Rebecca Cullum

Genome-wide relationship between histone H3 lysine 4 mono- and tri-methylation and transcription factor binding

We characterized the relationship of H3K4me1 and H3K4me3 at distal and proximal regulatory elements by comparing ChIP-seq profiles for these histone modifications and for two functionally different transcription factors: STAT1 in the immortalized HeLa S3 cell line, with and without interferon-gamma (IFNG) stimulation; and FOXA2 in mouse adult liver tissue. In unstimulated and stimulated HeLa cells, respectively, we determined approximately 270,000 and approximately 301,000 H3K4me1-enriched regions, and approximately 54,500 and approximately 76,100 H3K4me3-enriched regions. In mouse adult liver, we determined approximately 227,000 and approximately 34,800 H3K4me1 and H3K4me3 regions. Seventy-five percent of the approximately 70,300 STAT1 binding sites in stimulated HeLa cells and 87% of the approximately 11,000 FOXA2 sites in mouse liver were distal to known gene TSS; in both cell types, approximately 83% of these distal sites were associated with at least one of the two histone modifications, and H3K4me1 was associated with over 96% of marked distal sites. After filtering against predicted transcription start sites, 50% of approximately 26,800 marked distal IFNG-stimulated STAT1 binding sites, but 95% of approximately 5800 marked distal FOXA2 sites, were associated with H3K4me1 only. Results for HeLa cells generated additional insights into transcriptional regulation involving STAT1. STAT1 binding was associated with 25% of all H3K4me1 regions in stimulated HeLa cells, suggesting that a single transcription factor can interact with an unexpectedly large fraction of regulatory regions. Strikingly, for a large majority of the locations of stimulated STAT1 binding, the dominant H3K4me1/me3 combinations were established before activation, suggesting mechanisms independent of IFNG stimulation and high-affinity STAT1 binding.

Global analysis of in vivo Foxa2-binding sites in mouse adult liver using massively parallel sequencing

Foxa2 (HNF3β) is a one of three, closely related transcription factors that are critical to the development and function of the mouse liver. We have used chromatin immunoprecipitation and massively parallel Illumina 1G sequencing (ChIP–Seq) to create a genome-wide profile of in vivo Foxa2-binding sites in the adult liver. More than 65% of the ∼11.5 k genomic sites associated with Foxa2 binding, mapped to extended gene regions of annotated genes, while more than 30% of intragenic sites were located within first introns. 20.5% of all sites were further than 50 kb from any annotated gene, suggesting an association with novel gene regions. QPCR analysis demonstrated a strong positive correlation between peak height and fold enrichment for Foxa2-binding sites. We measured the relationship between Foxa2 and liver gene expression by overlapping Foxa2-binding sites with a SAGE transcriptome profile, and found that 43.5% of genes expressed in the liver were also associated with Foxa2 binding. We also identified potential Foxa2-interacting transcription factors whose motifs were enriched near Foxa2-binding sites. Our comprehensive results for in vivo Foxa2-binding sites in the mouse liver will contribute to resolving transcriptional regulatory networks that are important for adult liver function.

Rare Copy Number Variants Contribute to Congenital Left-Sided Heart Disease

Left-sided congenital heart disease (CHD) encompasses a spectrum of malformations that range from bicuspid aortic valve to hypoplastic left heart syndrome. It contributes significantly to infant mortality and has serious implications in adult cardiology. Although left-sided CHD is known to be highly heritable, the underlying genetic determinants are largely unidentified. In this study, we sought to determine the impact of structural genomic variation on left-sided CHD and compared multiplex families (464 individuals with 174 affecteds (37.5%) in 59 multiplex families and 8 trios) to 1,582 well-phenotyped controls. 73 unique inherited or de novo CNVs in 54 individuals were identified in the left-sided CHD cohort. After stringent filtering, our gene inventory reveals 25 new candidates for LS-CHD pathogenesis, such as SMC1A, MFAP4, and CTHRC1, and overlaps with several known syndromic loci. Conservative estimation examining the overlap of the prioritized gene content with CNVs present only in affected individuals in our cohort implies a strong effect for unique CNVs in at least 10% of left-sided CHD cases. Enrichment testing of gene content in all identified CNVs showed a significant association with angiogenesis. In this first family-based CNV study of left-sided CHD, we found that both co-segregating and de novo events associate with disease in a complex fashion at structural genomic level. Often viewed as an anatomically circumscript disease, a subset of left-sided CHD may in fact reflect more general genetic perturbations of angiogenesis and/or vascular biology.

Locus co-occupancy, nucleosome positioning, and H3K4me1 regulate the functionality of FOXA2-, HNF4A-, and PDX1-bound loci in islets and liver

The liver and pancreas share a common origin and coexpress several transcription factors. To gain insight into the transcriptional networks regulating the function of these tissues, we globally identify binding sites for FOXA2 in adult mouse islets and liver, PDX1 in islets, and HNF4A in liver. Because most eukaryotic transcription factors bind thousands of loci, many of which are thought to be inactive, methods that can discriminate functionally active binding events are essential for the interpretation of genome-wide transcription factor binding data. To develop such a method, we also generated genome-wide H3K4me1 and H3K4me3 localization data in these tissues. By analyzing our binding and histone methylation data in combination with comprehensive gene expression data, we show that H3K4me1 enrichment profiles discriminate transcription factor occupied loci into three classes: those that are functionally active, those that are poised for activation, and those that reflect pioneer-like transcription factor activity. Furthermore, we demonstrate that the regulated presence of H3K4me1-marked nucleosomes at transcription factor occupied promoters and enhancers controls their activity, implicating both tissue-specific transcription factor binding and nucleosome remodeling complex recruitment in determining tissue-specific gene expression. Finally, we apply these approaches to generate novel insights into how FOXA2, PDX1, and HNF4A cooperate to drive islet- and liver-specific gene expression.

The next generation: Using new sequencing technologies to analyse gene regulation

Next generation sequencing (NGS) has pushed back the limitations of prior sequencing technologies to advance genomic knowledge infinitely by allowing cost‐effective, rapid sequencing to become a reality. Genome‐wide transcriptional profiling can be achieved using NGS with either Tag‐Seq, in which short tags of cDNA represent a gene, or RNA‐Seq, in which the entire transcriptome is sequenced. Furthermore, the level and diversity of miRNA within different tissues or cell types can be monitored by specifically sequencing small RNA. The biological mechanisms underlying differential gene regulation can also be explored by coupling chromatin immunoprecipitation with NGS (ChIP‐Seq). Using this methodology genome‐wide binding sites for transcription factors, RNAP II, epigenetic modifiers and the distribution of modified histones can be assessed. The superior, high‐resolution data generated by adopting this sequencing technology allows researchers to distinguish the precise genomic location bound by a protein and correlate this with observed gene expression patterns. Additional methods have also been established to examine other factors influencing gene regulation such as DNA methylation or chromatin conformation on a genome‐wide scale. Within any research setting, these techniques can provide relevant data and answer numerous questions about gene expression and regulation. The advances made by pairing NGS with strategic experimental protocols will continue to impact the research community.

Hippo Signaling Influences HNF4A and FOXA2 Enhancer Switching during Hepatocyte Differentiation

Summary Cell fate acquisition is heavily influenced by direct interactions between master regulators and tissue-specific enhancers. However, it remains unclear how lineage-specifying transcription factors, which are often expressed in both progenitor and mature cell populations, influence cell differentiation. Using in vivo mouse liver development as a model, we identified thousands of enhancers that are bound by the master regulators HNF4A and FOXA2 in a differentiation-dependent manner, subject to chromatin remodeling, and associated with differentially expressed target genes. Enhancers exclusively occupied in the embryo were found to be responsive to developmentally regulated TEAD2 and coactivator YAP1. Our data suggest that Hippo signaling may affect hepatocyte differentiation by influencing HNF4A and FOXA2 interactions with temporal enhancers. In summary, transcription factor-enhancer interactions are not only tissue specific but also differentiation dependent, which is an important consideration for researchers studying cancer biology or mammalian development and/or using transformed cell lines.

An interactive analysis and exploration tool for epigenomic data

In this design study, we present an analysis and abstraction of the data and tasks related to the domain of epigenomics, and the design and implementation of an interactive tool to facilitate data analysis and visualization in this domain. Epigenomic data can be grouped into subsets either by k‐means clustering or by querying for combinations of presence or absence of signal (on/off) in different epigenomic experiments. These steps can easily be interleaved and the comparison of different workflows is explicitly supported. We took special care to contain the exponential expansion of possible on/off combinations by creating a novel querying interface. An interactive heat map facilitates the exploration and comparison of different clusters. We validated our iterative design by working closely with two groups of biologists on different biological problems. Both groups quickly found new insight into their data as well as claimed that our tool would save them several hours or days of work over using existing tools.

SOX9 modulates the expression of key transcription factors required for heart valve development.

Heart valve formation initiates when endothelial cells of the heart transform into mesenchyme and populate the cardiac cushions. The transcription factor SOX9 is highly expressed in the cardiac cushion mesenchyme, and is essential for heart valve development. Loss of Sox9 in mouse cardiac cushion mesenchyme alters cell proliferation, embryonic survival, and valve formation. Despite this important role, little is known about how SOX9 regulates heart valve formation or its transcriptional targets. Therefore, we mapped putative SOX9 binding sites by ChIP-Seq in E12.5 heart valves, a stage at which the valve mesenchyme is actively proliferating and initiating differentiation. Embryonic heart valves have been shown to express a high number of genes that are associated with chondrogenesis, including several extracellular matrix proteins and transcription factors that regulate chondrogenesis. Therefore, we compared regions of putative SOX9 DNA binding between E12.5 heart valves and E12.5 limb buds. We identified context-dependent and context-independent SOX9-interacting regions throughout the genome. Analysis of context-independent SOX9 binding suggests an extensive role for SOX9 across tissues in regulating proliferation-associated genes including key components of the AP-1 complex. Integrative analysis of tissue-specific SOX9-interacting regions and gene expression profiles on Sox9-deficient heart valves demonstrated that SOX9 controls the expression of several transcription factors with previously identified roles in heart valve development, including Twist1, Sox4, Mecom and Pitx2. Together, our data identify SOX9-coordinated transcriptional hierarchies that control cell proliferation and differentiation during valve formation. Summary: Transcriptional targets of SOX9 in developing tissues mapped by ChIP-Seq reveal that SOX9 mediates cell proliferation as well as differentiation during embryonic heart valve formation.

Genome‐wide microRNA and messenger RNA profiling in rodent liver development implicates mir302b and mir20a in repressing transforming growth factor‐beta signaling

MicroRNAs (miRNAs) are recently discovered small RNA molecules that regulate developmental processes, such as proliferation, differentiation, and apoptosis; however, the identity of miRNAs and their functions during liver development are largely unknown. Here we investigated the miRNA and gene expression profiles for embryonic day (E)8.5 endoderm, E14.5 Dlk1+ liver cells (hepatoblasts), and adult liver by employing Illumina sequencing. We found that miRNAs were abundantly expressed at all three stages. Using K‐means clustering analysis, 13 miRNA clusters with distinct temporal expression patterns were identified. mir302b, an endoderm‐enriched miRNA, was identified as an miRNA whose predicted targets are expressed highly in E14.5 hepatoblasts but low in the endoderm. We validated the expression of mir302b in the endoderm by whole‐mount in situ hybridization. Interestingly, mir20a, the most highly expressed miRNA in the endoderm library, was also predicted to regulate some of the same targets as mir302b. We found that through targeting Tgfbr2, mir302b and mir20a are able to regulate transforming growth factor beta (TGFβ) signal transduction. Moreover, mir302b can repress liver markers in an embryonic stem cell differentiation model. Collectively, we uncovered dynamic patterns of individual miRNAs during liver development, as well as miRNA networks that could be essential for the specification and differentiation of liver progenitors. (HEPATOLOGY 2013)

Twist1 transcriptional targets in the developing atrio-ventricular canal of the mouse.

Malformations of the cardiovascular system are the most common type of birth defect in humans, frequently affecting the formation of valves and septa. During heart valve and septa formation, cells from the atrio-ventricular canal (AVC) and outflow tract (OFT) regions of the heart undergo an epithelial-to-mesenchymal transformation (EMT) and invade the underlying extracellular matrix to give rise to endocardial cushions. Subsequent maturation of newly formed mesenchyme cells leads to thin stress-resistant leaflets. TWIST1 is a basic helix-loop-helix transcription factor expressed in newly formed mesenchyme cells of the AVC and OFT that has been shown to play roles in cell survival, cell proliferation and differentiation. However, the downstream targets of TWIST1 during heart valve formation remain unclear. To identify genes important for heart valve development downstream of TWIST1, we performed global gene expression profiling of AVC, OFT, atria and ventricles of the embryonic day 10.5 mouse heart by tag-sequencing (Tag-seq). Using this resource we identified a novel set of 939 genes, including 123 regulators of transcription, enriched in the valve forming regions of the heart. We compared these genes to a Tag-seq library from the Twist1 null developing valves revealing significant gene expression changes. These changes were consistent with a role of TWIST1 in controlling differentiation of mesenchymal cells following their transformation from endothelium in the mouse. To study the role of TWIST1 at the DNA level we performed chromatin immunoprecipitation and identified novel direct targets of TWIST1 in the developing heart valves. Our findings support a role for TWIST1 in the differentiation of AVC mesenchyme post-EMT in the mouse, and suggest that TWIST1 can exert its function by direct DNA binding to activate valve specific gene expression.