Edwin Cheung

An oestrogen-receptor-α-bound human chromatin interactome

Genomes are organized into high-level three-dimensional structures, and DNA elements separated by long genomic distances can in principle interact functionally. Many transcription factors bind to regulatory DNA elements distant from gene promoters. Although distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not been investigated in a genome-wide manner. Here we describe the development of a new strategy, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) for the de novo detection of global chromatin interactions, with which we have comprehensively mapped the chromatin interaction network bound by oestrogen receptor α (ER-α) in the human genome. We found that most high-confidence remote ER-α-binding sites are anchored at gene promoters through long-range chromatin interactions, suggesting that ER-α functions by extensive chromatin looping to bring genes together for coordinated transcriptional regulation. We propose that chromatin interactions constitute a primary mechanism for regulating transcription in mammalian genomes.

Extensive Promoter-centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation

Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.

Integrative model of genomic factors for determining binding site selection by estrogen receptor-α.

A major question in transcription factor (TF) biology is why a TF binds to only a small fraction of motif eligible binding sites in the genome. Using the estrogen receptor‐α as a model system, we sought to explicitly define parameters that determine TF‐binding site selection. By examining 12 genetic and epigenetic parameters, we find that an energetically favorable estrogen response element (ERE) motif sequence, co‐occupancy by the TF FOXA1, the presence of the H3K4me1 mark and an open chromatin configuration in the pre‐ligand state provide specificity for ER binding. These factors can model estrogen‐induced ER binding with high accuracy (ROC‐AUC=0.95 and 0.88 using different genomic backgrounds). Moreover, when assessed in another estrogen‐responsive cell line, this model was highly predictive for ERα binding (ROC‐AUC=0.86). Variance in binding site selection between MCF‐7 and T47D resides in sites with suboptimal ERE motifs, but modulated by the chromatin configuration. These results suggest a definable interplay between sequence motifs and local chromatin in selecting TF binding.

Smads orchestrate specific histone modifications and chromatin remodeling to activate transcription.

Smads are intracellular transducers for TGF‐β superfamily ligands, but little is known about the mechanism by which complexes of receptor‐phosphorylated Smad2 and Smad4 regulate transcription. Using an in vitro transcription system, we have discovered that, unlike most transcription factors that are sufficient to recruit the basal transcription machinery and therefore activate transcription on both naked DNA and chromatin templates, the Smads only activate transcription from chromatin templates. We demonstrate that Smad2‐mediated transcription requires the histone acetyltransferase, p300. Smad2‐recruited p300 exhibits an altered substrate specificity, specifically acetylating nucleosomal histone H3 at lysines 9 and 18, and these modifications are also detected on an endogenous Smad2‐dependent promoter in a ligand‐induced manner. Furthermore, we show that endogenous Smad2 interacts with the SWI/SNF ATPase, Brg1, in a TGF‐β‐dependent manner, and demonstrate that Brg1 is recruited to Smad2‐dependent promoters and is specifically required for TGF‐β‐induced expression of endogenous Smad2 target genes. Our data indicate that the Smads define a new class of transcription factors that absolutely require chromatin to assemble the basal transcription machinery and activate transcription.

AP-2γ regulates oestrogen receptor-mediated long-range chromatin interaction and gene transcription.

Oestrogen receptor α (ERα) is key player in the progression of breast cancer. Recently, the cistrome and interactome of ERα were mapped in breast cancer cells, revealing the importance of spatial organization in oestrogen-mediated transcription. However, the underlying mechanism of this process is unclear. Here, we show that ERα binding sites (ERBS) identified from the Chromatin Interaction Analysis-Paired End DiTag of ERα are enriched for AP-2 motifs. We demonstrate the transcription factor, AP-2γ, which has been implicated in breast cancer oncogenesis, binds to ERBS in a ligand-independent manner. Furthermore, perturbation of AP-2γ expression impaired ERα DNA binding, long-range chromatin interactions, and gene transcription. In genome-wide analyses, we show that a large number of AP-2γ and ERα binding events converge together across the genome. The majority of these shared regions are also occupied by the pioneer factor, FoxA1. Molecular studies indicate there is functional interplay between AP-2γ and FoxA1. Finally, we show that most ERBS associated with long-range chromatin interactions are colocalized with AP-2γ and FoxA1. Together, our results suggest AP-2γ is a novel collaborative factor in ERα-mediated transcription.

AP-2γ regulates oestrogen receptor-mediated long-range chromatin interaction and gene transcription: Long-range gene regulation by AP-2γ

Oestrogen receptor α (ERα) is key player in the progression of breast cancer. Recently, the cistrome and interactome of ERα were mapped in breast cancer cells, revealing the importance of spatial organization in oestrogen‐mediated transcription. However, the underlying mechanism of this process is unclear. Here, we show that ERα binding sites (ERBS) identified from the Chromatin Interaction Analysis‐Paired End DiTag of ERα are enriched for AP‐2 motifs. We demonstrate the transcription factor, AP‐2γ, which has been implicated in breast cancer oncogenesis, binds to ERBS in a ligand‐independent manner. Furthermore, perturbation of AP‐2γ expression impaired ERα DNA binding, long‐range chromatin interactions, and gene transcription. In genome‐wide analyses, we show that a large number of AP‐2γ and ERα binding events converge together across the genome. The majority of these shared regions are also occupied by the pioneer factor, FoxA1. Molecular studies indicate there is functional interplay between AP‐2γ and FoxA1. Finally, we show that most ERBS associated with long‐range chromatin interactions are colocalized with AP‐2γ and FoxA1. Together, our results suggest AP‐2γ is a novel collaborative factor in ERα‐mediated transcription.

Integration of Regulatory Networks by NKX3-1 Promotes Androgen-Dependent Prostate Cancer Survival

ABSTRACT The NKX3-1 gene is a homeobox gene required for prostate tumor progression, but how it functions is unclear. Here, using chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) we showed that NKX3-1 colocalizes with the androgen receptor (AR) across the prostate cancer genome. We uncovered two distinct mechanisms by which NKX3-1 controls the AR transcriptional network in prostate cancer. First, NKX3-1 and AR directly regulate each other in a feed-forward regulatory loop. Second, NKX3-1 collaborates with AR and FoxA1 to mediate genes in advanced and recurrent prostate carcinoma. NKX3-1- and AR-coregulated genes include those found in the “protein trafficking” process, which integrates oncogenic signaling pathways. Moreover, we demonstrate that NKX3-1, AR, and FoxA1 promote prostate cancer cell survival by directly upregulating RAB3B, a member of the RAB GTPase family. Finally, we show that RAB3B is overexpressed in prostate cancer patients, suggesting that RAB3B together with AR, FoxA1, and NKX3-1 are important regulators of prostate cancer progression. Collectively, our work highlights a novel hierarchical transcriptional regulatory network between NKX3-1, AR, and the RAB GTPase signaling pathway that is critical for the genetic-molecular-phenotypic paradigm in androgen-dependent prostate cancer.

A transcriptional repressor co-regulatory network governing androgen response in prostate cancers

Transcriptional corepressors are frequently aberrantly over‐expressed in prostate cancers. However, their crosstalk with the Androgen receptor (AR), a key player in prostate cancer development, is unclear. Using ChIP‐Seq, we generated extensive global binding maps of AR, ERG, and commonly over‐expressed transcriptional corepressors including HDAC1, HDAC2, HDAC3, and EZH2 in prostate cancer cells. Surprisingly, our results revealed that ERG, HDACs, and EZH2 are directly involved in androgen‐regulated transcription and wired into an AR centric transcriptional network via a spectrum of distal enhancers and/or proximal promoters. Moreover, we showed that similar to ERG, these corepressors function to mediate repression of AR‐induced transcription including cytoskeletal genes that promote epithelial differentiation and inhibit metastasis. Specifically, we demonstrated that the direct suppression of Vinculin expression by ERG, EZH2, and HDACs leads to enhanced invasiveness of prostate cancer cells. Taken together, our results highlight a novel mechanism by which, ERG working together with oncogenic corepressors including HDACs and the polycomb protein, EZH2, could impede epithelial differentiation and contribute to prostate cancer progression, through directly modulating the transcriptional output of AR.

Regulation of Estrogen Receptor-mediated Long Range Transcription via Evolutionarily Conserved Distal Response Elements

Nuclear signaling by estrogens rapidly induces the global recruitment of estrogen receptors (ERs) to thousands of highly specific locations in the genome. Here, we have examined whether ER binding sites that are located distal from the transcription start sites of estrogen target genes are functionally relevant. Similar to ER binding sites near the proximal promoter region, ER binding sites located at distal locations are occupied by ERs after estrogen stimulation. And, like proximal bound ERs, ERs occupied at distal sites can recruit coactivators and the RNA polymerase transcription machinery and mediate specific structural changes to chromatin. Furthermore, ERs occupied at the distal sites are capable of communicating with ERs bound at the promoter region, possibly via long range chromosome looping. In functional analysis, disruption of the response elements in the distal ER binding sites abrogated ER binding and significantly reduced transcriptional response. Finally, sequence comparison of the response elements at the distal sites suggests a high level of conservation across different species. Together, our data indicate that distal ER binding sites are bona fide transcriptional enhancers that are involved in long range chromosomal interaction, transcription complex formation, and distinct structural modifications of chromatin across large genomic spans.

Genomic analyses of hormone signaling and gene regulation.

Many cellular signaling pathways ultimately control specific patterns of gene expression in the nucleus through a variety of signal-regulated transcription factors (TFs), including nuclear hormone receptors (NRs). The advent of genomic technologies for examining signal-regulated transcriptional responses and TF binding on a genomic scale has dramatically increased our understanding of the cellular programs that control hormonal signaling and gene regulation. Studies of TFs, especially NRs, using genomic approaches have revealed novel and unexpected features of hormone-regulated transcription, and a global view is beginning to emerge. In this review, we discuss the genomic methodologies that have been applied to the study of hormone-regulated gene expression, the results that have been obtained from using them, and the future prospects for these approaches. Given the wealth of information about hormone-dependent gene regulation by NRs, we have focused this review on the knowledge gained from genomic studies of their function.