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Mol Cell Biol. Dec 2010; 30(24): 5741–5751.
Published online Oct 18, 2010. doi:  10.1128/MCB.00870-10
PMCID: PMC3004278
cis-Regulatory Remodeling of the SCL Locus during Vertebrate Evolution[down-pointing small open triangle]
Berthold Göttgens,1 Rita Ferreira,1 Maria-José Sanchez,2 Shoko Ishibashi,3 Juan Li,1 Dominik Spensberger,1 Pascal Lefevre,4 Katrin Ottersbach,1 Michael Chapman,1 Sarah Kinston,1 Kathy Knezevic,1 Maarten Hoogenkamp,4 George A. Follows,1 Constanze Bonifer,4 Enrique Amaya,3 and Anthony R. Green1*
Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom,1 Centro Andaluz de Biologia del Desarrollo, CSIC-Universidad Pablo de Olavide, Seville, Spain,2 Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom,3 Section of Experimental Haematology, University of Leeds, St James's University Hospital, Leeds LS9 7TF, United Kingdom4
*Corresponding author. Mailing address: Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom. Phone: 004401223336820. Fax: 00441223762670. E-mail: arg1000/at/cam.ac.uk
Equal contribution.
Received July 27, 2010; Revised August 21, 2010; Accepted October 3, 2010.
Development progresses through a sequence of cellular identities which are determined by the activities of networks of transcription factor genes. Alterations in cis-regulatory elements of these genes play a major role in evolutionary change, but little is known about the mechanisms responsible for maintaining conserved patterns of gene expression. We have studied the evolution of cis-regulatory mechanisms controlling the SCL gene, which encodes a key transcriptional regulator of blood, vasculature, and brain development and exhibits conserved function and pattern of expression throughout vertebrate evolution. SCL cis-regulatory elements are conserved between frog and chicken but accrued alterations at an accelerated rate between 310 and 200 million years ago, with subsequent fixation of a new cis-regulatory pattern at the beginning of the mammalian radiation. As a consequence, orthologous elements shared by mammals and lower vertebrates exhibit functional differences and binding site turnover between widely separated cis-regulatory modules. However, the net effect of these alterations is constancy of overall regulatory inputs and of expression pattern. Our data demonstrate remarkable cis-regulatory remodelling across the SCL locus and indicate that stable patterns of expression can mask extensive regulatory change. These insights illuminate our understanding of vertebrate evolution.
Spatiotemporal changes in gene expression patterns represent one of the main driving forces of evolution (reviewed in references 10 and 41), with morphological divergence being linked to mutations of specific cis-regulatory elements in a growing number of examples (3, 21, 29, 35, 42). Conversely, evolutionarily conserved features are thought to reflect conserved gene expression patterns, but little is known about the mechanisms responsible for such stability. Most evidence comes from invertebrate studies and points to disparate mechanisms. For example, in the urochordate Ciona, orthologous muscle-specific enhancers are highly conserved across 270 million years (7), whereas orthologues of the Drosophila even-skipped stripe 2 enhancer exhibit multiple differences in transcription factor binding sites and yet have maintained the same spatiotemporal activity (33, 34). In vertebrates, the cis-regulatory elements of many regulatory genes are not conserved in comparisons between mammals and lower vertebrates (1, 14, 49), and little is known about the mechanisms responsible for maintaining stable gene expression patterns.
The SCL gene encodes a basic helix-loop-helix (bHLH) transcription factor with pleiotropic and nonredundant roles in the development of the hematopoietic (40, 43), vascular (20, 36), and central nervous systems (5, 20). Systematic dissection of cis-regulatory mechanisms operating at the murine SCL locus has identified multiple elements, each of which directs expression to a subdomain of the normal SCL expression pattern in transgenic mice. The two promoters (1a and 1b) have no hematopoietic activity (46), but three distal elements (kb −4, +19, and +40 with respect to the start of exon 1a) that are active in blood and/or endothelium have been identified. The −4 element directs expression to endothelial cells (46), the +19 to hematopoietic stem cells/progenitors (44, 45), and the +40 to erythroid cells (15). Each of these elements is characterized by a particular set of transcription factor binding sites. Promoter 1a (here referred as promoter) contains two functionally important GATA sites (4), the −4 element contains two ETS sites (24), the +19 enhancer contains an ETS/ETS/GATA motif (22), and the +40 element contains two GATA/E-box motifs (39). Mutation or deletion of a single one of these motifs leads to a loss of function of the enhancer (4, 22, 24, 39, 46).
The pattern of SCL expression is highly conserved throughout vertebrate evolution (23, 26, 28, 36, 46, 51). In this work, we have used stringent functional assays to characterize the cis-regulatory mechanisms responsible for maintaining the stability of this expression pattern. Our results reveal the existence of extensive cis-regulatory remodelling, which affects multiple elements across the SCL locus. This unexpected plasticity has major implications for our understanding of vertebrate evolution.
Sequence analysis.
The human, mouse, and chicken SCL sequences and annotation were downloaded from our web server (http://hscl.cimr.cam.ac.uk/genomic_datasets.html). Frog SCL sequences were identified by BLAT analysis of the Xenopus tropicalis draft genome sequence at http://genome.ucsc.edu/. Global sequence alignments were performed using the lagan (http://lagan.stanford.edu/lagan_web/index.shtml) and chaos/dialign (http://dialign.gobics.de/chaos-dialign-submission) programs. Local sequence alignments were performed using ClustalW (48) installed locally and displayed using Genedoc (http://www.psc.edu/biomed/genedoc/). Genomic sequences were scanned for occurrences of transcription factor binding site consensus sequences using our own software program, TFBSsearch (13).
Transgenic mouse analysis.
Chicken transgenic constructs were prepared using standard recombinant DNA technology. cSCL P/lac contained chicken sequences from −0.9 to +1.6 in exon 3 linked to a LacZ reporter gene. The cSCL-4/SV/lac, cSV/lac/SCL+19, and cSV/lac/SCL+40 constructs contained sequences from kb −2.1 to −0.9, +6.1 to +10.9, and +21.6 to +22.3, respectively, linked to the simian virus 40 (SV40) minimal promoter from pGL2-Promoter (Promega) and a LacZ reporter gene. Mouse transgenic constructs have been described (22, 39, 46). Transgenic mice were generated and analyzed as described previously (44). Both F0 embryos and stable transgenic mouse lines were used for the analysis. All mice were kept under specific-pathogen-free conditions, and all procedures were performed under the project license approved by the UK Home Office.
Transfection assays.
Chicken core promoter fragments (188 and 142 bp) were amplified by PCR and cloned into the BglII-XhoI sites of the pGL2-Basic luciferase vector (Promega). The ETS mutant was generated by PCR using specific primers containing the mutation (TTCC to CTCT for the first ETS site and GGAA to GGCC for the second ETS site). The chicken SCL +19 region was subcloned from SV/lac/cSCL+19 into the SalI-BamHI sites of the pGL2-Promoter luciferase vector (Promega). The opossum 200-bp core promoter fragment and the platypus 250-bp core promoter fragment were PCR amplified from genomic DNA of opossum kidney cells and platypus BAC clone 200B24, respectively. Mutations were generated using the QuikChangeII XL site-directed mutagenesis kit (Stratagene). For luciferase assays, 416B cells were transfected and assayed as described previously (15).
In vivo DMS footprinting.
The chicken cell lines DT40 (B cells), MEP (multipotent progenitors), and HD37 (erythroid) were cultured as described previously (8, 30), and in vivo dimethylsulfate (DMS) footprinting was performed as detailed previously (19). Briefly, living cells or genomic DNA was incubated at room temperature in 0.2% DMS solution in phosphate-buffered saline (PBS) for 5 min. The reaction was stopped with multiple washes with ice-cold PBS. Following cell lysis and DNA extraction, the DNA was cleaved with 0.1 M piperidine at 90°C for 10 min. Cleavage sites were analyzed by ligation-mediated PCR (LM-PCR). PCR products were labeled by primer extension using 32P-labeled nested primers and were analyzed on 6% denaturing polyacrylamide gels as described previously (19). Linker and primer sequences are available on request. Experiments were performed on 3 separate occasions with material from 2 independent DMS-treated cell and genomic DNA preparations.
Chromatin immunoprecipitation.
Chromatin immunoprecipitation (ChIP) was performed as described previously (15), using rabbit anti-SCL antibody (17) (a kind gift from Chris Drake, Charleston, SC) and nonspecific rabbit IgG (Sigma). Enrichment was determined using SYBR green real-time quantitative PCR (Stratagene) as described previously (15) and normalized against nonspecific IgG. Primers specific for the 3′ untranslated region (3′UTR) of the chicken SCL gene and for the promoter of the hepatocyte-specific gene ApoVLDL2 (32) were used as negative controls.
Xenopus transgenics.
The SV/Venus and SV/Venus/cSCL+19 constructs were generated by replacement of the LacZ reporter gene in SV/Lac and SV/Lac/cSCL+19 by the Venus sequence from Venus/pCS2 (38) (a kind gift from Atsushi Miyawaki, Saitama, Japan). Double transgenic Xenopus laevis embryos were generated using restriction enzyme-mediated integration (27, 31), using either SV/Venus or SV/Venus/cSCL+19 and γ-crystallin-mCherry, which was derived from γ-crystallin-green fluorescent protein (GFP) (6). The plasmids were linearized with SalI prior to the transgenesis protocol. Embryos expressing mCherry in the lens were fixed at stage 37/38, and Venus expression was assessed by whole-mount in situ hybridization using an antisense enhanced green fluorescent protein (EGFP) probe (47). Some of the embryos were allowed to develop to the larval stage (stage 47) in order to assess for Venus expression in the circulating blood.
Comparison of the SCL loci in different vertebrates.
Comparative genome-based approaches are commonly used to identify cis-regulatory elements, based on the rationale that functionally important sequences remain unaltered through evolution while other sequences are allowed to change. Comparison of the human (Homo sapiens), murine (Mus musculus), chicken (Gallus gallus), frog (Xenopus tropicalis), and zebrafish (Danio rerio) SCL loci shows that flanking genes are largely conserved, with MAP17 situated immediately downstream of SCL in all four species (Fig. (Fig.1A).1A). Long-range (global) DNA sequence alignment of the murine and human loci demonstrated that the promoter, as well as all three hematopoietic enhancers, was highly conserved. In contrast, alignment of the murine and chicken loci revealed significant sequence homology corresponding to the promoter but failed to identify peaks corresponding to the murine −4, +19, and +40 enhancers (Fig. (Fig.1B).1B). Failure to identify homologues for regulatory elements by global sequence alignment is a well-known phenomenon in distal vertebrate comparisons (18). However, it is not clear whether this reflects the inability of sequence alignment algorithms to detect small but critical motifs in large stretches of otherwise highly diverged sequence or whether a large proportion of human/mouse regulatory elements are mammalian inventions with no lower vertebrate counterparts. Since the SCL cis-regulatory elements have been extensively characterized, we proceed to make use of the SCL locus as a model system to address this question.
FIG. 1.
FIG. 1.
Structure and sequence conservation of vertebrate SCL locus. (A) Structures of the human (H), mouse (M), chicken (C), frog (F), and zebrafish (Z) SCL loci. All loci are drawn to the same scale (numbers represent kb). Boxes represent exons, and arrows (more ...)
The chicken SCL promoter has hematopoietic activity.
Since the SCL promoter was the only cis-regulatory element to be identified by long-range DNA alignment, we initially focused on the structure and function of this conserved region. Comparative sequence analysis (human/mouse/chicken/frog) revealed an unexpected pattern (Fig. (Fig.2A).2A). In addition to blocks of homology conserved between all 4 species (black shading), the chicken sequence was more closely related to the frog sequence (turquoise shading) than to the mammalian sequences. This observation was surprising, since chickens and mammals separated 310 million years ago whereas chickens and frogs have evolved separately for more than 350 million years (see Fig. S1 at http://hscl.cimr.cam.ac.uk/Gottgens_et_al_MCB_Supplemental_Information.pdf). Moreover, the additional stretches of conservation restricted to chicken and frog contained ETS and GATA motifs reminiscent of the combination found in the mammalian +19 hematopoietic/endothelial enhancer (22). These data suggested that the promoter element of lower vertebrates might have additional functionality that compensates for the apparent lack of distal hematopoietic/endothelial enhancers.
FIG. 2.
FIG. 2.
The chicken SCL promoter has hematopoietic activity. (A) Four-way sequence alignment of the SCL promoter, indicating conservation across all species (black shading), human/mouse conservation only (gray shading), and chicken/frog conservation only (turquoise (more ...)
We have shown previously that individual murine SCL cis-regulatory elements reproducibly direct lacZ expression to subdomains of the normal SCL expression pattern (see Fig. S2 at http://hscl.cimr.cam.ac.uk/Gottgens_et_al_MCB_Supplemental_Information.pdf) (15, 22, 24, 39, 46). Therefore, we used transgenic reporter assays to compare the functions of the mouse and chicken promoter regions. A murine promoter construct directed expression to the midbrain and spinal cord (46), as did an equivalent chicken construct. However, the chicken construct was also active in endothelial cells, aortic clusters, and a subpopulation of hematopoietic cells in fetal liver (Fig. (Fig.2B2B and Table Table1;1; see also Fig. S3 and S4A at http://hscl.cimr.cam.ac.uk/Gottgens_et_al_MCB_Supplemental_Information.pdf). Consistent with these results, the chicken core promoter was highly active in stable transfection assays using the murine hematopoietic progenitor cell line 416B, whereas the equivalent murine core promoter was minimally active (Fig. (Fig.2C).2C). Deletion of a 5′ fragment, containing the additional ETS and GATA motifs conserved between chicken and frog, did not affect activity in 416B cells, but mutation of the two 3′ ETS sites abolished activity. Consistent with the transfection data, in vivo footprinting studies demonstrated that the 3′ GATA/GATA/ETS/ETS motifs were indeed occupied in the SCL-expressing chicken hematopoietic cells lines MEP (hematopoietic progenitors) and HD37 (erythroblast) but not in the SCL-negative cell line DT40 (B cell) (Fig. (Fig.2D;2D; see also supplemental Fig. 4B at the above URL). Moreover, the promoter of murine LYL-1, an SCL paralogue, contains GATA and ETS sites and is active in hematopoietic progenitors and endothelium (11, 12), suggesting that these attributes were present in the ancestral gene that gave rise to SCL and LYL-1. Taken together, our results indicate the following: (i) in the common ancestor of mammals and lower vertebrates, the SCL promoter exhibited hematopoietic and endothelial activity, and (ii) this activity was related to conserved 3′ ETS motifs subsequently lost from orthologous mammalian elements.
TABLE 1.
TABLE 1.
Activity of mouse and chicken SCL regulatory elements in transgenic embryos
Murine SCL −4 and +40 enhancers are functionally related to orthologous chicken enhancers.
These data raised the possibility that in the SCL loci of lower vertebrates, the promoter region might be sufficient to provide hematopoietic/endothelial activity and that homologues of the murine distal hematopoietic/endothelial enhancers (−4, +19, and +40 elements) might be absent. To test this idea, we took advantage of our previous identification of transcription factor binding sites essential for activities of SCL regulatory elements (15, 22, 24, 39, 44-46) and performed targeted searches of the chicken locus for motif combinations known to be required for activities of the murine −4, +40, and +19 elements.
Directed individual alignments indeed revealed putative orthologous elements within the chicken locus. Two ETS sites were identified upstream of chicken and frog SCL promoters (Fig. (Fig.33 A). Compared to functional ETS sites within the mouse −4 enhancer, both sites in chicken and frog were situated in extended blocks (7 to 11 bp) of sequence identity, a feature of functional motifs (13, 16). In transgenic reporter assays, the murine −4 enhancer was strongly active in all endothelial cells and in dorsal aorta clusters and targeted a proportion of fetal liver hematopoietic cells. In contrast, although activity of the chicken element was similar in fetal liver, endothelial activity was detectable only in rare cells (Fig. (Fig.3B3B and Table Table1;1; see also Fig. S3 at http://hscl.cimr.cam.ac.uk/Gottgens_et_al_MCB_Supplemental_Information.pdf).
FIG. 3.
FIG. 3.
The murine SCL −4 and SCL +40 enhancers are functionally related to the orthologous chicken enhancers. (A) Four-way sequence alignment of the SCL −4 region between human, mouse, chicken, and frog (shading as for Fig. Fig. (more ...)
A similar approach identified a pair of GATA/E-box composite motifs downstream of the chicken and frog MAP17 genes and in the same orientation as those in the mouse +40 enhancer (Fig. (Fig.3C).3C). The putative chicken element was functional in transgenic analyses and gave rise to midbrain staining in a pattern identical to that of the mouse element. The mouse +40 enhancer and its chicken orthologue exhibited similar activities in fetal liver, but the expression of the chicken element in circulating erythro- cytes was much lower than that of mouse +40 (9/10 and 2/11, respectively) (Fig. (Fig.3D3D and Table Table1;1; see also Fig. S3 at http://hscl.cimr.cam.ac.uk/Gottgens_et_al_MCB_Supplemental_Information.pdf). These data demonstrate that the chicken SCL locus contains orthologous enhancers functionally related to the murine −4 and +40 elements.
The murine SCL +19 enhancer and its chicken orthologue use different regulatory codes.
Identifying an orthologue for the mouse +19 enhancer was even more challenging. This mouse element contains a composite ETS/ETS/GATA motif with each individual binding site required for enhancer function (22), yet none of the 8 GATA motifs in the chicken SCL 3′ flanking sequence (4.8 kb from the chicken SCL poly(A) site to the equivalent of mouse SCL +23 [23]) was accompanied by two ETS motifs within any 50-nucleotide window (see Fig. S5A at http://hscl.cimr.cam.ac.uk/Gottgens_et_al_MCB_Supplemental_Information.pdf). However, one of these 8 GATA sites showed extended conservation (10/11 conserved nucleotides) with the mammalian +19 GATA site (Fig. (Fig.4A;4A; see also Fig. S5B at the above URL). This was the only 10/11 match to the extended GATA motif identified within the syntenic region of the chicken locus, a 30-kb fragment ranging from the poly(A) site of the SIL gene to the first exon of the CYP4X gene. Furthermore, the region 3′ of the frog SCL gene contained a GATA site with an 8/11 match to this particular chicken GATA motif, and strikingly, both chicken and frog GATA sites were preceded by an E-box motif in an 8-bp block of sequence identity (Fig. (Fig.4A).4A). Transgenic analysis confirmed the presence of a functional chicken enhancer. Whereas the mouse +19 enhancer was active in fetal liver, endothelium, and dorsal aorta clusters, the chicken enhancer was active mainly in circulating erythroid cells and fetal liver (Fig. (Fig.4B4B and Table Table1;1; see also Fig. S3 at http://hscl.cimr.cam.ac.uk/Gottgens_et_al_MCB_Supplemental_Information.pdf). The GATA and E-box motifs were both required for full activity of the chicken enhancer in stable transfection assays (Fig. (Fig.4C)4C) and in transgenic embryos (Table (Table11 and Fig. Fig.4D).4D). In vivo footprinting and chromatin immunoprecipitation (Fig. 4E and F) demonstrated occupancy of the E box and binding of the SCL protein to the chicken element in chicken hematopoietic cells. Consistent with the higher expression in circulating erythroid cells, the occupancy of the E box by SCL was higher in HD37 (erythroid) cells than in MEP (progenitor) cells. The bipartite GATA/E-box motif is commonly found in erythroid cell-specific regulatory elements (50), where it is bound by a multiprotein complex containing SCL and GATA-1. Due to the unavailability of antibody reagents against relevant chicken transcription factors other than chicken SCL, binding by chicken GATA-1 to the bipartite motif could not be proven directly. However, the cumulative evidence presented here strongly suggests that the chicken +19 enhancer operates through the typical bipartite GATA/E-box motif bound by SCL and GATA-1 in erythroid cells, because (i) the GATA site is conserved between chicken and frogs, (ii) the endogenous chicken SCL binds to the E-box portion of the bipartite motif, and (iii) mutation studies showed that both the E-box and GATA motifs were essential for erythroid activity of the chicken +19 enhancer when tested in both cell lines and transgenic mice.
FIG. 4.
FIG. 4.
The murine SCL +19 enhancer and its chicken orthologue use different regulatory codes. (A) Four-way sequence alignment of the SCL +19 region between human, mouse, chicken, and frog (shading as for Fig. Fig.2A).2A). Putative transcription (more ...)
The distinctive activity of the chicken +19 equivalent was confirmed in transgenic frogs. We have previously demonstrated that the mouse +19 enhancer directs expression in transgenic frogs to cells in the dorsolateral plate that are fated to give rise to hematopoietic progenitors and endothelium (22). In marked contrast, the chicken orthologue of the +19 element was active in the ventral blood islands of stage 37/38 embryos, the site of primitive erythropoiesis, and in the vast majority of circulating erythroid cells in stage 47 tadpoles (Fig. (Fig.5;5; see also Movies S1 and S2 at http://hscl.cimr.cam.ac.uk/Video%20S1.mov and http://hscl.cimr.cam.ac.uk/Video%20S2.mov, respectively). The results obtained using both transgenic mice and transgenic frogs are concordant with the erythroid activity of the chicken element in mice and also with the known erythroid activity of the E-box/GATA motif. Our data therefore demonstrate that the chicken SCL locus does contain a 3′ enhancer orthologous to the mouse +19 element but, remarkably, the two enhancers use different regulatory codes, the GATA/E-box erythroid code (50) and the hematopoietic stem cell/endothelial ETS/ETS/GATA code (25).
FIG. 5.
FIG. 5.
The chicken orthologue to the murine SCL +19 enhancer drives reporter gene expression to the ventral blood island in Xenopus embryos. The chicken SCL +19 enhancer drives reporter gene expression to the ventral blood island in Xenopus transgenics. (more ...)
Timing of cis-regulatory remodelling: the birth of a hematopoietic stem cell enhancer.
Our results demonstrate that the cis-regulatory elements regulating SCL expression in chicken are strikingly different from those operating in eutherian mammals. To better define the evolutionary timing of these changes, we cloned, sequenced, and annotated a BAC carrying the platypus (Ornithorhynchus anatinus) SCL locus (see Fig. S6 at http://hscl.cimr.cam.ac.uk/Gottgens_et_al_MCB_Supplemental_Information.pdf) and compared the SCL loci of platypus and opossum to those of eutherian mammals. The region between SCL and MAP17 in both platypus and opossum (Monodelphis domestica) contained the conserved ETS/ETS/GATA motif found in the +19 enhancer homologues of eutherian mammals (Fig. (Fig.6A).6A). In contrast, the SCL promoters of platypus and opossum contained two of the four ETS motifs found in chicken and frog (Fig. (Fig.6B).6B). Moreover, compared to the chicken and murine promoters, the platypus and opossum promoters showed intermediate activity in stable transfection assays in the 416B hematopoietic progenitor cell line, and mutation of the 5′ binding sites (ETS, ETS, and GATA) of the platypus promoter reduced its activity to the level of the mouse element's (Fig. (Fig.6C).6C). These data indicate that the emergence of the mammalian +19 structure, which directs expression to the vast majority of hematopoietic stem cells (44), preceded the complete loss of hematopoietic function in the SCL promoter and in a window between 310 million and 200 million years ago.
FIG. 6.
FIG. 6.
Binding site turnover across widely dispersed regulatory elements (A and B) 6-way sequence alignment of the SCL +19 (A) or SCL promoter (B) between human, mouse, opossum, platypus, chicken, and frog (shading as for Fig. Fig.2A).2A). Putative (more ...)
Over the last 2 decades, the concept of alteration in cis-regulatory elements, rather than coding sequences, has gained increasing support as a key driver for the evolution of morphological differences (reviewed in references 10 and 41). The majority of studies so far have focused on the analysis of cis-regulatory elements of invertebrate genes, alterations of which cause striking morphological changes in closely related species (3, 21, 29, 35, 42), with only a few examples in vertebrates (3). Compared to the evolution of altered gene expression patterns, much less is known about the mechanisms responsible for highly conserved patterns of expression. The latter might reflect highly conserved cis-regulatory mechanisms; some invertebrate data support this idea (3, 21, 29, 35, 42), but little is known about the mechanisms operating in vertebrates. In this study, we have used comparative genomic sequence analysis to identify the chicken SCL cis-regulatory elements. Where long-rage DNA alignments failed to identify conserved regions, we used our previous characterization of transcription factor binding sites essential for the activity of murine SCL regulatory elements to design targeted searches. To analyze the function of the chicken cis-regulatory elements and avoid possible functional redundancy, each element was analyzed independently. The function of each element was analyzed in vitro, using luciferase assays, in different hematopoietic cell lines and in vivo using transgenic mice and frogs. Both approaches showed that despite the conservation of gene expression patterns between mice and chickens, each independent element shows considerable functional differences compared with its mouse orthologue.
Several aspects of our data are of general significance. First, they demonstrate that a highly conserved expression pattern may hide unexpected cis-regulatory plasticity (Fig. (Fig.6D).6D). The functions of individual SCL elements are different in mammals and lower vertebrates. In some cases, the altered function reflects binding site turnover between distinct elements—the mammalian promoter has lost ETS sites, but these have been acquired by the mammalian +19 enhancer. In acquiring its ETS sites, the mammalian +19 enhancer has lost an E box present in the equivalent ancestral enhancer and has switched from an erythroid element to a hematopoietic stem cell/endothelial enhancer. The ancestral SCL locus contained two enhancers with erythroid GATA/E-box motifs (orthologous to the murine +19 and +40 enhancers), and this redundancy may have permitted the +19 equivalent to lose its E box without deleterious consequences. In the mouse, the +40 is the major element driving activity to the circulating erythroid cells, whereas in the chicken, the element corresponding to the +19 fulfils this role, raising the possibility that the chicken +40 orthologue functions primarily in the midbrain. Despite the multiple differences in the cis regulation of the mouse and chicken, the net effect is a constancy of regulatory inputs and spatiotemporal transcriptional output. However, this constancy conceals significant cis-regulatory alterations throughout the locus, with orthologous elements from different species exhibiting structural and functional differences. These observations also demonstrate that fixation of mammal-specific regulatory elements (2, 37) is not necessarily accompanied by the acquisition of new transcriptional inputs or changes in the overall expression pattern of a gene but may reflect “rewiring” within a locus. It is unclear how new transcription factor binding sites “appear” within an element, although it has recently been hypothesized that in addition to conventional evolutionary processes, specific binding sites may also be transferred between elements through recombination-based mechanisms (9).
Secondly, the data emphasize that when using comparative genomic sequence analysis to identify regulatory elements, an absence of evidence is not evidence of absence. Sequence alignments were unable to detect enhancers that could be identified given detailed knowledge of functionally important motifs. The +19 enhancer provides a particularly dramatic example, where a few conserved nucleotides flanking a consensus motif provided the clue to locating the chicken equivalent. Conversely, sequence conservation between orthologous regulatory elements may not equate to conserved function. The SCL promoter region exhibits significant sequence conservation throughout vertebrate evolution, and yet the mammalian promoter lacks hematopoietic/endothelial activity displayed by the SCL promoter from lower vertebrates.
Thirdly, our results have revealed an unexpected pattern of sequence conservation: the chicken sequence for each SCL regulatory element is more closely related to its frog orthologue than to its mammalian equivalent. This unexpected pattern of sequence conservation is not a general feature of the SCL locus, since alignments of the SCL coding region and the conserved SCL poly(A) region all show the expected pattern of sequence conservation (see Table S1 and Fig. S7 at http://hscl.cimr.cam.ac.uk/Gottgens_et_al_MCB_Supplemental_Information.pdf). These data indicate that after the divergence of birds and mammals, multiple SCL cis-regulatory elements accrued mutations at an accelerated rate in mammalian ancestors. The same “reversed” pattern of frog/chicken/mammal sequence conservation was identified for a GATA-2 hematopoietic/endothelial enhancer but not for Runx-1 or Hex/PRH (see Table S1 and Fig. S8 at the above URL), suggesting that cis-regulatory remodelling coupled with accelerated evolution of regulatory elements may be a more widespread phenomenon. We postulate that this accelerated acquisition of mutations in multiple cis-regulatory elements may have been triggered by the creation of a redundant element. The platypus and opossum sequences demonstrate that the switch of the +19 equivalent from a GATA/E-box to an ETS/ETS/GATA motif was an early event. This switch would have resulted in redundancy with the ancestral orthologues of both the promoter and the −4 enhancer, thus freeing other elements to accumulate alterations more rapidly without deleterious consequences. The ancestral SCL promoter is active in brain as well as in blood/endothelium, and so there may also be adaptive value in disentangling the individual components of such a hybrid bifunctional element, thus allowing unconstrained evolution of the individual components. We have shown that analogous departures from the expected pattern of regulatory element sequence conservation occur at other loci (e.g., GATA-2) but, depending on the timing of the initial trigger, will not always be revealed by frog/chicken/mammal comparisons.
In summary, our article contains a detailed characterization of the evolution of multiple elements across a key vertebrate regulatory locus, using stringent transgenic functional assays for individual cis elements. This analysis revealed remarkable remodeling of cis-regulatory elements across the locus despite constancy of transcriptional inputs and output (i.e., expression pattern). Moreover, an unexpected pattern of sequence conservation in regulatory elements (but not in coding regions) suggests an accelerated accumulation of mutations between 310 million and 200 million years ago, which our results suggest is probably the consequence of the formation of a new but redundant element. Given that redundancy of regulatory elements has been observed for many other vertebrate gene loci, cis-regulatory remodeling is likely to be a general phenomenon, suggesting that the comprehensive functional data presented here are of broad relevance for our understanding of vertebrate gene regulation and evolution.
Acknowledgments
We thank Michelle Hammett, Jacinta Carter, and Sandie Piltz for generating transgenic mice, Amy Chaney and Rebecca Kelley for sectioning embryos, and Pat Simpson and James C. Smith for constructive comments on the manuscript. We are also indebted to Jane Rogers, Darren Grafham, and the entire sequencing teams 40 and 47 at the Wellcome Trust Sanger Institute, Chris Drake for the chicken anti-SCL antibody, and Atsushi Miyawaki for the Venus/pSC2 construct.
Work in our laboratories is supported by the Wellcome Trust, the Leukemia Research Fund, the Medical Research Council, and the Leukemia and Lymphoma Society of America.
Footnotes
[down-pointing small open triangle]Published ahead of print on 18 October 2010.
1. Anand, S., W. C. Wang, D. R. Powell, S. A. Bolanowski, J. Zhang, C. Ledje, A. B. Pawashe, C. T. Amemiya, and C. S. Shashikant. 2003. Divergence of Hoxc8 early enhancer parallels diverged axial morphologies between mammals and fishes. Proc. Natl. Acad. Sci. U. S. A. 100:15666-15669. [PubMed]
2. Bejerano, G., M. Pheasant, I. Makunin, S. Stephen, W. J. Kent, J. S. Mattick, and D. Haussler. 2004. Ultraconserved elements in the human genome. Science 304:1321-1325. [PubMed]
3. Belting, H. G., C. S. Shashikant, and F. H. Ruddle. 1998. Modification of expression and cis-regulation of Hoxc8 in the evolution of diverged axial morphology. Proc. Natl. Acad. Sci. U. S. A. 95:2355-2360. [PubMed]
4. Bockamp, E. O., F. McLaughlin, A. M. Murrell, B. Gottgens, L. Robb, C. G. Begley, and A. R. Green. 1995. Lineage-restricted regulation of the murine SCL/TAL-1 promoter. Blood 86:1502-1514. [PubMed]
5. Bradley, C. K., E. A. Takano, M. A. Hall, J. R. Gothert, A. R. Harvey, C. G. Begley, and J. A. van Eekelen. 2006. The essential haematopoietic transcription factor Scl is also critical for neuronal development. Eur. J. Neurosci. 23:1677-1689. [PubMed]
6. Bronchain, O. J., K. O. Hartley, and E. Amaya. 1999. A gene trap approach in Xenopus. Curr. Biol. 9:1195-1198. [PubMed]
7. Brown, C. D., D. S. Johnson, and A. Sidow. 2007. Functional architecture and evolution of transcriptional elements that drive gene coexpression. Science 317:1557-1560. [PubMed]
8. Caldwell, R. B., P. Fiedler, U. Schoetz, and J. M. Buerstedde. 2007. Gene function analysis using the chicken B-cell line DT40. Methods Mol. Biol. 408:193-210. [PubMed]
9. Cameron, R. A., and E. H. Davidson. 2009. Flexibility of transcription factor target site position in conserved cis-regulatory modules. Dev. Biol. 336:122-135. [PubMed]
10. Carroll, S. B. 2008. Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution. Cell 134:25-36. [PubMed]
11. Chan, W. Y., G. A. Follows, G. Lacaud, J. E. Pimanda, J. R. Landry, S. Kinston, K. Knezevic, S. Piltz, I. J. Donaldson, L. Gambardella, F. Sablitzky, A. R. Green, V. Kouskoff, and B. Gottgens. 2007. The paralogous hematopoietic regulators Lyl1 and Scl are coregulated by Ets and GATA factors, but Lyl1 cannot rescue the early Scl−/− phenotype. Blood 109:1908-1916. [PubMed]
12. Chapman, M. A., F. J. Charchar, S. Kinston, C. P. Bird, D. Grafham, J. Rogers, F. Grutzner, J. A. Graves, A. R. Green, and B. Gottgens. 2003. Comparative and functional analyses of LYL1 loci establish marsupial sequences as a model for phylogenetic footprinting. Genomics 81:249-259. [PubMed]
13. Chapman, M. A., I. J. Donaldson, J. Gilbert, D. Grafham, J. Rogers, A. R. Green, and B. Gottgens. 2004. Analysis of multiple genomic sequence alignments: a web resource, online tools, and lessons learned from analysis of mammalian SCL loci. Genome Res. 14:313-318. [PubMed]
14. Chiu, C. H., C. Amemiya, K. Dewar, C. B. Kim, F. H. Ruddle, and G. P. Wagner. 2002. Molecular evolution of the HoxA cluster in the three major gnathostome lineages. Proc. Natl. Acad. Sci. U. S. A. 99:5492-5497. [PubMed]
15. Delabesse, E., S. Ogilvy, M. A. Chapman, S. G. Piltz, B. Gottgens, and A. R. Green. 2005. Transcriptional regulation of the SCL locus: identification of an enhancer that targets the primitive erythroid lineage in vivo. Mol. Cell. Biol. 25:5215-5225. [PMC free article] [PubMed]
16. Donaldson, I. J., M. Chapman, S. Kinston, J. R. Landry, K. Knezevic, S. Piltz, N. Buckley, A. R. Green, and B. Gottgens. 2005. Genome-wide identification of cis-regulatory sequences controlling blood and endothelial development. Hum. Mol. Genet. 14:595-601. [PubMed]
17. Drake, C. J., S. J. Brandt, T. C. Trusk, and C. D. Little. 1997. TAL1/SCL is expressed in endothelial progenitor cells/angioblasts and defines a dorsal-to-ventral gradient of vasculogenesis. Dev. Biol. 192:17-30. [PubMed]
18. Fisher, S., E. A. Grice, R. M. Vinton, S. L. Bessling, and A. S. McCallion. 2006. Conservation of RET regulatory function from human to zebrafish without sequence similarity. Science 312:276-279. [PubMed]
19. Follows, G. A., H. Tagoh, P. Lefevre, D. Hodge, G. J. Morgan, and C. Bonifer. 2003. Epigenetic consequences of AML1-ETO action at the human c-FMS locus. EMBO J. 22:2798-2809. [PubMed]
20. Gering, M., A. R. Rodaway, B. Gottgens, R. K. Patient, and A. R. Green. 1998. The SCL gene specifies haemangioblast development from early mesoderm. EMBO J. 17:4029-4045. [PubMed]
21. Gompel, N., B. Prud'homme, P. J. Wittkopp, V. A. Kassner, and S. B. Carroll. 2005. Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila. Nature 433:481-487. [PubMed]
22. Gottgens, B., L. M. Barton, M. A. Chapman, A. M. Sinclair, B. Knudsen, D. Grafham, J. G. Gilbert, J. Rogers, D. R. Bentley, and A. R. Green. 2002. Transcriptional regulation of the stem cell leukemia gene (SCL)—comparative analysis of five vertebrate SCL loci. Genome Res. 12:749-759. [PubMed]
23. Gottgens, B., L. M. Barton, J. G. Gilbert, A. J. Bench, M. J. Sanchez, S. Bahn, S. Mistry, D. Grafham, A. McMurray, M. Vaudin, E. Amaya, D. R. Bentley, A. R. Green, and A. M. Sinclair. 2000. Analysis of vertebrate SCL loci identifies conserved enhancers. Nat. Biotechnol. 18:181-186. [PubMed]
24. Gottgens, B., C. Broccardo, M. J. Sanchez, S. Deveaux, G. Murphy, J. R. Gothert, E. Kotsopoulou, S. Kinston, L. Delaney, S. Piltz, L. M. Barton, K. Knezevic, W. N. Erber, C. G. Begley, J. Frampton, and A. R. Green. 2004. The scl +18/19 stem cell enhancer is not required for hematopoiesis: identification of a 5′ bifunctional hematopoietic-endothelial enhancer bound by Fli-1 and Elf-1. Mol. Cell. Biol. 24:1870-1883. [PMC free article] [PubMed]
25. Gottgens, B., A. Nastos, S. Kinston, S. Piltz, E. C. Delabesse, M. Stanley, M. J. Sanchez, A. Ciau-Uitz, R. Patient, and A. R. Green. 2002. Establishing the transcriptional programme for blood: the SCL stem cell enhancer is regulated by a multiprotein complex containing Ets and GATA factors. EMBO J. 21:3039-3050. [PubMed]
26. Green, A. R., T. Lints, J. Visvader, R. Harvey, and C. G. Begley. 1992. SCL is coexpressed with GATA-1 in hemopoietic cells but is also expressed in developing brain. Oncogene 7:653-660. [PubMed]
27. Ishibashi, S., K. L. Kroll, and E. Amaya. 2008. A method for generating transgenic frog embryos. Methods Mol. Biol. 461:447-466. [PubMed]
28. Jaffredo, T., K. Bollerot, D. Sugiyama, R. Gautier, and C. Drevon. 2005. Tracing the hemangioblast during embryogenesis: developmental relationships between endothelial and hematopoietic cells. Int. J. Dev. Biol. 49:269-277. [PubMed]
29. Jeong, S., M. Rebeiz, P. Andolfatto, T. Werner, J. True, and S. B. Carroll. 2008. The evolution of gene regulation underlies a morphological difference between two Drosophila sister species. Cell 132:783-793. [PubMed]
30. Kontaraki, J., H. H. Chen, A. Riggs, and C. Bonifer. 2000. Chromatin fine structure profiles for a developmentally regulated gene: reorganization of the lysozyme locus before trans-activator binding and gene expression. Genes Dev. 14:2106-2122. [PubMed]
31. Kroll, K. L., and E. Amaya. 1996. Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122:3173-3183. [PubMed]
32. Lefevre, P., J. Witham, C. E. Lacroix, P. N. Cockerill, and C. Bonifer. 2008. The LPS-induced transcriptional upregulation of the chicken lysozyme locus involves CTCF eviction and noncoding RNA transcription. Mol. Cell 32:129-139. [PMC free article] [PubMed]
33. Ludwig, M. Z., C. Bergman, N. H. Patel, and M. Kreitman. 2000. Evidence for stabilizing selection in a eukaryotic enhancer element. Nature 403:564-567. [PubMed]
34. Ludwig, M. Z., A. Palsson, E. Alekseeva, C. M. Bergman, J. Nathan, and M. Kreitman. 2005. Functional evolution of a cis-regulatory module. PLoS Biol. 3:e93. [PMC free article] [PubMed]
35. McGregor, A. P., V. Orgogozo, I. Delon, J. Zanet, D. G. Srinivasan, F. Payre, and D. L. Stern. 2007. Morphological evolution through multiple cis-regulatory mutations at a single gene. Nature 448:587-590. [PubMed]
36. Mead, P. E., C. M. Kelley, P. S. Hahn, O. Piedad, and L. I. Zon. 1998. SCL specifies hematopoietic mesoderm in Xenopus embryos. Development 125:2611-2620. [PubMed]
37. Mikkelsen, T. S., M. J. Wakefield, B. Aken, C. T. Amemiya, J. L. Chang, S. Duke, M. Garber, A. J. Gentles, L. Goodstadt, A. Heger, J. Jurka, M. Kamal, E. Mauceli, S. M. Searle, T. Sharpe, M. L. Baker, M. A. Batzer, P. V. Benos, K. Belov, M. Clamp, A. Cook, J. Cuff, R. Das, L. Davidow, J. E. Deakin, M. J. Fazzari, J. L. Glass, M. Grabherr, J. M. Greally, W. Gu, T. A. Hore, G. A. Huttley, M. Kleber, R. L. Jirtle, E. Koina, J. T. Lee, S. Mahony, M. A. Marra, R. D. Miller, R. D. Nicholls, M. Oda, A. T. Papenfuss, Z. E. Parra, D. D. Pollock, D. A. Ray, J. E. Schein, T. P. Speed, K. Thompson, J. L. VandeBerg, C. M. Wade, J. A. Walker, P. D. Waters, C. Webber, J. R. Weidman, X. Xie, M. C. Zody, J. A. Graves, C. P. Ponting, M. Breen, P. B. Samollow, E. S. Lander, and K. Lindblad-Toh. 2007. Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences. Nature 447:167-177. [PubMed]
38. Nagai, T., K. Ibata, E. S. Park, M. Kubota, K. Mikoshiba, and A. Miyawaki. 2002. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20:87-90. [PubMed]
39. Ogilvy, S., R. Ferreira, S. G. Piltz, J. M. Bowen, B. Gottgens, and A. R. Green. 2007. The SCL +40 enhancer targets the midbrain together with primitive and definitive hematopoiesis and is regulated by SCL and GATA proteins. Mol. Cell. Biol. 27:7206-7219. [PMC free article] [PubMed]
40. Porcher, C., W. Swat, K. Rockwell, Y. Fujiwara, F. W. Alt, and S. H. Orkin. 1996. The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 86:47-57. [PubMed]
41. Prud'homme, B., N. Gompel, and S. B. Carroll. 2007. Emerging principles of regulatory evolution. Proc. Natl. Acad. Sci. U. S. A. 104(Suppl.)1:8605-8612. [PubMed]
42. Prud'homme, B., N. Gompel, A. Rokas, V. A. Kassner, T. M. Williams, S. D. Yeh, J. R. True, and S. B. Carroll. 2006. Repeated morphological evolution through cis-regulatory changes in a pleiotropic gene. Nature 440:1050-1053. [PubMed]
43. Robb, L., N. J. Elwood, A. G. Elefanty, F. Kontgen, R. Li, L. D. Barnett, and C. G. Begley. 1996. The scl gene product is required for the generation of all hematopoietic lineages in the adult mouse. EMBO J. 15:4123-4129. [PubMed]
44. Sanchez, M., B. Gottgens, A. M. Sinclair, M. Stanley, C. G. Begley, S. Hunter, and A. R. Green. 1999. An SCL 3′ enhancer targets developing endothelium together with embryonic and adult haematopoietic progenitors. Development 126:3891-3904. [PubMed]
45. Silberstein, L., M. J. Sanchez, M. Socolovsky, Y. Liu, G. Hoffman, S. Kinston, S. Piltz, M. Bowen, L. Gambardella, A. R. Green, and B. Gottgens. 2005. Transgenic analysis of the stem cell leukemia +19 stem cell enhancer in adult and embryonic hematopoietic and endothelial cells. Stem Cells. 23:1378-1388. [PubMed]
46. Sinclair, A. M., B. Gottgens, L. M. Barton, M. L. Stanley, L. Pardanaud, M. Klaine, M. Gering, S. Bahn, M. Sanchez, A. J. Bench, J. L. Fordham, E. Bockamp, and A. R. Green. 1999. Distinct 5′ SCL enhancers direct transcription to developing brain, spinal cord, and endothelium: neural expression is mediated by GATA factor binding sites. Dev. Biol. 209:128-142. [PubMed]
47. Sive, H. L., R. M. Grainger, and R. M. Harland. 2000. Early development of Xenopus laevis: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
48. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680. [PMC free article] [PubMed]
49. Tumpel, S., M. Maconochie, L. M. Wiedemann, and R. Krumlauf. 2002. Conservation and diversity in the cis-regulatory networks that integrate information controlling expression of Hoxa2 in hindbrain and cranial neural crest cells in vertebrates. Dev. Biol. 246:45-56. [PubMed]
50. Wadman, I. A., H. Osada, G. G. Grutz, A. D. Agulnick, H. Westphal, A. Forster, and T. H. Rabbitts. 1997. The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI proteins. EMBO J. 16:3145-3157. [PubMed]
51. Zhang, X. Y., and A. R. Rodaway. 2007. SCL-GFP transgenic zebrafish: in vivo imaging of blood and endothelial development and identification of the initial site of definitive hematopoiesis. Dev. Biol. 307:179-194. [PubMed]
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