Automated discovery of tissue-targeting enhancers and transcription factors from binding motif and gene function data.
PLoS computational biology
2014; 10 (1)
The Enhancer Landscape during Early Neocortical Development Reveals Patterns of Dense Regulation and Co-option.
2013; 9 (8)
Identifying enhancers regulating gene expression remains an important and challenging task. While recent sequencing-based methods provide epigenomic characteristics that correlate well with enhancer activity, it remains onerous to comprehensively identify all enhancers across development. Here we introduce a computational framework to identify tissue-specific enhancers evolving under purifying selection. First, we incorporate high-confidence binding site predictions with target gene functional enrichment analysis to identify transcription factors (TFs) likely functioning in a particular context. We then search the genome for clusters of binding sites for these TFs, overcoming previous constraints associated with biased manual curation of TFs or enhancers. Applying our method to the placenta, we find 33 known and implicate 17 novel TFs in placental function, and discover 2,216 putative placenta enhancers. Using luciferase reporter assays, 31/36 (86%) tested candidates drive activity in placental cells. Our predictions agree well with recent epigenomic data in human and mouse, yet over half our loci, including 7/8 (87%) tested regions, are novel. Finally, we establish that our method is generalizable by applying it to 5 additional tissues: heart, pancreas, blood vessel, bone marrow, and liver.
View details for DOI 10.1371/journal.pcbi.1003449
View details for PubMedID 24499934
Foxa2 and H2A.Z Mediate Nucleosome Depletion during Embryonic Stem Cell Differentiation
2012; 151 (7): 1608-1616
Genetic studies have identified a core set of transcription factors and target genes that control the development of the neocortex, the region of the human brain responsible for higher cognition. The specific regulatory interactions between these factors, many key upstream and downstream genes, and the enhancers that mediate all these interactions remain mostly uncharacterized. We perform p300 ChIP-seq to identify over 6,600 candidate enhancers active in the dorsal cerebral wall of embryonic day 14.5 (E14.5) mice. Over 95% of the peaks we measure are conserved to human. Eight of ten (80%) candidates tested using mouse transgenesis drive activity in restricted laminar patterns within the neocortex. GREAT based computational analysis reveals highly significant correlation with genes expressed at E14.5 in key areas for neocortex development, and allows the grouping of enhancers by known biological functions and pathways for further studies. We find that multiple genes are flanked by dozens of candidate enhancers each, including well-known key neocortical genes as well as suspected and novel genes. Nearly a quarter of our candidate enhancers are conserved well beyond mammals. Human and zebrafish regions orthologous to our candidate enhancers are shown to most often function in other aspects of central nervous system development. Finally, we find strong evidence that specific interspersed repeat families have contributed potentially key developmental enhancers via co-option. Our analysis expands the methodologies available for extracting the richness of information found in genome-wide functional maps.
View details for DOI 10.1371/journal.pgen.1003728
View details for PubMedID 24009522
PESNPdb: A comprehensive database of SNPs studied in association with pre-eclampsia
2012; 33 (12): 1055-1057
Nucleosome occupancy is fundamental for establishing chromatin architecture. However, little is known about the relationship between nucleosome dynamics and initial cell lineage specification. Here, we determine the mechanisms that control global nucleosome dynamics during embryonic stem (ES) cell differentiation into endoderm. Both nucleosome depletion and de novo occupation occur during the differentiation process, with higher overall nucleosome density after differentiation. The variant histone H2A.Z and the winged helix transcription factor Foxa2 both act to regulate nucleosome depletion and gene activation, thus promoting ES cell differentiation, whereas DNA methylation promotes nucleosome occupation and suppresses gene expression. Nucleosome depletion during ES cell differentiation is dependent on Nap1l1-coupled SWI/SNF and INO80 chromatin remodeling complexes. Thus, both epigenetic and genetic regulators cooperate to control nucleosome dynamics during ES cell fate decisions.
View details for DOI 10.1016/j.cell.2012.11.018
View details for Web of Science ID 000312890300023
View details for PubMedID 23260146
Foxa1 and Foxa2 Are Essential for Sexual Dimorphism in Liver Cancer
2012; 148 (1-2): 72-83
Pre-eclampsia is a pregnancy specific disorder that can be life threatening for mother and child. Multiple studies have been carried out in an attempt to identify SNPs that contribute to the genetic susceptibility of the disease. Here we describe PESNPdb (http://bejerano.stanford.edu/pesnpdb), a database aimed at centralizing SNP and study details investigated in association with pre-eclampsia. We also describe a Placenta Disorders ontology that utilizes information from PESNPdb. The main focus of PESNPdb is to help researchers study the genetic complexity of pre-eclampsia through a user-friendly interface that encourages community participation.
View details for DOI 10.1016/j.placenta.2012.09.016
View details for Web of Science ID 000312171900015
View details for PubMedID 23084601
The nucleosome map of the mammalian liver
NATURE STRUCTURAL & MOLECULAR BIOLOGY
2011; 18 (6): 742-U145
Hepatocellular carcinoma (HCC) is sexually dimorphic in both rodents and humans, with significantly higher incidence in males, an effect that is dependent on sex hormones. The molecular mechanisms by which estrogens prevent and androgens promote liver cancer remain unclear. Here, we discover that sexually dimorphic HCC is completely reversed in Foxa1- and Foxa2-deficient mice after diethylnitrosamine-induced hepatocarcinogenesis. Coregulation of target genes by Foxa1/a2 and either the estrogen receptor (ER?) or the androgen receptor (AR) was increased during hepatocarcinogenesis in normal female or male mice, respectively, but was lost in Foxa1/2-deficient mice. Thus, both estrogen-dependent resistance to and androgen-mediated facilitation of HCC depend on Foxa1/2. Strikingly, single nucleotide polymorphisms at FOXA2 binding sites reduce binding of both FOXA2 and ER? to their targets in human liver and correlate with HCC development in women. Thus, Foxa factors and their targets are central for the sexual dimorphism of HCC.
View details for DOI 10.1016/j.cell.2011.11.026
View details for Web of Science ID 000299540700015
View details for PubMedID 22265403
Species-Specific Strategies Underlying Conserved Functions of Metabolic Transcription Factors
2011; 25 (4): 694-706
Binding to nucleosomal DNA is critical for 'pioneer' transcription factors such as the winged-helix transcription factors Foxa1 and Foxa2 to regulate chromatin structure and gene activation. Here we report the genome-wide map of nucleosome positions in the mouse liver, with emphasis on transcriptional start sites, CpG islands, Foxa2 binding sites and their correlation with gene expression. Despite the heterogeneity of liver tissue, we could clearly discern the nucleosome pattern of the predominant liver cell, the hepatocyte. By analyzing nucleosome occupancy and the distributions of heterochromatin protein 1 (Hp1), CBP (also known as Crebbp) and p300 (Ep300) in Foxa1- and Foxa2-deficient livers, we find that the maintenance of nucleosome position and chromatin structure surrounding Foxa2 binding sites is independent of Foxa1 and Foxa2.
View details for DOI 10.1038/nsmb.2060
View details for Web of Science ID 000291308000018
View details for PubMedID 21623366
Cell-Specific Determinants of Peroxisome Proliferator-Activated Receptor gamma Function in Adipocytes and Macrophages
MOLECULAR AND CELLULAR BIOLOGY
2010; 30 (9): 2078-2089
The winged helix protein FOXA2 and the nuclear receptor peroxisome proliferator-activated receptor-? (PPAR?) are highly conserved, regionally expressed transcription factors (TFs) that regulate networks of genes controlling complex metabolic functions. Cistrome analysis for Foxa2 in mouse liver and PPAR? in mouse adipocytes has previously produced consensus-binding sites that are nearly identical to those used by the corresponding TFs in human cells. We report here that, despite the conservation of the canonical binding motif, the great majority of binding regions for FOXA2 in human liver and for PPAR? in human adipocytes are not in the orthologous locations corresponding to the mouse genome, and vice versa. Of note, TF binding can be absent in one species despite sequence conservation, including motifs that do support binding in the other species, demonstrating a major limitation of in silico binding site prediction. Whereas only approximately 10% of binding sites are conserved, gene-centric analysis reveals that about 50% of genes with nearby TF occupancy are shared across species for both hepatic FOXA2 and adipocyte PPAR?. Remarkably, for both TFs, many of the shared genes function in tissue-specific metabolic pathways, whereas species-unique genes fail to show enrichment for these pathways. Nonetheless, the species-unique genes, like the shared genes, showed the expected transcriptional regulation by the TFs in loss-of-function experiments. Thus, species-specific strategies underlie the biological functions of metabolic TFs that are highly conserved across mammalian species. Analysis of factor binding in multiple species may be necessary to distinguish apparent species-unique noise and reveal functionally relevant information.
View details for DOI 10.1210/me.2010-0454
View details for Web of Science ID 000288894500013
View details for PubMedID 21292830
Extracting transcription factor targets from ChIP-Seq data
NUCLEIC ACIDS RESEARCH
2009; 37 (17)
The nuclear receptor peroxisome proliferator activator receptor gamma (PPARgamma) is the target of antidiabetic thiazolidinedione drugs, which improve insulin resistance but have side effects that limit widespread use. PPARgamma is required for adipocyte differentiation, but it is also expressed in other cell types, notably macrophages, where it influences atherosclerosis, insulin resistance, and inflammation. A central question is whether PPARgamma binding in macrophages occurs at genomic locations the same as or different from those in adipocytes. Here, utilizing chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we demonstrate that PPARgamma cistromes in mouse adipocytes and macrophages are predominantly cell type specific. In thioglycolate-elicited macrophages, PPARgamma colocalizes with the hematopoietic transcription factor PU.1 in areas of open chromatin and histone acetylation, near a distinct set of immune genes in addition to a number of metabolic genes shared with adipocytes. In adipocytes, the macrophage-unique binding regions are marked with repressive histone modifications, typically associated with local chromatin compaction and gene silencing. PPARgamma, when introduced into preadipocytes, bound only to regions depleted of repressive histone modifications, where it increased DNA accessibility, enhanced histone acetylation, and induced gene expression. Thus, the cell specificity of PPARgamma function is regulated by cell-specific transcription factors, chromatin accessibility, and histone marks. Our data support the existence of an epigenomic hierarchy in which PPARgamma binding to cell-specific sites not marked by repressive marks opens chromatin and leads to local activation marks, including histone acetylation.
View details for DOI 10.1128/MCB.01651-09
View details for Web of Science ID 000276420500002
View details for PubMedID 20176806
CRTC2 (TORC2) Contributes to the Transcriptional Response to Fasting in the Liver but Is Not Required for the Maintenance of Glucose Homeostasis
2009; 10 (1): 55-62
ChIP-Seq technology, which combines chromatin immunoprecipitation (ChIP) with massively parallel sequencing, is rapidly replacing ChIP-on-chip for the genome-wide identification of transcription factor binding events. Identifying bound regions from the large number of sequence tags produced by ChIP-Seq is a challenging task. Here, we present GLITR (GLobal Identifier of Target Regions), which accurately identifies enriched regions in target data by calculating a fold-change based on random samples of control (input chromatin) data. GLITR uses a classification method to identify regions in ChIP data that have a peak height and fold-change which do not resemble regions in an input sample. We compare GLITR to several recent methods and show that GLITR has improved sensitivity for identifying bound regions closely matching the consensus sequence of a given transcription factor, and can detect bona fide transcription factor targets missed by other programs. We also use GLITR to address the issue of sequencing depth, and show that sequencing biological replicates identifies far more binding regions than re-sequencing the same sample.
View details for DOI 10.1093/nar/gkp536
View details for Web of Science ID 000271569100035
View details for PubMedID 19553195
Foxa1 and Foxa2 regulate bile duct development in mice
JOURNAL OF CLINICAL INVESTIGATION
2009; 119 (6): 1537-1545
The liver contributes to glucose homeostasis by promoting either storage or production of glucose, depending on the physiological state. The cAMP response element-binding protein (CREB) is a principal regulator of genes involved in coordinating the hepatic response to fasting, but its mechanism of gene activation remains controversial. We derived CRTC2 (CREB-regulated transcription coactivator 2, previously TORC2)-deficient mice to assess the contribution of this cofactor to hepatic glucose metabolism in vivo. CRTC2 mutant hepatocytes showed reduced glucose production in response to glucagon, which correlated with decreased CREB binding to several gluconeogenic genes. However, despite attenuated expression of CREB target genes, including PEPCK, G6Pase, and PGC-1alpha, no hypoglycemia was observed in mutant mice. Collectively, these results provide genetic evidence supporting a role for CRTC2 in the transcriptional response to fasting, but indicate only a limited contribution of this cofactor to the maintenance of glucose homeostasis.
View details for DOI 10.1016/j.cmet.2009.06.006
View details for Web of Science ID 000267874200010
View details for PubMedID 19583954
Cis-regulatory modules in the mammalian liver: composition depends on strength of Foxa2 consensus site
NUCLEIC ACIDS RESEARCH
2008; 36 (12): 4149-4157
The forkhead box proteins A1 and A2 (Foxa1 and Foxa2) are transcription factors with critical roles in establishing the developmental competence of the foregut endoderm and in initiating liver specification. Using conditional gene ablation during a later phase of liver development, we show here that deletion of both Foxa1 and Foxa2 (Foxa1/2) in the embryonic liver caused hyperplasia of the biliary tree. Abnormal bile duct formation in Foxa1/2-deficient liver was due, at least in part, to activation of IL-6 expression, a proliferative signal for cholangiocytes. The glucocorticoid receptor is a negative regulator of IL-6 transcription; in the absence of Foxa1/2, the glucocorticoid receptor failed to bind to the IL-6 promoter, causing enhanced IL-6 expression. Thus, after liver specification, Foxa1/2 are required for normal bile duct development through prevention of excess cholangiocyte proliferation. Our data suggest that Foxa1/2 function as terminators of bile duct expansion in the adult liver through inhibition of IL-6 expression.
View details for DOI 10.1172/JCI38201
View details for Web of Science ID 000266601000019
View details for PubMedID 19436110
SnapShot: Forkhead transcription factors II
2007; 131 (1)
SnapShot: Forkhead transcription factors I
2007; 130 (6)
Foxa2 is a critical transcription factor that controls liver development and plays an important role in hepatic gluconeogensis in adult mice. Here, we use genome-wide location analysis for Foxa2 to identify its targets in the adult liver. We then show by computational analyses that Foxa2 containing cis-regulatory modules are not constructed from a random assortment of binding sites for other transcription factors expressed in the liver, but rather that their composition depends on the strength of the Foxa2 consensus site present. Genes containing a cis-regulatory module with a medium or weak Foxa2 consensus site are much more liver-specific than the genes with a strong consensus site. We not only provide a better understanding of the mechanisms of Foxa2 regulation but also introduce a novel method for identification of different cis-regulatory modules involving a single factor.
View details for DOI 10.1093/nar/gkn366
View details for Web of Science ID 000257578600027
View details for PubMedID 18556755