Transcriptional Regulation of the Stem CellLeukemia Gene (SCL) — Comparative Analysisof Five Vertebrate SCL LociBerthold Go¨ttgens,1,4,5Linda M. Barton,1,4Michael A. Chapman,1Angus M. Sinclair,1Bjarne Knudsen,2Darren Grafham,3James G.R. Gilbert,3Jane Rogers,3David R. Bentley,3and Anthony R. Green11Cambridge Institute for Medical Research, Cambridge University, Cambridge, CB2 2XY, United Kingdom;2Bioinformatics Research Center, University of Aarhus, DK-8000 Denmark;3The Wellcome Trust Sanger Institute,Cambridge, CB101SA United KingdomThe stem cell leukemia (SCL) gene encodes a bHLH transcription factor with a pivotal role in hematopoiesis andvasculogenesis and a pattern of expression that is highly conserved between mammals and zebrafish. Here wereport the isolation and characterization of the zebrafish SCL locus together with the identification of threeneighboring genes, IER5, MAP17, and MUPP1. This region spans 68 kb and comprises the longest zebrafish genomicsequence currently available for comparison with mammalian, chicken, and pufferfish sequences. Our data showconserved synteny between zebrafish and mammalian SCL and MAP17 loci, thus suggesting the likely genomicdomain necessary for the conserved pattern of SCL expression. Long-range comparative sequenceanalysis/phylogenetic footprinting was used to identify noncoding conserved sequences representing candidatetranscriptional regulatory elements. The SCL promoter/enhancer, exon 1, and the poly(A) region were highlyconserved, but no homology to other known mouse SCL enhancers was detected in the zebrafish sequence. Acombined homology/structure analysis of the poly(A) region predicted consistent structural features, suggestinga conserved functional role in mRNA regulation. Analysis of the SCL promoter/enhancer revealed five motifs,which were conserved from zebrafish to mammals, and each of which is essential for the appropriate pattern orlevel of SCL transcription.[The following individuals kindly provided reagents, samples, or unpublished information as indicated in thepaper: N. Tanese.]The SCL gene (also known as Tal1) encodes a basic helix-loop-helix (bHLH) transcription factor with a critical role in hema-topoiesis and vasculogenesis. It was identified by virtue of itsdisruption in T-cell acute leukemia and rearrangements of theSCL locus are perhaps the most frequent molecular pathologyassociated with this tumor (Begley and Green 1999; Orkin etal. 1999). Targeted mutation of the SCL gene has shown thatit is essential for the development of all hemopoietic lineages(Porcher et al. 1996; Robb et al. 1996), and also for normalyolk sac angiogenesis (Visvader et al. 1998). Ectopic SCL ex-pression in zebrafish embryos specifies hemangioblast devel-opment from early mesoderm, with a consequent excessiveproduction of blood and endothelial progenitors, and canalso partially rescue endothelial and hemopoietic phenotypesof the cloche mutant (Gering et al. 1998; Liao et al. 1998). Acrucial role for SCL in haemopoietic and endothelial develop-ment has also been revealed in an analysis of the in vitrodifferentiation potential of SCLⳮ/ⳮembryonic stem cells(Robertson et al. 2000).SCL is normally expressed in hemopoietic cells, endothe-lium, and within specific regions of the central nervous sys-tem (CNS). This pattern of expression is highly conservedthroughout vertebrates from mammals to teleost fish (Greenet al. 1992; Kallianpur et al. 1994; Gering et al. 1998; Liao etal. 1998; Mead et al. 1998; Sinclair et al. 1999; Drake andFleming 2000). Murine and human SCL expression is tightlyregulated and involves two lineage-specific promoters (Le-cointe et al. 1994; Bockamp et al. 1995, 1997; Bockamp et al.1998). In addition, a detailed analysis of the chromatin struc-ture of the mouse SCL locus identified a number of DNaseIhypersensitive sites associated with enhancer or silencer ac-tivity (Go¨ttgens et al. 1997). More recently, studies usingtransgenic mice have identified five separate enhancers,which direct reporter gene expression in vivo to endothelium,midbrain, hindbrain/spinal cord, or hemopoietic progenitorcells, all subdomains of the normal SCL expression pattern(Sanchez et al. 1999; Sinclair et al. 1999; Go¨ttgens et al. 2000).We have recently cloned and sequenced the SCL locusfrom human, mouse, chicken, and pufferfish (Go¨ttgens et al.2000, 2001; Barton et al. 2001). Comparative sequence analy-sis of the human and mouse loci showed that all known regu-latory regions are highly conserved, and revealed a number ofadditional conserved noncoding regions that represent can-didate gene regulatory elements (Go¨ttgens et al. 2001). Inclu-sion of chicken sequences into multiple sequence alignmentsallowed us to prioritize some of these regions for functionalstudies, but failed to detect chicken homologs for otherknown enhancers (Go¨ttgens et al. 2000). The pufferfish SCLlocus exhibits considerable genomic compression, and a 10.5-4These authors contributed equally to this work.5Corresponding author.E-MAIL [email protected]; FAX 44-1223-762670.Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.45502.Letter12:749–759 ©2002 by Cold Spring Harbor Laboratory Press ISSN 1088-9051/01 $5.00; www.genome.org Genome Research 749www.genome.orgkb region containing the SCL geneand extending to the immediateflanking genes was sufficient to pro-duce appropriate expression in ze-brafish embryos (Barton et al. 2001).These results suggest that all of theregulatory elements necessary forconserved embryonic expression arepresent in this construct. However,this approach would not detect regu-latory elements necessary for expres-sion in adult tissues and is also lim-ited by the paucity of informationon the pattern of SCL expression inpufferfish.Zebrafish represent a powerfulmodel organism for studies of verte-brate development (Driever et al.1994), and zebrafish studies haveprovided considerable insight intohematopoiesis (Amatruda and Zon1999). Plans for a zebrafish genomesequencing project are well advanced(Duyk and Schmitt 2001), but littleinformation is available on the utilityof zebrafish/mammalian genomic se-quence comparisons (for examples ofconserved enhancer sequences, seeBeckers et al. 1996; Zerucha et al.2000). The pattern of SCL expressionis highly conserved between mam-mals and zebrafish (Gering et al.1998; Liao et al. 1998; Elefanty et al.1999; Sinclair et al. 1999) and wetherefore reasoned that comparisonof the
View Full Document
Unlocking...