Leukemia (1999) 13, 750–759  1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu Chromatin structure and transcriptional regulation of the stem cell leukaemia (SCL) gene in mast cells

JL Fordham, B Go¨ttgens, F McLaughlin and AR Green

University of Cambridge, Department of Haematology, MRC Centre, Hills Road, Cambridge CB2 2QH, UK

The stem cell leukaemia (SCL) gene is a member of the basic that ectopic expression of SCL in thymocytes gives rise to T helix–loop–helix family of transcription factors and is essential cell tumours.7–10 for the development of all haematopoietic lineages. SCL is The murine SCL gene has 7 exons spanning 15 kb of DNA. expressed in pluripotent haematopoietic stem cells and also Ј following commitment to the erythroid, mast and megakary- Two promoters are present in alternate 5 exons and exhibit ocytic lineages. The mechanisms responsible for this pattern lineage-restricted activity in different haematopoietic cell of expression are poorly understood, but are likely to illuminate types.11–13 A third promoter in exon 4 has also been reported the molecular basis for stem cell development and lineage in the human SCL gene.14 SCL expression is normally restric- commitment. Here we present the first description of the regu- ted to haematopoietic tissues, endothelial cells, the heart and lation of the SCL gene in mast cells. In this study we systemati- 15–17 cally analysed the chromatin structure of a 45 kb region of the distinct regions in the brain. Within the haematopoietic murine SCL locus in mast cells. The pattern of DNase 1 and system SCL is expressed in committed erythroid, mast and restriction endonuclease hypersensitive sites in mast cells was megakaryocytic cells as well as in primitive myeloid cells and distinct from, but overlapped with, the pattern previously multipotent progenitors.15–20 SCL appears to perform different described in erythroid and primitive myeloid cells. Each poten- functions within distinct haematopoietic lineages. Expression tial regulatory element was tested using transient reporter of antisense SCL constructs in a multipotent cell line resulted assays to assess their functional significance in mast cells. 21 These studies identified two potent enhancers, one of which in reduced proliferation and self-renewal whereas the results was downstream of the SCL gene. Further characterisation of of similar experiments showed an inhibition in erythroid dif- this 3Ј enhancer demonstrated that it required the presence of ferentiation of a committed erythroid cell line.22 Over- two distinct DNase 1 hypersensitive sites for full activity, and expression of exogenous SCL impaired macrophage differen- that it was capable of stimulating transcription from both pro- tiation of M1 cells suggesting that down-regulation of SCL Ј moter 1a and 1b. Since the 3 enhancer is active in both expression may not only accompany but also be essential for erythroid and mast cells, it will now be important to see 23 whether it is independently activated in these lineages, or normal myeloid differentiation. Growth factor-induced whether it is also active in haematopoietic stem cells. erythroid differentiation of a multipotent progenitor cell line Keywords: SCL gene; mast cells; hypersensitive sites was accompanied by up-regulation of SCL mRNA, whereas induced granulocyte/monocyte differentiation resulted in extinction of SCL expression.24 Similarly, constitutive over- Introduction expression of normal, mutant or truncated SCL polypeptides in myeloid progenitor cell lines suggested that SCL modulates A central question in haematopoietic development concerns several physiological processes including proliferation, differ- 25 the mechanisms whereby a pluripotent stem cell reproducibly entiation and apoptosis. generates multiple distinct differentiated cell types. The com- Knockout studies have demonstrated a critical role for SCL plexity of lineage commitment involves a highly controlled in haematopoietic stem cell formation or function. Mice lack- developmental progression through successive stages of differ- ing SCL protein died at 9.5 days post-coitus and exhibited a 26,27 entiation. It is clear that these processes are regulated in part complete absence of all haematopoietic cells. In addition, by key transcription factors. As a consequence, it is of parti- SCL null ES cells failed to give rise to haematopoietic colony- cular importance to identify such regulators and understand forming cells in vitro and also did not contribute to haemato- 28,29 how they themselves are controlled – what regulates the regu- poietic tissues in vivo. Recent zebrafish experiments sug- lators? To address these questions we have chosen to study gest that SCL acts at an even earlier stage, since ectopic SCL the stem cell leukaemia (SCL) gene, which encodes a basic expression specified haemangioblast development from early 30 helix–loop–helix (bHLH) transcription factor, is expressed in mesoderm at the expense of other mesodermal cell fates. pluripotent haematopoietic stem cells and functions as a The concept that SCL plays an important role in vasculogen- pivotal regulator of haematopoiesis. esis is consistent with recent reports that SCL is required for 31 The SCL gene (also known as TAL-1) was originally ident- normal yolk sac formation and that SCL can partially rescue ified as a result of its activation by chromosome rearrange- both vascular and haematopoietic defects in the zebrafish 32 ments affecting chromosomal band 1p32, which is where the mutant, cloche. gene is located, in T cell acute lymphoblastic leukaemia (T- The complex pattern of SCL expression raises important ALL).1–4 Genomic rearrangements of the SCL gene have been questions about the molecular mechanisms underlying lin- reported in approximately 30% of patients with paediatric T- eage-restricted SCL expression in different cell types. We have ALL5,6 and it has been suggested that additional mechanisms therefore identified a number of regulatory elements respon- may be responsible for ectopic expression in a further 30% of sible for lineage-specific expression of the murine SCL gene + 33 these patients.4 Transgenic studies in mice have also shown in erythroid and CD34 myeloid cells. However, nothing is known about SCL enhancers directing expression to mast cells. Mast cells play a central role in host responses to both allergens and parasites34,35 but relatively little is known about Correspondence: AR Green; Fax: 44 01223 336827 the transcriptional programs of mast cells and only a few mast Received 4 September 1998; accepted 20 February 1999 cell-specific elements have been reported.36–42 SCL transcriptional regulation in mast cells JL Fordham et al 751 In this paper we have therefore identified and mapped Tris/HCl pH 7.4, 100 mM EDTA, 100 mM NaCl, 1% SDS) to DNase 1 hypersensitive sites within a 45 kb region of the the aliquots and incubated with proteinase K (1 mg/ml) at murine SCL locus in mast cells. Transient reporter assays have 55°C for 2.5 h. The DNA was isolated following proteinase been used to assign functional activity to these elements. Two K digestion/phenol extraction and ethanol precipitation. The exhibited potent enhancer activity in mast cells. Further probes used were the same as for DNase 1 hypersensitive site characterisation of one of these (the 3Ј enhancer) demon- analysis (Figure 1) except for the probe; p1 – 170 bp KpnI/SpeI. strated that it required the presence of two distinct DNase 1 hypersensitive sites for full activity and that it was capable of stimulating transcription from both SCL promoter 1a and 1b Preparation of reporter constructs within mast cells. Luciferase reporter constructs were generated in pGL-2 basic as previously described.33 The +0.2E2b construct was gener- Materials and methods ated by cloning an approximately 1 kb Vent polymerase (New England Biolabs, Beverly, MA, USA) PCR fragment containing Cell lines promoter lb and exon IIb into the SmaI site (maintains the SmaI site) between promoter 1a and 1b and an XhoI within The factor-independent murine mast cell lines 6− and the previously described −0.2E3.33 The PCR construct was MST96.47B3 have both been previously described.13,18,43 The subsequently sequenced. 6− cell lines (obtained from Dr S Hasthrope, Peter MacCallum Institute, Melbourne, Australia) and MST96.47B3 (kindly pro- vided by Dr A Elefanty, The Walter and Eliza Hall Institute of Transient transfections Medical Research, Melbourne, Australia) were grown in RPMI 1640 plus 10% foetal calf serum. All experiments were perfor- Transient transfections were carried out as described11 with med using cells in the exponential phase of growth. the exception that only 5 ␮g of the pEF-BOS lacZ vector was used for normalisation of each pulse. Also, in order to adjust for the large size differences between the reporter constructs Analysis of DNase 1 hypersensitivity used in this study, the molar amount of reporter construct added to each pulse was kept constant using 5 ␮g of pGL-2 Approximately 3 × 108 cells were rinsed in phosphate-buff- basic and proportionally more of the larger constructs. The ered saline (PBS) and resuspended in 20 ml of homogenisation total amount of DNA added to each pulse was kept constant buffer (10 mM Tris pH 7.4, 15 mM NaCl, 0.15 mM spermine, by adding the required amount of the plasmid pks-bluescript 0.5 mM spermidine, 1 mM EDTA, 0.1 mM EGTA, 0.2% NP-40, (Stratagene, La Jolla, CA, USA). Luciferase and ␤-galactosidase 5% sucrose). After Dounce homogenisation, the homogenate assays were carried out as described.11 The relative light units was spun through a 10% sucrose layer and resuspended in presented are the mean of at least four independent experi- buffer (10 mM Tris pH 7.4, 15 mM NaCl, 60 mM KC1, 0.15 mM ments and the results obtained were confirmed using a second spermine, 0.5 mM spermidine). After adding DNase 1 (Sigma, DNA preparation for each construct. Poole, UK) (0.8–16 ␮g), samples were incubated at 37°Cin the presence of 50 mM MgCl2 and 50 mM CaCl2 for 10 min. Reactions were stopped by adding 12.5 mM EDTA and 1% Results SDS and the DNA was isolated following proteinase K digestion/phenol extraction and ethanol precipitation. After Mast cells possess a distinct lineage-restricted pattern digestion with restriction enzymes, the DNA was analysed by of DNase 1 hypersensitive sites Southern blotting. Probes used were as follows: fragment p2 – a 220 bp Sau3AI/ApaI fragment 50 bp upstream of exon la; Lineage-specific expression of the SCL promoter has been p3–900 bp SmaI/SacI fragment, p4–180 bp SacI/SmaI frag- studied in erythroid, early myeloid and T cells lines.11,12,44 ment, p5 – a 1200 bp HindIII fragment 260 bp upstream of However, nothing is known about the molecular mechanisms the EcoRI site in exon VI; p6 – an 400 bp SacI/ScaI fragment underlying lineage-restricted expression of SCL in mast cells. extending 3Ј of SCL exon VI. We therefore elected to study two well characterised murine mast cell lines, Mst 96.47B3 and 6−, which express the mast cell-specific Fc⑀ receptor I, GATA-1, GATA-2 and comparable Restriction endonuclease accessibility assay levels of SCL.13 DNase 1 hypersensitive site analysis was used to identify putative regulatory elements in a region of 45 kb The method for the restriction endonuclease accessibility spanning the murine SCL locus. assay was kindly provided by Prof Doug Higgs (Institute of Figure 1a is a schematic representation of the strategy used Molecular Medicine, Oxford). Approximately 1 × 108 cells to analyse the SCL locus. Probe p2 was used to identify hyper- were spun down and washed twice with PBS. The pellet was sensitive sites 5Ј of the promoter region. As shown in Figure resuspended in 4 ml of high salt lysis buffer (50 mM KCl, 1b, probe p2 was hybridised to a filter containing DNase 1-

10 mM MgSO4,3mM DTT, 5 mM HEPES, 0.05% NP40) and treated DNA subsequently digested with the ApaI restriction the cells left for 1 h at room temperature. Nuclei were pelleted enzyme. In addition to the germline fragment of approxi- by spinning at 1000 g for 5 min. The nuclei were washed in mately 23 kb (closed arrowhead), two additional bands were RSB buffer (10 mM NaCl, 10 mM Tris/HCl pH 7.4, 3 mM also observed at approximately −9 kb and −4 kb (open

MgCl26H2O) repelleted and divided into aliquots of approxi- arrowheads). All the numbering of the hypersensitive sites mately 1 × 107 cells. 0–400 units of restriction enzyme were refers to their position relative to the beginning of exon la. then incubated with the nuclei aliquots at 37°C for 1 h. The To identify DNase 1 hypersensitive sites within the body of incubation was stopped by the addition of tail buffer (50 mM the SCL gene, DNase 1-treated DNA was digested with either SCL transcriptional regulation in mast cells JL Fordham et al 752

Figure 1 Analysis of DNase 1 hypersensitive sites associated with the murine SCL gene. Hypersensitive site mapping was performed using the Mst cell line. Filled arrowheads, germline fragments; open arrowheads, fragments generated by DNase 1 cleavage. (a) Partial restriction map of the murine SCL locus illustrating the probes (p2–6) used for DNase 1 hypersensitive analysis. The exons of the SCL gene are indicated. Filled boxes represent coding exons. A, ApaI; K, KpnI; R, EcoRI; S, SacI. (b) 5Ј Hypersensitive sites detected by probe p2 using ApaI-digested DNase 1-treated DNA. (c) Hypersensitive sites within the SCL gene detected by probe p3 using SacI-digested DNase 1-treated DNA. (d) Hypersen- sitive site within the SCL gene detected by probe p4 using SacI-digested DNase 1-treated DNA. (e) Hypersensitive sites within the SCL gene detected by probe p5 using SacI-digested DNase 1-treated DNA. (f) 3Ј Hypersensitive sites detected by probe p6 using SacI-digested DNase 1- treated DNA. SCL transcriptional regulation in mast cells JL Fordham et al 753 SacIorEcoRI restriction enzymes and hybridised with probes sitive sites. In addition, there are AvaII recognition sites p3, p4 or p5. Hybridisation of SacI-digested DNA with p3 between the −8.6HS and the 8.1HS which do not cut, suggest- resulted in a germline band of 5 kb and bands of approxi- ing the existence of two distinct hypersensitive sites rather mately 2.0, 1.4 and 1 kb (Figure 1c). The 2 kb band represents than a single one. a doublet reflecting hypersensitive sites over both promoter In Figure 2d nuclei were incubated with MspI, the DNA 1a and 1b (data not shown). The 1.3 kb and 1 kb bands rep- isolated and then digested with ApaI. The DNA was Southern resent newly identified DNase 1 hypersensitive sites termed blotted and the filter hybridised with probe p2. A 23 kb germ- +0.7HS and +1HS. A blot containing SacI-digested DNA was line band was observed in the control lane but was very sensi- hybridised with probe p4 (Figure 1d). This detected a germline tive to MspI digestion. A 4 kb fragment represented the −4HS band of approximately 1.1 kb and a band of approximately (open arrowhead). The band at approximately 5 kb was 500 bp (open arrowhead), which corresponds to a hypersensi- observed irrespective of the initial restriction enzyme used tive site 3 kb downstream of promoter 1a (+3HS), which we and probably represents cross-hybridisation. have previously noted in erythroid cells. The diffuse nature of To confirm the DNase 1 hypersensitive sites identified this band is probably due to its small size together with the within the 5Ј portion of the body of the SCL gene, DNA was fact that DNase 1 cleaves over a region of 100–200 bp within extracted from HhaIorSau96A-treated nuclei, subsequently a given hypersensitive site. In support of this we observed a digested with SacI and hybridised with either probe p3 or p4. much tighter band for this hypersensitive site in 6− and Mst In Figure 2e a 5 kb germline band was accompanied by three cells when we use restriction endonuclease accessibility smaller bands of 2 kb, 1.7 kb and 1 kb representing hypersen- analysis (Figure 2g and data not shown). Hybridisation of sitive sites over promoter 1a, promoter 1b and a hypersensi- EcoRI-digested DNA with probe p5 (Figure 1e) gave a germ- tive site 1 kb downstream of promoter 1a (+1HS). In Figure 2f line band of 10 kb and an 8 kb band (open arrowhead) corre- two additional bands could be seen (open arrowheads) corre- sponding to a DNase 1 hypersensitive site 7 kb downstream sponding to hypersensitive sites at promoter 1a (2 kb band) of promoter 1a, termed +7HS. The faint bands observed in and the +0.7HS (1.3 kb band). There are no Sau96A sites some of the ‘no DNase 1’ control lanes (Figure 1c and f) are within promoter 1b or the +1HS which explains why there are likely to reflect endogenous DNase activity during incubation no bands corresponding to these sites detected. However, the of the nuclei at 37°C. sequence of this region demonstrates the existence of Sau96A DNase 1 hypersensitive sites downstream of the SCL gene and HhaI sites between +0.7HS and +1HS which remain were sought using SacI-digested and DNase 1-treated DNA uncut thus demonstrating the existence of two spatially dis- which was hybridised with probe p6 (Figure 1f). In addition tinct regions of hypersensitive chromatin (data not shown). In to a 5 kb germline band, two bands of approximately 1.3 and addition, appropriately digested DNA samples representing 2.3 kb were evident (open arrowheads) representing DNase 1 the +1HS and +0.7HS have been run on the same gel and hypersensitive sites mapping 17 kb and 18 kb downstream of show clear differences in migration (data not shown). promoter 1a, +17HS and +18HS, respectively. In Figure 2 g a filter containing HhaI + SacI-digested DNA To confirm the reproducibility of this pattern of DNase 1 was hybridised with probe p4 to yield a 1.1 kb germline band hypersensitive sites we analysed a second mast cell line, 6−, and a 0.5 kb band corresponding to the +3HS. In order to using a restriction endonuclease accessibility assay.45 The survey the 3Ј portion of the body of the SCL gene, nuclei were technique is essentially similar to DNase 1 hypersensitive site incubated with HhaI, DNA subsequently digested with EcoRI analysis with the exception that isolated nuclei were incu- and hybridised with probe p5 (Figure 2h). A 10 kb germline bated with a restriction enzyme instead of DNase 1. We have band was seen together with an 8 kb band representing the found this technique to be faster and less technically +7HS. demanding than DNase 1 hypersensitive site analysis. In Finally, to confirm the DNase 1 hypersensitive sites ident- addition, the use of several ‘frequent-cutter’ restriction ified downstream of the SCL gene, DNA from ScrFI or HaeIII- endonucleases permits the precise mapping of the DNase 1 treated nuclei was digested with SacI, and Southern filters hypersensitive sites if the sequence of the region is known. hybridised with probe p6. In addition to the 5 kb germline Figure 2a is a schematic representation of the restriction fragment, bands at approximately 1.2 kb (Figure 2i, open digestions and DNA probes used to analyse the SCL locus in arrowhead) and 2.3 kb (Figure 2j, open arrowhead) were also 6− cells. In Figure 2b isolated nuclei were incubated with evident corresponding to the +17HS and +18HS, respectively. AvaII and the DNA subsequently isolated and digested with The additional faint bands observed in Figure 2i are likely to KpnI. The DNA was then Southern blotted and the filter reflect ‘background cutting’ due to digestion of DNA from hybridised with probe p1. In addition to the germline fragment lysed nuclei during the incubation step at 37°C and were of 6.7 kb (closed arrowhead), a 1.5 kb band (open arrowhead) not reproducible. was also observed corresponding to a hypersensitive site A summary of the nuclease hypersensitive sites is depicted 8.6 kb upstream of exon 1a (−8.6HS) (Figure 2b). In Figure 2c schematically in Figure 3. Importantly, we consistently nuclei were digested with RsaI, the DNA subsequently observed the same pattern using the different techniques in digested with KpnI and run on the same gel as in Figure 2b. two independently generated mast cell lines. Furthermore, the Southern blot filters were hybridised with probe p1. The pattern overlaps with but is distinct from that previously seen expected germline fragment of 6.7 kb was observed (closed in erythroid and primitive myeloid cell lines.33 arrowhead) with a faint but reproducible band at approxi- mately 1 kb representing a hypersensitive site 8.1 kb upstream − Lineage-restricted regulatory elements modulate SCL of promoter 1a ( 8.1HS). The band at approximately 3 kb seen transcription in mast cells in all lanes in Figures 2b and 2c is likely to represent cross- hybridisation or a KpnI polymorphism within the SCL locus. To investigate the function of the putative SCL regulatory There are 14 AvaII and 20 RsaI recognition sites spread elements in mast cells we have used a panel of luciferase throughout the 6.7 kb KpnI fragment (data not shown), illus- reporter constructs and assessed their activity following transi- trating that the bands we observe represent specific hypersen- ent transfection into Mst cells. SCL transcriptional regulation in mast cells JL Fordham et al 754

Figure 2 Analysis of restriction endonuclease hypersensitive sites associated with the murine SCL gene in 6− cells. Restriction enzyme hypersensitive site analysis of 6− mast cells. Filled arrowheads, germline fragments; open arrowheads, fragments generated by restriction enzyme cleavage. (a) Partial restriction map of the murine SCL locus illustrating the probes (p1–6) used for hypersensitive analysis. The exons of the SCL gene are indicated. Filled boxes represent coding exons. A, ApaI; K, KpnI; R, EcoRI; S, SacI. (b–j) Nuclei were exposed to the restriction enzyme labelled above each panel, DNA subsequently extracted and digested with an appropriate second restriction enzyme (see a) and Southern blots analysed using the probes indicated below each panel. SCL transcriptional regulation in mast cells JL Fordham et al 755

Figure 3 Summary of DNase 1 and restriction endonuclease hypersensitive sites associated with the murine SCL gene. Hypersensitive sites identified in Mst and 6− mast cell lines are shown as arrows. K, KpnI; B, BamHI; A, ApaII; S, SacI.

Initial studies focused on the regions surrounding promoter construct is also comparable to a construct containing just 1a and promoter 1b. Both core promoter 1b (construct promoter 1a and 1b.13 These results suggest that the region +0.2E1b) and promoter 1a (construct −0.2E1a) were active in containing the +3HS acts as a potent enhancer. Mst cells, consistent with previous findings13 (Figure 4). By The SCL gene exhibits complex alternate splicing patterns contrast, transient reporter assays revealed activity of only pro- involving the 5Ј untranslated exons and which results in exon moter 1a in erythroid cells, and only 1b in primitive 3 being the first exon that is shared between transcripts orig- myeloid cells.12 inating at either promoters 1a or promoter 1b. Since distant However, a construct containing both promoters with the regulatory elements may potentially act on promoter 1a, pro- luciferase gene fused into exon 3 (−0.2E3), and which there- moter 1b or both, we tested the effects of DNA fragments con- fore contained the +0.7HS, +1HS and +3HS, was approxi- taining the various hypersensitive sites on the activity of the mately 10-fold more active than either of the promoters alone. −0.2E3 cassette. DNA fragments containing the −8.6/−8.1HS, This effect may reflect altered RNA processing and/or stability +7HS, and +17/+18HS were subcloned downstream of the resulting from the inclusion of introns in the −0.2E3 construct, luciferase gene in −0.2E3. The −4HS was tested by including although this seems unlikely because the parental pGL-2 basic 7 kb of sequence upstream of exon 1a (construct −7.0E3). vector already includes an intron. Interestingly, removal of the Transient luciferase reporter assays were then performed in +3HS (construct −0.2E2b) reduced activity to the level of the Mst cells to assess the effect of all these hypersensitive sites 1b promoter alone (Figure 4). The activity of the −0.2E2b on the activity of −0.2E3 (Figure 5).

Figure 4 Transient transfection assays of SCL promoter constructs. Transient transfections were performed using Mst cells. SCL promoter constructs and the additional hypersensitive sites are depicted on the left. Luciferase values are the mean (±s.d) of at least four independent electroporations using two different DNA preparations. Fold activation values represent luciferase activity (normalised against ␤-gal values) relative to background luciferase activity obtained using pGL-2 basic. SCL transcriptional regulation in mast cells JL Fordham et al 756

Figure 5 Transient transfection analysis of hypersensitive site func- tion. Transient transfections were performed using Mst cells. The con- structs and the additional hypersensitive sites (HS) that are not present within the −0.2E3 cassette are indicated on the left. Fold activation values represent luciferase activity (normalised against ␤-gal values) relative to luciferase activity obtained using −0.2E3.

Luciferase activities are presented relative to the −0.2E3 construct which is already 450-fold more active than pGL-2 basic. The addition of either the −8.6/−8.1HS, or −4HS had little or no effect on luciferase activity. Addition of the +7HS resulted in an approximately two-fold drop in activity which may represent a modest silencing effect. By contrast, the addition of +17/+18HS resulted in a 3.5-fold increase in − luciferase activity over 0.2E3 and a 1350-fold increase rela- + + tive to pGL2 basic. The activity of the +17/+18 HS is lineage Figure 6 The 17HS and 18HS element function co-operatively. (a) Schematic representation of the +17HS and +18HS constructs gen- restricted since this construct exhibited no activity in a T cell erated to address the enhancer activity of each hypersensitive site indi- 33 (non-SCL expressing) or primitive myeloid cell line. vidually. (b) Transient transfections were performed using Mst cells. The constructs and the additional hypersensitive sites (HS) that are not present with in the −0.2E3 cassette are indicated on the left. Fold + + activation values represent the luciferase activity (normalised against Both the 17HS and 18HS are required for full ␤ − activity of the 3Ј enhancer in mast cells -gal values) relative to luciferase activity obtained using 0.2E3.

Since the 3Ј enhancer contains two hypersensitive sites it was series of constructs containing promoter 1a, promoter 1b, or important to assess whether the +17HS, the +18HS or both the SV40 early promoter in pGL2 with the +17/+18HS element were responsible for the enhancer activity in mast cells. A ser- cloned downstream of the luciferase gene.33 The constructs ies of constructs was therefore generated to map more pre- were transiently transfected into Mst cells and their activities cisely the enhancer element. The constructs are illustrated in measured by luciferase reporter assays (Figure 7). Interestingly, Figure 6a. Three constructs were generated, a 3 kb HindIII the +17/+18HS was able to enhance approximately three-fold fragment which contains both the +17HS and +18HS transcription from both promoter 1a and promoter 1b and also (−0.2E3/a), a BglII/BamHI fragment containing the +17HS produced a four-fold increase in transcription from the SV40 (−0.2E3/b), and a BamHI/SacI fragment containing the +18HS promoter. Thus, in a mast cell environment, but not in an (−0.2E3/c). The activity of the constructs was measured fol- erythroid environment33 the 3Ј enhancer was capable of lowing transient transfection into Mst cells (Figure 6b). The acting on both promoter 1a and promoter 1b. −0.2E3/+17,18 and −0.2E3/a constructs both exhibited approximately 3.5-fold greater activity than −0.2E3 alone. Interestingly, the +17HS alone (−0.2E3/b) displayed no Discussion enhancing activity and the +18HS alone (−0.2E3/c) gave rise to only a two-fold stimulation relative to −0.2E3. These data SCL is expressed in multipotent progenitors and also in com- demonstrate that both the +17HS and the +18HS were mitted erythroid, mast and megakaryocytic cells15–17,24 but required for full activity of the 3Ј enhancer. SCL expression is extinguished during differentiation along other lineages.23,24 This expression pattern suggested that SCL might play an important role in haematopoiesis, a concept The 3Ј enhancer activates both promoter 1a and that was confirmed by the generation of SCL null mice which promoter 1b failed to develop all haematopoietic lineages.28,29 We have elected to dissect the mechanisms which control SCL The −0.2E3 cassette contained both promoter 1a and pro- expression throughout haematopoiesis in order to illuminate moter 1b, and so the 3Ј enhancer might potentially interact the molecular basis for stem cell development and lineage with one or both SCL promoters. In order to address promoter commitment. specificity of the 3Ј enhancer we took advantage of a further Mast cells are crucial for protective responses to parasitic SCL transcriptional regulation in mast cells JL Fordham et al 757 open chromatin 8.6 and 8.1 kb upstream of promoter 1a (data not shown). The results presented here demonstrate the presence of a consistent pattern of hypersensitive sites in mast cells, which overlaps with but was distinct from the patterns previously described in erythroid and primitive myeloid cells. In parti- cular, the −8.6HS, −8.1HS and +7HS were present in mast, erythroid and primitive myeloid cells. By contrast, the +3HS, +17HS and +18HS were found in mast and erythroid and the −4HS was found in mast and primitive myeloid cells. A second hypersensitive site observed 3 kb upstream of pro- moter 1a (−3HS) in primitive myeloid cells is not present in mast cells. The significance of these distinct patterns of hyper- sensitive sites in different lineages is not clear, but suggests that distinct combinations of regulatory elements may be responsible for modulating SCL expression in different haema- topoietic cell types. Functional assays of the various hypersensitive sites ident- ified two regions with particularly powerful enhancer activity in mast cells. The first of these was a region containing the Ј Figure 7 Promoter specificity of the SCL 3 enhancer. Transient +3HS in the body of the SCL gene. This region functioned in analysis in Mst cells of the +17/+18HS in conjunction with SCL pro- moter 1a (−0.2E1a), SCL promoter 1b (+0.2E1b) and SV40 early pro- a similar manner in both erythroid and primitive myeloid 33 moter (SV40). Fold activation values represent the luciferase activity cells. Interestingly, the human SCL gene contains a hyper- (normalised against ␤-gal values) relative to luciferase activity sensitive site at approximately the same position46 and com- obtained using the appropriate promoter alone. parison of the human and mouse sequence has revealed a highly conserved stretch of 150 bp immediately upstream of exon 3.47 These data therefore suggest the presence of a con- infections and are also the major cellular mediator for many served intronic enhancer which functions in multiple haema- allergic reactions. Identification of SCL enhancers that target topoietic lineages. mast cell progenitors and mature mast cells will shed light on The second potent enhancer was downstream of the SCL the mechanisms responsible for maintaining SCL expression gene. The position of this 3Ј enhancer was marked by two following lineage commitment. Such enhancers would also hypersensitive sites, +17HS and +18HS. DNA fragments con- allow targeted expression of exogenous genes within the mast taining both hypersensitive sites were required for full cell lineage, and would therefore provide powerful tools for enhancer activity. We have previously shown that a the 3Ј experimental and therapeutic manipulation of mast cells. We enhancer upregulates the −0.2E3 promoter cassette in have previously characterised the molecular basis for the erythroid cells but not in T cells or primitive myeloid cells.33 activity of SCL promoter 1a and promoter 1b in mast cells.13 Furthermore the +17 and +18 DNase 1 hypersensitive sites However, nothing is known about distant SCL regulatory were not detectable in primitive myeloid cells or T cells.33 elements in mast cells. These data strongly suggest that the enhancer exhibits cell- In this paper we have undertaken a systematic analysis of type specificity. Within a given cell type, we speculate that DNase 1 hypersensitive sites within a region of approximately the promoter specificity of the enhancer may depend on 45 kb spanning the SCL gene. This has allowed us to compare whether the promoters being tested are potentially active and the pattern of DNase 1 hypersensitive sites in mast cells with therefore permissive for enhancer function. Thus, both pro- the patterns we have previously observed in two other haema- moter 1a and 1b are active in mast cells (and therefore able topoietic lineages, namely erythroid and primitive myeloid to respond to the enhancer), whereas the core promoter 1b is cells.33 Furthermore, we have now used restriction endonucle- inactive in erythroid cells (and therefore not responsive to the ase accessibility to provide high resolution mapping of the enhancer). These data therefore raise the possibility that lin- precise location of the regions of open chromatin in mast eage-restricted transcription factors needed for promoter cells. activity may also be required for promoter/enhancer interac- Our results have identified two hypersensitive sites (+0.7HS tion. Promoter 1a and 1b contain Sp1 sites and promoter 1a and +1HS) previously unrecognised in other SCL-expressing contains two GATA sites. It may therefore be relevant that cell types. Given the relatively low resolution of DNase 1 both GATA-1 and Sp1 have been shown to participate in pro- hypersensitive site studies, it will be important to assess tein–protein interactions which may mediate the physical whether these two sites are in fact present in other SCL- association of promoters with enhancers.48–50 expressing lineages, or whether they are indeed mast cell The data presented here together with our previous studies restricted. It is also interesting to note that the intron between show that the 3Ј enhancer is active in mast and erythroid cells exons 1b and 2b which contains the +0.7HS and +1HS, also but is inactive in primitive myeloid cells and T cells.33 How- contains a 400 bp stretch of sequence that is well conserved ever, the SCL gene itself is expressed in haematopoietic stem between man and mouse (data not shown). cells and its expression is subsequently maintained following The −10HS described previously33 was also mapped using commitment to erythroid, mast and megakaryocytic lineages DNase 1 hypersensitive site analysis with a relatively low res- but down-regulated following commitment to myeloid and olution. Here, we have used restriction endonuclease accessi- lymphoid lineages.51 It will now be important to address bility to precisely map a doublet of hypersensitive sites at whether the 3Ј enhancer is activated independently in the −8.6/−8.1 kb. 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