid fragment which comprises an SCL gene regulatory element, said fragment including one or more SCL gene hypersensitive (HS) sites and being free of SCL coding sequence and/or promoter for said SCL gene, wherein said regulatory element can alter transcription from a said SCL gene if said gene is operably linked thereto.
2. An isolated nucleic acid fragment according to claim 1 which includes a single said SCL gene HS site.
3. An isolated nucleic acid fragment according to claim 1 which includes two or more said SCL gene HS sites.
4. An isolated nucleic acid fragment according to claim 3 which includes 2, 3, 4, 5, 6 or 7 said SCL gene HS
sites.
5. An isolated nucleic acid fragment according to any one of the preceding claims wherein the HS site maps downstream of exon 1a of the SCL gene in the native situation.
6. An isolated nucleic acid fragment according to any one of claims 1 to 5 wherein said SCL gene regulatory element is chromatin dependent.
7. An isolated nucleic acid fragment according to any one of the preceding claims which is substantially free of nucleic acid of the species of origin other than said SCL gene HS site(s).

8. An isolated nucleic acid fragment according to any one of claims 1 to 6 which comprises nucleic acid which, in the species of origin, is adjacent a said SCL gene HS
site.
9. An isolated nucleic acid fragment according to claim 8 wherein said nucleic acid is between 2 said SCL gene HS
sites.
10. An isolated nucleic acid fragment according to any one of claims 1 to 9 wherein said regulatory element is substantially free of nucleic acid which flanks the regulatory element in the native situation.
11. An isolated nucleic acid fragment according to claim 10 which comprises no more than about 1.5kbp of the nucleic acid which flanks a said regulatory element in the native situation.
12. An isolated nucleic acid fragment wherein the HS
site consists substantially of a sequence shown in Figure 8 or 9 or a sequence complementary to a sequence shown in Figure 8 or 9.
13. An isolated nucleic acid fragment according to any one of claims 1 to 11 wherein the HS site comprises a sequence shown in Figure 8 or 9.
14. An isolated nucleic acid fragment which includes a sequence capable of annealing to a sequence shown in Figure 8 or 9 under stringent hybridisation conditions which fragment is free of sequence complementary to said SCL coding sequence and/or promoter for said SCL gene.
15. An isolated nucleic acid fragment which includes a sequence complementary to an SCL gene HS site as defined in claim 1, which fragment is free of sequence complementary to said SCL coding sequence and/or promoter for said SCL gene.
16. An isolated nucleic acid fragment according to claim 15, wherein the sequence complementary to an SCL gene regulatory element includes a sequence complementary to a sequence shown in Figure 8 or 9.
17. Use of an isolated nucleic acid fragment for controlling expression o~ a gene in a cell wherein the isolated nucleic acid fragment comprises an SCL gene regulatory element, said fragment including one or more SCL gene hypersensitive sites, which regulatory element can alter transcription from a said SCL gene if said gene is operably linked thereto.
18. Use according to claim 17 for controlling expression of an SCL gene.
19. Use according to claim 17 for controlling expression of a non-SCL gene.
20. Use according to any one of claims 17 to 19 for controlling expression of a gene in haematopoietic cells, endothelial cells, neuronal cells or yolk sac cells.
21. Use of an isolated nucleic acid fragment for the manufacture of a preparation for use in controlling expression of a gene in a cell wherein the isolated nucleic acid fragment comprises an SCL gene regulatory element, said fragment including one or more SCL gene hypersensitive sites, which regulatory element can alter transcription from a said SCL gene if said gene is operably linked thereto.
22. Use according to claim 21 wherein the preparation is for use in controlling expression of an SCL gene.
23. Use according to claim 21 wherein the preparation is for use in controlling expression of a non-SCL gene.
24. Use according to any one of claims 21 to 23 wherein the preparation is a medicament.
25. An oligonucleotide which is capable of modulating transcription from a promoter and which includes a fragment of one or more of: (i) a sequence shown in Figure 8 or 9; or (ii) a sequence complementary to a sequence shown in Figure 8 or 9; or (iii) a sequence which can anneal, with either a sequence shown in Figure 8 or 9 or a sequence complementary to a sequence shown in Figure 8 or 9, under stringent hybridisation conditions.
25. An oligonucleotide according to claim 25 wherein the fragment is at least 10 nucleotides in length.
27. A nucleic acid construct which comprises an isolated nucleic acid fragment according to any one of claims 1 to 11 operably linked to a promoter.
28. A nucleic acid construct according to claim 27 wherein the promoter is that of an SCL gene.
29. A nucleic acid construct according to claim 27 wherein the promoter is not of an SCL gene.
30. A nucleic acid construct which comprises ar isolated nucleic acid fragment according to any one of claims 1 to 11 operably linked to a promoter and/or a heterologous gene.
31. A nucleic acid vector which comprises an isolated nucleic acid fragment according to any one of claims 1 to 16.
32. A nucleic acid vector according to claim 31 wherein the isolated nucleic acid fragment is operably linked to a promoter and/or heterologous gene.
33. A host cell which has a nucleic acid vector according to claim 31 or claim 32.
34. A host cell according to claim 33 wherein the vector is integrated into the genome of said host cell.
35. A method which comprises the step of introducing a vector according to claim 31 or claim 32 into a host cell.
36. A method of expressing a heterologous gene which comprises the steps of: (i) introducing a vector according to claim 31 or claim 32 into a host cell; and (ii) causing or allowing said heterologous gene to be expressed under the control of said regulatory element.
37. An organism containing a host cell according to claim 33 or claim 34.
38. An organism according to claim 37 which is a mammal.
39. Use of an isolated nucleic acid fragment for identifying a modulator which can modulate activity of the regulatory element contained within said isolate wherein the isolated nucleic acid fragment comprises an SCL gene regulatory element, said fragment including one or more SCL gene hypersensitive sites, which regulatory element can alter transcription from a said SCL gene if said gene is operably linked thereto.
40. A modulator identified according to claim 39, 41. A pharmaceutical composition containing a therapeutically effective amount of (i) an isolated nucleic acid fragment according to any one of claims 1 to 16 or (ii) a modulator according to claim 40, said composition including one or more of a pharmaceutically acceptable excipient, carrier, buffer, stabiliser, preservative or antioxidant.
42. A kit comprising an isolated nucleic acid fragment according to any one of claims 1 to 16 or a modulator according to claim 40.