Transcription Factor Occupancy Research Articles

Genome-wide ChIP-Seq reveals a dramatic shift in the binding of the transcription factor erythroid Kruppel-like factor during erythrocyte differentiation

AbstractErythropoiesis is dependent on the activity of transcription factors, including the erythroid-specific erythroid Kruppel-like factor (EKLF). ChIP followed by massively parallel sequencing (ChIP-Seq) is a powerful, unbiased method to map trans-factor occupancy. We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts. We correlated these results with the nuclear distribution of EKLF, RNA-Seq analysis of the transcriptome, and the occupancy of other erythroid transcription factors. In progenitor cells, EKLF is found predominantly at the periphery of the nucleus, where EKLF primarily occupies the promoter regions of genes and acts as a transcriptional activator. In erythroblasts, EKLF is distributed throughout the nucleus, and erythroblast-specific EKLF occupancy is predominantly in intragenic regions. In progenitor cells, EKLF modulates general cell growth and cell cycle regulatory pathways, whereas in erythroblasts EKLF is associated with repression of these pathways. The EKLF interactome shows very little overlap with the interactomes of GATA1, GATA2, or TAL1, leading to a model in which EKLF directs programs that are independent of those regulated by the GATA factors or TAL1.

Integrated genome-wide analysis of transcription factor occupancy, RNA polymerase II binding and steady-state RNA levels identify differentially regulated functional gene classes.

Routine methods for assaying steady-state mRNA levels such as RNA-seq and micro-arrays are commonly used as readouts to study the role of transcription factors (TFs) in gene expression regulation. However, cellular RNA levels do not solely depend on activity of TFs and subsequent transcription by RNA polymerase II (Pol II), but are also affected by RNA turnover rate. Here, we demonstrate that integrated analysis of genome-wide TF occupancy, Pol II binding and steady-state RNA levels provide important insights in gene regulatory mechanisms. Pol II occupancy, as detected by Pol II ChIP-seq, was found to correlate better with TF occupancy compared to steady-state RNA levels and is thus a more precise readout for the primary transcriptional mechanisms that are triggered by signal transduction. Furthermore, analysis of differential Pol II occupancy and RNA-seq levels identified genes with high Pol II occupancy and relatively low RNA levels and vice versa. These categories are strongly enriched for genes from different functional classes. Our results demonstrate a complementary value in Pol II chip-seq and RNA-seq approaches for better understanding of gene expression regulation.

Dynamics of the epigenetic landscape during erythroid differentiation after GATA1 restoration

Interplays among lineage-specific nuclear proteins, chromatin modifying enzymes, and the basal transcription machinery govern cellular differentiation, but their dynamics of action and coordination with transcriptional control are not fully understood. Alterations in chromatin structure appear to establish a permissive state for gene activation at some loci, but they play an integral role in activation at other loci. To determine the predominant roles of chromatin states and factor occupancy in directing gene regulation during differentiation, we mapped chromatin accessibility, histone modifications, and nuclear factor occupancy genome-wide during mouse erythroid differentiation dependent on the master regulatory transcription factor GATA1. Notably, despite extensive changes in gene expression, the chromatin state profiles (proportions of a gene in a chromatin state dominated by activating or repressive histone modifications) and accessibility remain largely unchanged during GATA1-induced erythroid differentiation. In contrast, gene induction and repression are strongly associated with changes in patterns of transcription factor occupancy. Our results indicate that during erythroid differentiation, the broad features of chromatin states are established at the stage of lineage commitment, largely independently of GATA1. These determine permissiveness for expression, with subsequent induction or repression mediated by distinctive combinations of transcription factors.

Transcription Factor AP1 Potentiates Chromatin Accessibility and Glucocorticoid Receptor Binding

Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with coregulators. The role of these interactions in determining selective binding of GR to regulatory elements remains unclear. Recent findings indicate that a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing, we identify the activator protein 1 (AP1) as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. We propose a model in which the basal occupancy of transcription factors acts to prime chromatin and direct inducible transcription factors to select regions in the genome.

Genome-wide Analysis of Simultaneous GATA1/2, RUNX1, FLI1, and SCL Binding in Megakaryocytes Identifies Hematopoietic Regulators

SummaryHematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors—GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL—in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes.

Adipogenic hotspots: where the action is

EMBO J 30 8, 1459–1472 (2011); published online March222011 [PMC free article] [PubMed] Transcription factors (TFs) orchestrate cell-fate decisions by programming the expression of many genes, including those of other TFs that further drive lineage specification. C/EBP proteins and glucocorticoid receptor (GR) activate transcription of the master regulator of adipocyte differentiation, PPARγ. A study in this issue of The EMBO Journal sheds light on how C/EBPs, GR, STAT5, and RXR cooperate during early adipogenesis to globally remodel the epigenome. Adipocytes, the major lipid-containing component of adipose tissue, benefit metabolism by coordinating lipid and glucose homoeostasis. They are post-mitotic, terminally differentiated cells derived from mesenchymal precursors. In cell culture models, temporary exposure of preadipocytes to a mix of insulin, glucocorticoid, and an inducer of cAMP signaling triggers adipogenesis, changing the expression of hundreds of structural genes, and a variety of TFs (Farmer, 2006). Upon their induction, C/EBPs and PPARγ occupy sites near most of the genes that are upregulated during adipogenesis, including their own and each other's genes, forming a transcription network that helps to explain how they establish and maintain the adipocyte phenotype (Lefterova et al, 2008; Nielsen et al, 2008). Comparative analysis of chromatin state maps at different stages of adipogenesis has recently provided further insight into an adipogenic transcription network (Mikkelsen et al, 2010; Steger et al, 2010). Thousands of putative preadipoctye- and adipocyte-specific cis-regulatory regions were identified through examination of several histone modifications associated with transcriptionally active or repressed chromatin. Bioinformatics identified enriched TF motifs, and PLZF, SRF (Mikkelsen et al, 2010) and GR (Steger et al, 2010) were validated as sequence-specific regulators of adipogenesis. Mandrup and colleagues now present a genome-wide analysis of chromatin remodeling events occurring during adipocyte differentiation (Siersbaek et al, 2011). By monitoring differential sensitivity to DNase I, they detected dynamic changes to chromatin, identified enriched TF motifs at DNase I hypersensitive sites (DHSs), and validated binding of C/EBP proteins, GR, RXR, and STAT5 to the regions remodeled at early time points. Remarkably, these TFs co-localize at over 900 regions of open chromatin, which they termed ‘hotspots'. Most of the hotspots are transiently open only during the early stages of adipogenesis, and many of these correspond to enhancer regions marked by transient histone acetylation, TF binding, and coactivator recruitment that define an epigenomic transition state during adipogenesis (Figure 1A) (Steger et al, 2010). Figure 1 Chromatin opening during adipogenesis. Temporary exposure of preadipocytes to adipogenic stimuli induces DNase I hypersensitive ‘hotspots' with characteristics of enhancers including binding of multiple TFs, coactivator recruitment (e.g. p300), ... Interestingly, chromatin sites that open early and remain open through adipogenesis are frequently occupied by PPARγ in adipocytes (Figure 1B), suggesting that early adipogenic TFs may facilitate its binding. It is important to note, however, that most PPARγ-binding sites lie outside of early DHSs, leading to the possibility that PPARγ does not require pioneer factors to open up chromatin before gaining access to these targets (Figure 1C). Indeed, forced expression of PPARγ in preadipocytes caused binding to regions normally occupied only in adipocytes, which was accompanied by increased DNA accessibility and histone acetylation (Lefterova et al, 2010). It is intriguing to compare adipogenic hotspots with another recently identified DNA element termed highly occupied target (HOT) regions (Gerstein et al, 2010; modENCODE Consortium et al, 2010). HOT regions bind many different TFs (at least 15 in Caenorhabditis elegans) but are not enriched in the known motifs to which they bind, suggesting that the interactions are indirect. Adipogenic hotspots appear to be functionally distinct from HOT regions, as they display enrichment for motifs and tissue-specific expression of targets. Yet, the existence of hotspots and HOT regions indicates that genomic binding of many TFs may not be distributed evenly across sites containing cognate binding motifs, but rather, concentrated at a smaller number of regions of open chromatin. This may in part explain the specificity of TF binding that goes far beyond simple recognition of high-affinity motifs in the genome (Carroll et al, 2005). Adipogenic hotspots likely regulate gene expression, but it is unknown whether they function solely in this regard. Could TF occupancy accompanied by increased chromatin accessibility and enriched histone modification define all active enhancers? An answer awaits development of high-throughput methods that determine the function of putative cis-regulatory elements. Indeed, although many DHSs co-localize with active histone modifications, thousands are unmarked, and it is possible that various combinations of chromatin features may signal distinct three-dimensional structures that positively or negatively regulate mRNA and/or non-coding RNA synthesis as well as DNA replication and repair. Future studies will need to determine all the action going on at the hotspots.

Regulation of the human ascorbate transporter SVCT2 exon 1b gene by zinc-finger transcription factors

The sodium-dependent vitamin C transporter (SVCT) 2 is crucial for ascorbate uptake in metabolically active and specialized tissues. This study focused on the gene regulation of SVCT2 exon 1b, which is ubiquitously expressed in human and mouse tissues. Although the human SVCT2 exon 1b promoter does not contain a classical TATA box, we found that it does contain a functional initiator that binds Yin Yang-1 (YY1) and interacts with upstream Sp1/Sp3 elements in the proximal promoter region. These elements in turn play a critical role in regulating YY1-mediated transcription of exon 1b. Formation of YY1/Sp complexes on the promoter is required for its optional function. YY1 with Sp1 or Sp3 synergistically enhanced exon 1b promoter activity as well as the endogenous SVCT2 protein expression. Further, in addition to Sp1/Sp3, both EGR-1 and EGR-2 were detected in the protein complexes that bound the three GC boxes bearing overlapping binding sites for EGR/WT1 and Sp1/3. The EGR family factors WT1 and MAZ were found to differentially regulate exon 1b promoter activity. These results show that differential occupancy of transcription factors on the GC-rich consensus sequences in the SVCT2 exon 1b promoter contributes to the regulation of cell and tissue expression of SVCT2.

Species-Specific Strategies Underlying Conserved Functions of Metabolic Transcription Factors

The winged helix protein FOXA2 and the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) 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 PPARγ 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 PPARγ 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 PPARγ. 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.

Chromatin remodeling complex NURF regulates thymocyte maturation

The maturation of T cells requires signaling from both cytokine and T-cell receptors to gene targets in chromatin, but how chromatin architecture influences this process is largely unknown. Here we show that thymocyte maturation post-positive selection is dependent on the nucleosome remodeling factor (NURF). Depletion of Bptf (bromodomain PHD finger transcription factor), the largest NURF subunit, in conditional mouse mutants results in developmental arrest beyond the CD4(+) CD8(int) stage without affecting cellular proliferation, cellular apoptosis, or coreceptor gene expression. In the Bptf mutant, specific subsets of genes important for thymocyte development show aberrant expression. We also observed defects in DNase I-hypersensitive chromatin structures at Egr1, a prototypical Bptf-dependent gene that is required for efficient thymocyte development. Moreover, chromatin binding of the sequence-specific factor Srf (serum response factor) to Egr1 regulatory sites is dependent on Bptf function. Physical interactions between NURF and Srf suggest a model in which Srf recruits NURF to facilitate transcription factor binding at Bptf-dependent genes. These findings provide evidence for causal connections between NURF, transcription factor occupancy, and gene regulation during thymocyte development.

SeqMINER: an integrated ChIP-seq data interpretation platform

In a single experiment, chromatin immunoprecipitation combined with high throughput sequencing (ChIP-seq) provides genome-wide information about a given covalent histone modification or transcription factor occupancy. However, time efficient bioinformatics resources for extracting biological meaning out of these gigabyte-scale datasets are often a limiting factor for data interpretation by biologists. We created an integrated portable ChIP-seq data interpretation platform called seqMINER, with optimized performances for efficient handling of multiple genome-wide datasets. seqMINER allows comparison and integration of multiple ChIP-seq datasets and extraction of qualitative as well as quantitative information. seqMINER can handle the biological complexity of most experimental situations and proposes methods to the user for data classification according to the analysed features. In addition, through multiple graphical representations, seqMINER allows visualization and modelling of general as well as specific patterns in a given dataset. To demonstrate the efficiency of seqMINER, we have carried out a comprehensive analysis of genome-wide chromatin modification data in mouse embryonic stem cells to understand the global epigenetic landscape and its change through cellular differentiation.

Genome-Wide Profiling of Epigenetic and Transcription Factor Regulation In Human Macrophage Differentiation

AbstractAbstract 3875The differentiation of human macrophages is accompanied by distinctive phenotypical changes and generally proceeds in the absence of proliferation. The molecular events governing this process are still poorly understood. Using ChIP-Seq technology we studied epigenetic changes as well as alterations in transcription factor occupancy during human monocyte differentiation and correlated these events with gene expression levels in hematopoietic cell types. We show that putative enhancer regions marked by histone H3 lysine4 monomethylation (H3K4me1) at different developmental stages (human progenitor cells, peripheral blood monocytes and in vitro differentiated macrophages) are enriched in distinct sets of transcription factor motifs corresponding to lineage-determining factors. Cell stage-specific histone methylation at promoter-distal sites corresponds with increased mRNA expression levels of neighboring genes. We generated global DNA-binding maps in monocytes and macrophages for two transcription factors (PU.1 and C/EBPβ) with a well established role in monocyte/macrophage differentiation. Comparison of human binding sites with corresponding mouse data revealed a surprisingly low level of conservation (∼10-15%) of PU.1-or C/EBPβ -bound sites between man and mouse, despite a highly conserved binding preference for both transcription factors. During monocytic differentiation, human macrophages primarily gained additional binding sites for both transcription factors (as well as promoter-distal H3K4me1). Interestingly, only neighboring genes with multiple binding events showed significantly increased, macrophage-specific mRNA expression as compared to monocytic as well as lymphocytic cell types. Human macrophage-specific H3K4me1-marked regions as well as macrophage-specific PU.1- and C/EBP-bound sites were characterized by overlapping sets of novel sequence motifs, suggesting that the combinatorial interaction of corresponding DNA-binding factors with PU.1 and C/EBPβ may be required for the establishment of human macrophage-specific enhancers. These data provide novel insights into PU.1 and C/EBPβ mediated gene regulation during human macrophage differentiation. Disclosures:No relevant conflicts of interest to declare.

An Active Chromatin Hub Involving the Three Alternate Promoter/First Exons of the Ankyrin-1 (ANK-1) Gene

AbstractAbstract 3862The Ankyrin-1 (ANK-1) gene is an example of the many mammalian genes with alternate promoters in which transcripts containing distinct first exons are fused to a common exon 2 in different cell types. The ANK-1E promoter/first exon is active in only erythroid cells and is located between a promoter/first exon located 40 kb upstream that is expressed exclusively in brain and muscle cells (ANK-1B) and a promoter/first exon located 16 kb downstream that is expressed in many cell types (ANK-1A). We have previously shown that the ANK-1E promoter/first exon is flanked by two DNase I hypersensitive sites (HS). The ANK-1E 5′HS is located in the promoter region, while two adjacent HS (3′HS 1&2) are located ∼6 kb downstream. We hypothesize that the erythroid-specific activation of the ANK-1E promoter/first exon involves both changes in transcription factor occupancy and in chromatin structure. Using whole-genome Chromatin Immune Precipitation (ChIPSeq) and whole-genome mRNA analysis (RNASeq), we have made a detailed study of Erythroid Krupple-like Factor (EKLF) occupancy and mRNA levels in sorted mouse erythroid progenitor cells and definitive erythroblasts. In erythroid progenitor cells, EKLF occupies the Ank-1E 5′HS, and the level of Ank-1E mRNA is relatively low. In erythroblasts, EKLF is not detected at either Ank-1E 5′HS or the Ank-1E 3′HS and the Ank-1 mRNA level is increased more than 2-fold, consistent with EKLF acting as a transcriptional repressor. We have also shown that the ANK-1E 5′HS and the 3′HS region are both occupied by the transcription factors GATA-1 and NF-E2, although EMSA assays have shown that NF-E2 does not bind to the 5′HS region, and that GATA-1 does not bind to the 3′HS region in vitro. Addition of the NF-E2 site in 3′HS1 to the ANK-1E promoter increases expression 4-fold indicating a potential interaction of these two regions. We have recently demonstrated that both the ANK-1E 5′HS and 3′HS2 act as barrier elements that prevent gene silencing in cultured erythroid cells and transgenic mice. Consistent with the functional observations, in erythroid cells both ANK-1E 5′HS and the 3′HS region are occupied by the barrier-associated proteins USF-1 and -2, PRMT-1 and -4, CTCF and SRC1, while the ANK-1A region is occupied by the barrier-associated proteins USF-2, PRMT-1 and -4, but not USF-1 and CTCF. We hypothesized that the HS flanking the ANK-1E promoter/first exon form a chromatin loop that brings both the transcription factors and barrier proteins into proximity. Using the chromatin conformation capture assay we demonstrated an interaction between the Ank-1E 5′HS and the 3′HS region. Analysis of the three dimensional structure of the 100 kb region upstream of the mouse Ank-1 exon 2 region was performed using the chromatin conformation capture carbon copy (5C) assay. We designed a set of 12 forward primers on the three Ank-1 promoter/first exons and the HS that flank them, and a set of 264 reverse primers to analyze all interactions in this region. In mouse fetal liver erythroid cells, the strongest interaction is between Ank-1E 5′HS and a region 15 kb 3′ to the Ank-1B region. In addition, the Ank-1B promoter/first exon region interacts with both the Ank-1A region and a region 30 kb downstream of the Ank-1B region forming a ‘rosette’ that includes Ank-1E. We propose a model in which the formation of an active chromatin hub in erythroid cells which is facilitated by erythroid transcription factors and stabilized by the barrier-associated proteins. Disclosures:No relevant conflicts of interest to declare.

The Importance of Being Specified: Cell Fate Decisions and Their Role in Cell Biology

The Importance of Being Specified: Cell Fate Decisions and Their Role in Cell Biology

Proinflammatory Cytokines Enhance Estrogen-dependent Expression of the Multidrug Transporter Gene ABCG2 through Estrogen Receptor and NFκB Cooperativity at Adjacent Response Elements

Constitutive activation of NFκB in estrogen receptor (ER)-positive breast cancer is associated with tumor recurrence and development of anti-estrogen resistance. Furthermore, a gene expression signature containing common targets for ER and NFκB has been identified and found to be associated with the more aggressive luminal B intrinsic subtype of ER-positive breast tumors. Here, we describe a novel mechanism by which ER and NFκB cooperate to up-regulate expression of one important gene from this signature, ABCG2, which encodes a transporter protein associated with the development of drug-resistant breast cancer. We and others have confirmed that this gene is regulated primarily by estrogen in an ER- and estrogen response element (ERE)-dependent manner. We found that whereas proinflammatory cytokines have little effect on this gene in the absence of 17β-estradiol, they can potentiate ER activity in an NFκB-dependent manner. ER allows the NFκB family member p65 to access a latent NFκB response element located near the ERE in the gene promoter. NFκB recruitment to the gene is, in turn, required to stabilize ER occupancy at the functional ERE. The result of this cooperative binding of ER and p65 at adjacent response elements leads to a major increase in both ABCG2 mRNA and protein expression. These findings indicate that estrogen and inflammatory factors can modify each other's activity through modulation of transcription factor accessibility and/or occupancy at adjacent response elements. This novel transcriptional mechanism could have important implications in breast cancer, where both inflammation and estrogen can promote cancer progression.

Architectural Epigenetics: Mitotic Retention of Mammalian Transcriptional Regulatory Information

Epigenetic regulatory information must be retained during mammalian cell division to sustain phenotype-specific and physiologically responsive gene expression in the progeny cells. Histone modifications, DNA methylation, and RNA-mediated silencing are well-defined epigenetic mechanisms that control the cellular phenotype by regulating gene expression. Recent results suggest that the mitotic retention of nuclease hypersensitivity, selective histone marks, as well as the lineage-specific transcription factor occupancy of promoter elements contribute to the epigenetic control of sustained cellular identity in progeny cells. We propose that these mitotic epigenetic signatures collectively constitute architectural epigenetics, a novel and essential mechanism that conveys regulatory information to sustain the control of phenotype and proliferation in progeny cells by bookmarking genes for activation or suppression.

High resolution models of transcription factor-DNA affinities improve in vitro and in vivo binding predictions.

Accurately modeling the DNA sequence preferences of transcription factors (TFs), and using these models to predict in vivo genomic binding sites for TFs, are key pieces in deciphering the regulatory code. These efforts have been frustrated by the limited availability and accuracy of TF binding site motifs, usually represented as position-specific scoring matrices (PSSMs), which may match large numbers of sites and produce an unreliable list of target genes. Recently, protein binding microarray (PBM) experiments have emerged as a new source of high resolution data on in vitro TF binding specificities. PBM data has been analyzed either by estimating PSSMs or via rank statistics on probe intensities, so that individual sequence patterns are assigned enrichment scores (E-scores). This representation is informative but unwieldy because every TF is assigned a list of thousands of scored sequence patterns. Meanwhile, high-resolution in vivo TF occupancy data from ChIP-seq experiments is also increasingly available. We have developed a flexible discriminative framework for learning TF binding preferences from high resolution in vitro and in vivo data. We first trained support vector regression (SVR) models on PBM data to learn the mapping from probe sequences to binding intensities. We used a novel -mer based string kernel called the di-mismatch kernel to represent probe sequence similarities. The SVR models are more compact than E-scores, more expressive than PSSMs, and can be readily used to scan genomics regions to predict in vivo occupancy. Using a large data set of yeast and mouse TFs, we found that our SVR models can better predict probe intensity than the E-score method or PBM-derived PSSMs. Moreover, by using SVRs to score yeast, mouse, and human genomic regions, we were better able to predict genomic occupancy as measured by ChIP-chip and ChIP-seq experiments. Finally, we found that by training kernel-based models directly on ChIP-seq data, we greatly improved in vivo occupancy prediction, and by comparing a TF's in vitro and in vivo models, we could identify cofactors and disambiguate direct and indirect binding.

The reactive tumor microenvironment: MUC1 signaling directly reprograms transcription of CTGF

The MUC1 cytoplasmic tail (MUC1.CT) conducts signals from spatial and extracellular cues (growth factor and cytokine stimulation) to evoke a reprogramming of the cellular transcriptional profile. Specific phosphorylated forms of the MUC1.CT achieve this function by differentially associating with transcription factors and redirecting their transcriptional regulatory capabilities at specific gene regulatory elements. The specificity of interaction between MUC1.CT and several transcription factors is dictated by the phosphorylation pattern of the 18 potential phosphorylation motifs within the MUC1.CT. To better appreciate the scope of differential gene expression triggered by MUC1.CT activation, we performed microarray gene expression analysis and ChIP-chip promoter analysis and identified the genome-wide transcriptional targets of MUC1.CT signaling in pancreatic cancer. On a global scale, MUC1.CT preferentially targets genes relating to invasion, angiogenesis and metastasis, suggesting that MUC1.CT signaling contributes to establishing a reactive tumor microenvironment during tumor progression to metastatic disease. We examined in detail the molecular mechanisms of MUC1.CT signaling that induces expression of connective tissue growth factor (CTGF/CCN2), a potent mediator of ECM remodeling and angiogenesis. We demonstrate a robust induction of CTGF synthesis and secretion in response to serum factors that is enabled only when MUC1 is highly expressed. We demonstrate the requirement of phosphorylation at distinct tyrosine motifs within the MUC1.CT for MUC1-induced CTGF expression and demonstrate a phosphorylation-specific localization of MUC1.CT to the CTGF promoter. We found that MUC1 reorganizes transcription factor occupancy of genomic regions upstream of the CTGF gene, directing β-catenin and mutant p53 to CTGF gene regulatory elements to promote CTGF expression and destabilizing the interaction at these regions of the transcriptional repressor, c-Jun. With this example we illustrate the capacity of MUC1.CT to mediate transcription factor activity in a context-dependent manner to achieve widespread and robust changes in gene expression and facilitate creation of the reactive tumor microenvironment.

Examination of transcriptional networks reveals an important role for TCFAP2C, SMARCA4, and EOMES in trophoblast stem cell maintenance

Trophoblast stem cells (TS cells), derived from the trophectoderm (TE) of blastocysts, require transcription factors (TFs) and external signals (FGF4, INHBA/NODAL/TGFB1) for self-renewal. While many reports have focused on TF networks that regulate embryonic stem cell (ES cell) self-renewal and pluripotency, little is know about TF networks that regulate self-renewal in TS cells. To further understand transcriptional networks in TS cells, we used chromatin immunoprecipitation with DNA microarray hybridization (ChIP-chip) analysis to investigate targets of the TFs-TCFAP2C, EOMES, ETS2, and GATA3-and a chromatin remodeling factor, SMARCA4. We then evaluated the transcriptional states of target genes using transcriptome analysis and genome-wide analysis of histone H3 acetylation (AcH3). Our results describe previously unknown transcriptional networks in TS cells, including TF occupancy of genes involved in ES cell self-renewal and pluripotency, co-occupancy of TCFAP2C, SMARCA4, and EOMES at a significant number of genes, and transcriptional regulatory circuitry within the five factors. Moreover, RNAi depletion of Tcfap2c, Smarca4, and Eomes transcripts resulted in a loss of normal colony morphology and down-regulation of TS cell-specific genes, suggesting an important role for TCFAP2C, SMARCA4, and EOMES in TS cell self-renewal. Through genome-wide mapping and global expression analysis of five TF target genes, our data provide a comprehensive analysis of transcriptional networks that regulate TS cell self-renewal.

The Current State of Chromatin Immunoprecipitation

The biological significance of interactions of nuclear proteins with DNA in the context of gene expression, cell differentiation, or disease has immensely been enhanced by the advent of chromatin immunoprecipitation (ChIP). ChIP is a technique whereby a protein of interest is selectively immunoprecipitated from a chromatin preparation to determine the DNA sequences associated with it. ChIP has been widely used to map the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin modifying enzymes on the genome or on a given locus. In spite of its power, ChIP has for a long time remained a cumbersome procedure requiring large numbers of cells. These limitations have sparked the development of modifications to shorten the procedure, simplify sample handling and make ChIP amenable to small numbers of cells. In addition, the combination of ChIP with DNA microarray and high-throughput sequencing technologies has in recent years enabled the profiling of histone modification, histone variants, and transcription factor occupancy on a genome-wide scale. This review highlights the variations on the theme of the ChIP assay, the various detection methods applied downstream of ChIP, and examples of their application.

High-throughput sequencing identifies STAT3 as the DNA-associated factor for p53-NF-κB-complex-dependent gene expression in human heart failure

BackgroundGenome-wide maps of DNA regulatory elements and their interaction with transcription factors may form a framework for understanding regulatory circuits and gene expression control in human disease, but how these networks, comprising transcription factors and DNA-binding proteins, form complexes, interact with DNA and modulate gene expression remains largely unknown.MethodsUsing microRNA-21 (mir-21), which is an example of genes that are regulated in heart failure, we performed chromatin immunoprecipitation (ChIP) assays to determine the occupancy of transcription factors at this genetic locus. Tissue ChIP was further performed using human hearts and genome-wide occupancies of these transcription factors were analyzed by high-throughput sequencing.ResultsWe show that the transcription factor p53 piggy-backs onto NF-κB/RELA and utilizes the κB-motif at a cis-regulatory region to control mir-21 expression. p53 behaves as a co-factor in this complex because despite a mutation in its DNA binding domain, mutant p53 was still capable of binding RELA and the cis-element, and inducing mir-21 expression. In dilated human hearts where mir-21 upregulation was previously demonstrated, the p53-RELA complex was also associated with this cis-element. Using high-throughput sequencing, we analyzed genome-wide binding sites for the p53-RELA complex in diseased and control human hearts and found a significant overrepresentation of the STAT3 motif. We further determined that STAT3 was necessary for the p53-RELA complex to associate with this cis-element and for mir-21 expression.ConclusionsOur results uncover a mechanism by which transcription factors cooperate in a multi-molecular complex at a cis-regulatory element to control gene expression.