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1.  Genome Wide Analysis Reveals Zic3 Interaction with Distal Regulatory Elements of Stage Specific Developmental Genes in Zebrafish 
PLoS Genetics  2013;9(10):e1003852.
Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches – ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.
Author Summary
The Zic3 transcription factor regulates early embryonic patterning, and the loss of its function leads to defects in left-right body asymmetry. Previous studies have only identified a small number of Zic3 targets, which renders the molecular mechanism underlying its activity insufficiently understood. Utilizing two genomics technologies, next generation sequencing and microarray, we profile the genome-wide binding sites of Zic3 and identified its target genes in the developing zebrafish embryo. Our results show that Zic3 regulates its target genes predominantly through regulatory elements located far from promoters. Among the targets of Zic3 are the Nodal and Wnt pathways known to regulate gastrulation and left-right body asymmetry, as well as neural pre-pattern genes regulating proliferation of neural progenitors. Using enhancer activity assay, we further show that genomic regions bound by Zic3 function as enhancers. Our study provides a genome-wide view of the regulatory landscape of Zic3 and its changes during vertebrate development.
doi:10.1371/journal.pgen.1003852
PMCID: PMC3814314  PMID: 24204288
2.  kmer-SVM: a web server for identifying predictive regulatory sequence features in genomic data sets 
Nucleic Acids Research  2013;41(Web Server issue):W544-W556.
Massively parallel sequencing technologies have made the generation of genomic data sets a routine component of many biological investigations. For example, Chromatin immunoprecipitation followed by sequence assays detect genomic regions bound (directly or indirectly) by specific factors, and DNase-seq identifies regions of open chromatin. A major bottleneck in the interpretation of these data is the identification of the underlying DNA sequence code that defines, and ultimately facilitates prediction of, these transcription factor (TF) bound or open chromatin regions. We have recently developed a novel computational methodology, which uses a support vector machine (SVM) with kmer sequence features (kmer-SVM) to identify predictive combinations of short transcription factor-binding sites, which determine the tissue specificity of these genomic assays (Lee, Karchin and Beer, Discriminative prediction of mammalian enhancers from DNA sequence. Genome Res. 2011; 21:2167–80). This regulatory information can (i) give confidence in genomic experiments by recovering previously known binding sites, and (ii) reveal novel sequence features for subsequent experimental testing of cooperative mechanisms. Here, we describe the development and implementation of a web server to allow the broader research community to independently apply our kmer-SVM to analyze and interpret their genomic datasets. We analyze five recently published data sets and demonstrate how this tool identifies accessory factors and repressive sequence elements. kmer-SVM is available at http://kmersvm.beerlab.org.
doi:10.1093/nar/gkt519
PMCID: PMC3692045  PMID: 23771147
3.  Mutations in the TGF-β Repressor SKI Cause Shprintzen-Goldberg Syndrome with Aortic Aneurysm 
Nature genetics  2012;44(11):1249-1254.
Increased transforming growth factor beta (TGF-β) signaling has been implicated in the pathogenesis of syndromic presentations of aortic aneurysm, including Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS)1-4. However, the location and character of many of the causal mutations in LDS would intuitively infer diminished TGF-β signaling5. Taken together, these data have engendered controversy regarding the specific role of TGF-β in disease pathogenesis. Shprintzen-Goldberg syndrome (SGS) has considerable phenotypic overlap with MFS and LDS, including aortic aneurysm6-8. We identified causative variation in 10 patients with SGS in the proto-oncogene SKI, a known repressor of TGF-β activity9,10. Cultured patient dermal fibroblasts showed enhanced activation of TGF-β signaling cascades and increased expression of TGF-β responsive genes. Morpholino-induced silencing of SKI paralogs in zebrafish recapitulated abnormalities seen in SGS patients. These data support the conclusion that increased TGF-β signaling is the mechanism underlying SGS and contributes to multiple syndromic presentations of aortic aneurysm.
doi:10.1038/ng.2421
PMCID: PMC3545695  PMID: 23023332
Aortic aneurysm; Shprintzen-Goldberg syndrome; Marfan syndrome; Loeys-Dietz syndrome; TGF-β signaling; SKI
4.  SOX10 Regulates Expression of the SH3-Domain Kinase Binding Protein 1 (Sh3kbp1) locus in Schwann Cells via an Alternative Promoter 
The transcription factor SOX10 has essential roles in neural crest-derived cell populations, including myelinating Schwann cells—specialized glial cells responsible for ensheathing axons in the peripheral nervous system. Importantly, SOX10 directly regulates the expression of genes essential for proper myelin function. To date, only a handful of SOX10 target loci have been characterized in Schwann cells. Addressing this lack of knowledge will provide a better understanding of Schwann cell biology and candidate loci for relevant diseases such as demyelinating peripheral neuropathies. We have identified a highly-conserved SOX10 binding site within an alternative promoter at the SH3-domain kinase binding protein 1 (Sh3kbp1) locus. The genomic segment identified at Sh3kbp1 binds to SOX10 and displays strong promoter activity in Schwann cells in vitro and in vivo. Mutation of the SOX10 binding site ablates promoter activity, and ectopic expression of SOX10 in SOX10-negative cells promotes the expression of endogenous Sh3kbp1. Combined, these data reveal Sh3kbp1 as a novel target of SOX10 and raise important questions regarding the function of SH3KBP1 isoforms in Schwann cells.
doi:10.1016/j.mcn.2011.10.004
PMCID: PMC3277675  PMID: 22037207
SOX10; Schwann cells; Comparative sequence analysis; transcriptional regulation; SH3KBP1; cin85
5.  A zebrafish SKIV2L2-enhancer trap line provides a useful tool for the study of peripheral sensory circuit development 
Gene expression patterns : GEP  2011;11(7):409-414.
The zebrafish is an ideal model for elucidating the cellular and molecular mechanisms that underlie development of the peripheral nervous system. A transgenic line that selectively labels all the sensory circuits would be a valuable tool for such investigations. In this study, we describe such a line: the enhancer trap zebrafish line Tg(SKIV2L2:gfp)j1775 which expresses green fluorescent protein (gfp) in the peripheral sensory ganglia. We show that this transgene marks all peripheral ganglia and sensory nerves, beginning at the time when the neurons are first extending their processes, but does not label the efferent nerves. The trapped reporter is inserted just upstream of a previously poorly described gene: lhfpl4 on LG6. The expression pattern of this gene by in situ hybridization reveals a different, but overlapping, pattern of expression compared to that of the transgene. This pattern also does not mimic that of the gene (skiv2l2), which provided the promoter element in the construct. These findings indicate that reporter expression is not dictated by an endogenous enhancer element, but instead arises through an unknown mechanism. Regardless, this reporter line should prove to be a valuable tool in the investigation of peripheral nervous system formation in the zebrafish.
doi:10.1016/j.gep.2011.06.002
PMCID: PMC3163734  PMID: 21742057
6.  Steroid hormone modulation of RET through two estrogen responsive enhancers in breast cancer 
Human Molecular Genetics  2011;20(19):3746-3756.
RET, a gene causatively mutated in Hirschsprung disease and cancer, has recently been implicated in breast cancer estrogen (E2) independence and tamoxifen resistance. RET displays both E2 and retinoic acid (RA)-dependent transcriptional modulation in E2-responsive breast cancers. However, the regulatory elements through which the steroid hormone transcriptional regulation of RET is mediated are poorly defined. Recent genome-wide chromatin immunoprecipitation-based studies have identified 10 putative E2 receptor-alpha (ESR1) and RA receptor alpha-binding sites at the RET locus, of which we demonstrate only two (RET −49.8 and RET +32.8) display significant E2 regulatory response when assayed independently in MCF-7 breast cancer cells. We demonstrate that endogenous RET expression and RET −49.8 regulatory activity are cooperatively regulated by E2 and RA in breast cancer cells. We identify key sequences that are required for RET −49.8 and RET +32.8 E2 responsiveness, including motifs known to be bound by ESR1, FOXA1 and TFAP2C. We also report that both RET −49.8 regulatory activity and endogenous RET expression are completely dependent on ESR1 for their (E2)-induction and that ESR1 is sufficient to mediate the E2-induced enhancer activity of RET −49.8 and RET +32.8. Finally, using zebrafish transgenesis, we also demonstrate that RET −49.8 directs reporter expression in the central nervous system and peripheral nervous system consistent with the endogenous ret expression. Taken collectively, these data suggest that RET transcription in breast cancer cells is modulated by E2 via ESR1 acting on multiple elements collectively.
doi:10.1093/hmg/ddr291
PMCID: PMC3168285  PMID: 21737465
7.  Intronic Cis-Regulatory Modules Mediate Tissue-Specific and Microbial Control of angptl4/fiaf Transcription 
PLoS Genetics  2012;8(3):e1002585.
The intestinal microbiota enhances dietary energy harvest leading to increased fat storage in adipose tissues. This effect is caused in part by the microbial suppression of intestinal epithelial expression of a circulating inhibitor of lipoprotein lipase called Angiopoietin-like 4 (Angptl4/Fiaf). To define the cis-regulatory mechanisms underlying intestine-specific and microbial control of Angptl4 transcription, we utilized the zebrafish system in which host regulatory DNA can be rapidly analyzed in a live, transparent, and gnotobiotic vertebrate. We found that zebrafish angptl4 is transcribed in multiple tissues including the liver, pancreatic islet, and intestinal epithelium, which is similar to its mammalian homologs. Zebrafish angptl4 is also specifically suppressed in the intestinal epithelium upon colonization with a microbiota. In vivo transgenic reporter assays identified discrete tissue-specific regulatory modules within angptl4 intron 3 sufficient to drive expression in the liver, pancreatic islet β-cells, or intestinal enterocytes. Comparative sequence analyses and heterologous functional assays of angptl4 intron 3 sequences from 12 teleost fish species revealed differential evolution of the islet and intestinal regulatory modules. High-resolution functional mapping and site-directed mutagenesis defined the minimal set of regulatory sequences required for intestinal activity. Strikingly, the microbiota suppressed the transcriptional activity of the intestine-specific regulatory module similar to the endogenous angptl4 gene. These results suggest that the microbiota might regulate host intestinal Angptl4 protein expression and peripheral fat storage by suppressing the activity of an intestine-specific transcriptional enhancer. This study provides a useful paradigm for understanding how microbial signals interact with tissue-specific regulatory networks to control the activity and evolution of host gene transcription.
Author Summary
Recent studies have revealed that the community of microorganisms residing in the intestine regulates fat storage. Microbes evoke this response in part by suppressing expression of the Angptl4 gene, which encodes a secreted inhibitor of fat storage. Although Angptl4 is expressed in multiple tissues, microbial suppression occurs only in the intestine. To determine how microbes control fat storage, we must elucidate the mechanisms underlying intestine-specific and microbial regulation of Angptl4 expression. Here, we take advantage of the unique features of the zebrafish model to define the regulatory DNA sequences controlling angptl4 expression. Our results reveal that different DNA regulatory regions within the angptl4 gene mediate expression of angptl4 in the intestine and other tissues. By assessing the evolution of angptl4 regulatory regions and subjecting them to structure-function analyses, we identify discrete DNA sequences that are required for intestinal expression. Strikingly, microbes suppress the activity of the intestine-specific regulatory region similar to the endogenous angptl4 gene. Therefore, intestinal microbes might regulate angptl4 production by suppressing the signaling pathway interpreted by an intestine-specific transcriptional regulatory region. Our results provide new mechanistic insights into how intestinal microbes might influence fat storage and contribute to the development of obesity.
doi:10.1371/journal.pgen.1002585
PMCID: PMC3315460  PMID: 22479192
8.  Identification of RNA binding motif proteins essential for cardiovascular development 
Background
We recently identified Rbm24 as a novel gene expressed during mouse cardiac development. Due to its tightly restricted and persistent expression from formation of the cardiac crescent onwards and later in forming vasculature we posited it to be a key player in cardiogenesis with additional roles in vasculogenesis and angiogenesis.
Results
To determine the role of this gene in cardiac development, we have identified its zebrafish orthologs (rbm24a and rbm24b), and functionally evaluated them during zebrafish embryogenesis. Consistent with our underlying hypothesis, reduction in expression of either ortholog through injection of morpholino antisense oligonucleotides results in cardiogenic defects including cardiac looping and reduced circulation, leading to increasing pericardial edema over time. Additionally, morphant embryos for either ortholog display incompletely overlapping defects in the forming vasculature of the dorsal aorta (DA), posterior caudal vein (PCV) and caudal vein (CV) which are the first blood vessels to form in the embryo. Vasculogenesis and early angiogenesis in the trunk were similarly compromised in rbm24 morphant embryos at 48 hours post fertilization (hpf). Subsequent vascular maintenance was impaired in both rbm24 morphants with substantial vessel degradation noted at 72 hpf.
Conclusion
Taken collectively, our functional data support the hypothesis that rbm24a and rbm24b are key developmental cardiac genes with unequal roles in cardiovascular formation.
doi:10.1186/1471-213X-11-62
PMCID: PMC3277282  PMID: 22011202
9.  SOX10 directly modulates ERBB3 transcription via an intronic neural crest enhancer 
Background
The ERBB3 gene is essential for the proper development of the neural crest (NC) and its derivative populations such as Schwann cells. As with all cell fate decisions, transcriptional regulatory control plays a significant role in the progressive restriction and specification of NC derived lineages during development. However, little is known about the sequences mediating transcriptional regulation of ERBB3 or the factors that bind them.
Results
In this study we identified three transcriptional enhancers at the ERBB3 locus and evaluated their regulatory potential in vitro in NC-derived cell types and in vivo in transgenic zebrafish. One enhancer, termed ERBB3_MCS6, which lies within the first intron of ERBB3, directs the highest reporter expression in vitro and also demonstrates epigenetic marks consistent with enhancer activity. We identify a consensus SOX10 binding site within ERBB3_MCS6 and demonstrate, in vitro, its necessity and sufficiency for the activity of this enhancer. Additionally, we demonstrate that transcription from the endogenous Erbb3 locus is dependent on Sox10. Further we demonstrate in vitro that Sox10 physically interacts with that ERBB3_MCS6. Consistent with its in vitro activity, we also show that ERBB3_MCS6 drives reporter expression in NC cells and a subset of its derivative lineages in vivo in zebrafish in a manner consistent with erbb3b expression. We also demonstrate, using morpholino analysis, that Sox10 is necessary for ERBB3_MCS6 expression in vivo in zebrafish.
Conclusions
Taken collectively, our data suggest that ERBB3 may be directly regulated by SOX10, and that this control may in part be facilitated by ERBB3_MCS6.
doi:10.1186/1471-213X-11-40
PMCID: PMC3124416  PMID: 21672228
10.  Efficient discovery of ASCL1 regulatory sequences through transgene pooling 
Genomics  2010;95(6):363-369.
Zebrafish transgenesis is a powerful and increasingly common strategy to assay vertebrate transcriptional regulatory control. Several challenges remain, however, to the broader application of this technique; they include increasing the rate with which transgenes can be analyzed and maximizing the informational value of the data generated. Presently, many rely on the injection of individual constructs and the analysis of resulting reporter expression in mosaic G0 embryos. Here, we contrast these approaches, examining whether injecting pooled transgene constructs can increase the efficiency with which regulatory sequences can be assayed, restricting analysis to the offspring of germ line transmitting transgenic zebrafish in an effort to reduce potential subjectivity. We selected a 64 kb interval encompassing the human ASCL1 locus as our model interval and report the analysis of 9 highly conserved putative enhancers therein. We identified 32 transgene-positive zebrafish, transmitting one or more independent constructs displaying ASCL1-like regulatory control. Through examination of embryos harboring one or more transgenes, we demonstrate that five of the nine sequences account for the observed control and describe their likely roles in ASCL1 regulation. These data demonstrate the utility of this approach and its potential for further adaptation and higher throughput application.
doi:10.1016/j.ygeno.2010.02.011
PMCID: PMC2904508  PMID: 20206680
11.  Peroxisome-proliferator-activated receptor-binding protein (PBP) is essential for the growth of active Notch4-immortalized mammary epithelial cells by activating SOX10 expression 
The Biochemical journal  2009;425(2):435-444.
PBP (peroxisome-proliferator-activated receptor-binding protein) [Med1 (mediator 1)/TRAP220 (thyroid-hormone-receptor-associated protein 220)] is essential for mammary gland development. We established a mammary epithelial cell line with a genotype of PBPLoxP/LoxP by expressing an active form of Notch4. Null mutation of PBP caused severe growth inhibition of the Notch4-immortalized mammary cells. We found that truncated PBP without the two LXXLL motifs could reverse the growth inhibition due to the deficiency of endogenous PBP, indicating that signalling through nuclear receptors is unlikely to be responsible for the growth inhibition as the result of PBP deficiency. Loss of PBP expression was shown to completely ablate the expression of SOX10 [Sry-related HMG (high-mobility group) box gene 10]. The re-expression of SOX10 was capable of reversing the growth inhibition due to PBP deficiency, whereas suppressed expression of SOX10 inhibited the growth of Notch4-immortalized mammary cells. Further studies revealed PBP is directly recruited to the enhancer of the SOX10 gene, indicating that SOX10 is a direct target gene of PBP. We conclude that PBP is essential for the growth of Notch4-immortalized mammary cells by activating SOX10 expression, providing a potential molecular mechanism through which PBP regulates the growth of mammary stem/progenitor cells.
doi:10.1042/BJ20091237
PMCID: PMC3005363  PMID: 19852756
mammary gland development; mammary progenitor cell; mediator complex; Notch4; peroxisome-proliferator-activated receptor (PPAR)-binding protein (PBP); Sry-related HMG box gene 10 (SOX10)
12.  A Rare Myelin Protein Zero (MPZ) Variant Alters Enhancer Activity In Vitro and In Vivo 
PLoS ONE  2010;5(12):e14346.
Background
Myelin protein zero (MPZ) is a critical structural component of myelin in the peripheral nervous system. The MPZ gene is regulated, in part, by the transcription factors SOX10 and EGR2. Mutations in MPZ, SOX10, and EGR2 have been implicated in demyelinating peripheral neuropathies, suggesting that components of this transcriptional network are candidates for harboring disease-causing mutations (or otherwise functional variants) that affect MPZ expression.
Methodology
We utilized a combination of multi-species sequence comparisons, transcription factor-binding site predictions, targeted human DNA re-sequencing, and in vitro and in vivo enhancer assays to study human non-coding MPZ variants.
Principal Findings
Our efforts revealed a variant within the first intron of MPZ that resides within a previously described SOX10 binding site is associated with decreased enhancer activity, and alters binding of nuclear proteins. Additionally, the genomic segment harboring this variant directs tissue-relevant reporter gene expression in zebrafish.
Conclusions
This is the first reported MPZ variant within a cis-acting transcriptional regulatory element. While we were unable to implicate this variant in disease onset, our data suggests that similar non-coding sequences should be screened for mutations in patients with neurological disease. Furthermore, our multi-faceted approach for examining the functional significance of non-coding variants can be readily generalized to study other loci important for myelin structure and function.
doi:10.1371/journal.pone.0014346
PMCID: PMC3002941  PMID: 21179557
13.  Oligodendroglial and Pan-neural Crest Expression of Cre Recombinase Directed by Sox10 Enhancer 
Genesis (New York, N.Y. : 2000)  2009;47(11):765-770.
Utilizing a recently identified Sox10 distal enhancer directing Cre expression, we report S4F:Cre, a transgenic mouse line capable of inducing recombination in oligodendroglia and all examined neural crest derived tissues. Assayed using R26R:LacZ reporter mice expression was detected in neural crest derived tissues including the forming facial skeleton, dorsal root ganglia, sympathetic ganglia, enteric nervous system, aortae and melanoblasts, consistent with Sox10 expression. LacZ reporter expression was also detected in non-neural crest derived tissues including the oligodendrocytes and the ventral neural tube. This line provides appreciable differences in Cre expression pattern from other transgenic mouse lines that mark neural crest populations, including additional populations defined by the expression of other SoxE proteins. The S4F:Cre transgenic line will thus serve as a powerful tool for lineage tracing, gene function characterization and genome manipulation in these populations.
doi:10.1002/dvg.20559
PMCID: PMC2835405  PMID: 19830815
14.  Gpnmb is a Melanoblast-Expressed, MITF-Dependent Gene 
Pigment cell & melanoma research  2008;22(1):99-110.
SUMMARY
Expression profile analysis clusters Gpnmb with known pigment genes, Tyrp1, Dct, and Si. During development, Gpnmb is expressed in a pattern similar to Mitf, Dct and Si with expression vastly reduced in Mitf mutant animals. Unlike Dct and Si, Gpnmb remains expressed in a discrete population of caudal melanoblasts in Sox10-deficient embryos. To understand the transcriptional regulation of Gpnmb we performed a whole genome annotation of 2,460,048 consensus MITF binding sites, and cross-referenced this with evolutionarily conserved genomic sequences at the GPNMB locus. One conserved element, GPNMB-MCS3, contained two MITF consensus sites, significantly increased luciferase activity in melanocytes and was sufficient to drive expression in melanoblasts in vivo. Deletion of the 5’-most MITF consensus site dramatically reduced enhancer activity indicating a significant role for this site in Gpnmb transcriptional regulation. Future analysis of the Gpnmb locus will provide insight into the transcriptional regulation of melanocytes and Gpnmb expression can be used as a marker for analyzing melanocyte development and disease progression.
SIGNIFICANCE
Comparative analysis of gene expression profiles using melanocyte lines derived from mice provides a powerful resource to explore genetic components of melanocyte development and pigment cell function. Using expression data, we identified Gpnmb as a new marker for early melanoblast development. We show that Gpnmb is dependent on Mitf for in vivo expression and marks a unique set of Sox10-independent melanoblasts. We identified an 89 basepair evolutionarily conserved genomic sequence at the Gpnmb locus that can enhance expression in melanocytes and tested MITF E-box consensus sequences for their involvement in melanocyte-restricted expression. Gpnmb and the panel of genes identified in this study will be valuable resources for understanding the genetic components involved in melanocyte development and diseases.
doi:10.1111/j.1755-148X.2008.00518.x
PMCID: PMC2714741  PMID: 18983539
Gpnmb; Mitf; Sox10; melanoblast; melanocyte; melanoma
15.  Asymmetrical distribution of non-conserved regulatory sequences at PHOX2B is reflected at the ENCODE loci and illuminates a possible genome-wide trend 
BMC Genomics  2009;10:8.
Background
Transcriptional regulatory elements are central to development and interspecific phenotypic variation. Current regulatory element prediction tools rely heavily upon conservation for prediction of putative elements. Recent in vitro observations from the ENCODE project combined with in vivo analyses at the zebrafish phox2b locus suggests that a significant fraction of regulatory elements may fall below commonly applied metrics of conservation. We propose to explore these observations in vivo at the human PHOX2B locus, and also evaluate the potential evidence for genome-wide applicability of these observations through a novel analysis of extant data.
Results
Transposon-based transgenic analysis utilizing a tiling path proximal to human PHOX2B in zebrafish recapitulates the observations at the zebrafish phox2b locus of both conserved and non-conserved regulatory elements. Analysis of human sequences conserved with previously identified zebrafish phox2b regulatory elements demonstrates that the orthologous sequences exhibit overlapping regulatory control. Additionally, analysis of non-conserved sequences scattered over 135 kb 5' to PHOX2B, provides evidence of non-conserved regulatory elements positively biased with close proximity to the gene. Furthermore, we provide a novel analysis of data from the ENCODE project, finding a non-uniform distribution of regulatory elements consistent with our in vivo observations at PHOX2B. These observations remain largely unchanged when one accounts for the sequence repeat content of the assayed intervals, when the intervals are sub-classified by biological role (developmental versus non-developmental), or by gene density (gene desert versus non-gene desert).
Conclusion
While regulatory elements frequently display evidence of evolutionary conservation, a fraction appears to be undetected by current metrics of conservation. In vivo observations at the PHOX2B locus, supported by our analyses of in vitro data from the ENCODE project, suggest that the risk of excluding non-conserved sequences in a search for regulatory elements may decrease as distance from the gene increases. Our data combined with the ENCODE data suggests that this may represent a genome wide trend.
doi:10.1186/1471-2164-10-8
PMCID: PMC2630312  PMID: 19128492
16.  Identification of Neural Crest and Glial Enhancers at the Mouse Sox10 Locus through Transgenesis in Zebrafish 
PLoS Genetics  2008;4(9):e1000174.
Sox10 is a dynamically regulated transcription factor gene that is essential for the development of neural crest–derived and oligodendroglial populations. Developmental genes often require multiple regulatory sequences that integrate discrete and overlapping functions to coordinate their expression. To identify Sox10 cis-regulatory elements, we integrated multiple model systems, including cell-based screens and transposon-mediated transgensis in zebrafish, to scrutinize mammalian conserved, noncoding genomic segments at the mouse Sox10 locus. We demonstrate that eight of 11 Sox10 genomic elements direct reporter gene expression in transgenic zebrafish similar to patterns observed in transgenic mice, despite an absence of observable sequence conservation between mice and zebrafish. Multiple segments direct expression in overlapping populations of neural crest derivatives and glial cells, ranging from pan-Sox10 and pan-neural crest regulatory control to the modulation of expression in subpopulations of Sox10-expressing cells, including developing melanocytes and Schwann cells. Several sequences demonstrate overlapping spatial control, yet direct expression in incompletely overlapping developmental intervals. We were able to partially explain neural crest expression patterns by the presence of head to head SoxE family binding sites within two of the elements. Moreover, we were able to use this transcription factor binding site signature to identify the corresponding zebrafish enhancers in the absence of overall sequence homology. We demonstrate the utility of zebrafish transgenesis as a high-fidelity surrogate in the dissection of mammalian gene regulation, especially those with dynamically controlled developmental expression.
Author Summary
The neural crest is a population of embryonic migratory stem cells. They form atop the future spinal cord and migrate throughout developing embryos and form many different cells, including the epidermal pigment cells, bone cells in the head, and nerve cells of the peripheral nervous system. In this study, we studied the genome elements responsible for expression of SOX10, a dynamically expressed gene that is essential for neural crest development. We isolated candidate regulatory elements for SOX10 by identifying the small percentage of genomic DNA around the gene that did not vary as avian and mammalian genomes changed though evolution. We tested these fragments for their ability to regulate gene expression in zebrafish, a model system that is highly efficient for DNA-mediated expression studies and embryology. We found that even though the genome sequences were not similar to the SOX10 gene in fish, the genomic fragments were able to recapitulate the dynamic expression of SOX10 during development. Through computational analysis of the sequences, we identified a transcription factor binding site signature that identified the corresponding zebrafish SOX10 regulatory elements. This study describes a paradigm for dissecting regulation of essential genes that display complex expression patterns during development.
doi:10.1371/journal.pgen.1000174
PMCID: PMC2518861  PMID: 18773071
17.  Efficient Array-Based Identification of Novel Cardiac Genes through Differentiation of Mouse ESCs 
PLoS ONE  2008;3(5):e2176.
Remarkably, although cardiac disease accounts for the largest proportion of adult mortality and morbidity in the industrialized world, the genetic programs controlling early cardiogenesis are largely incompletely understood. To better understand this process, we set out to identify genes whose expression is enriched within early cardiac fated populations, obtaining the transcriptional signatures of mouse embryonic stem cells (mESCs) at defined intervals during their differentiation along a cardiac path. We compared the RNA profiles of cardiac precursors cells (CPCs) with time-matched non-CPCs and undifferentiated mESCs, using a transgenic mESC line harboring an Nkx2-5 cardiac-specific regulatory sequence driving green fluorescent protein (GFP) to facilitate selection of CPCs. We identify 176 transcripts that are significantly elevated in their abundance within CPCs compared with other assayed populations, predicting that they will likely play a role in cardiogenesis. Of note, approximately 24% (43/176) of the cardiogenic candidate transcripts have known roles in cardiac function or development. Importantly, we evaluated the biological relevance of a significant subset 31/133 (23%) of the remaining candidate genes by in situ hybridization at multiple time points during development (embryonic day, E7.5–9.5) and report that all were expressed in key cardiac structures during cardiogenesis. Furthermore 9/31, of which many were previously uncharacterized, were detected as early as the formation of the cardiac crescent. These data demonstrate the potential power of integrating genomic approaches with mESC differentiation to illuminate developmental processes, and provides a valuable resource that may be mined to further elucidate the genetic programs underlying cardiogenesis.
doi:10.1371/journal.pone.0002176
PMCID: PMC2364653  PMID: 18478100
18.  Mouse ES cell–derived cardiac precursor cells are multipotent and facilitate identification of novel cardiac genes 
Although the differentiation of ES cells to cardiomyocytes has been firmly established, the extent to which corresponding cardiac precursor cells can contribute to other cardiac populations remains unclear. To determine the molecular and cellular characteristics of cardiac-fated populations derived from mouse ES (mES) cells, we isolated cardiac progenitor cells (CPCs) from differentiating mES cell cultures by using a reporter cell line that expresses GFP under the control of a cardiac-specific enhancer element of Nkx2-5, a transcription factor expressed early in cardiac development. This ES cell–derived CPC population initially expressed genetic markers of both stem cells and mesoderm, while differentiated CPCs displayed markers of 3 distinct cell lineages (cardiomyocytes, vascular smooth muscle cells, and endothelial cells) — Flk1 (also known as Kdr), c-Kit, and Nkx2-5, but not Brachyury — and subsequently expressed Isl1. Clonally derived CPCs also demonstrated this multipotent phenotype. By transcription profiling of CPCs, we found that mES cell–derived CPCs displayed a transcriptional signature that paralleled in vivo cardiac development. Additionally, these studies suggested the involvement of genes that we believe were previously unknown to play a role in cardiac development. Taken together, our data demonstrate that ES cell–derived CPCs comprise a multipotent precursor population capable of populating multiple cardiac lineages and suggest that ES cell differentiation is a valid model for studying development of multiple cardiac-fated tissues.
doi:10.1172/JCI33942
PMCID: PMC2214848  PMID: 18246200

Results 1-18 (18)