Expanded encyclopaedias of DNA elements in the human and mouse genomes.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

Reduction of ZFX levels decreases histone H4 acetylation and increases Pol2 pausing at target promoters.

The ZFX transcriptional activator binds to CpG island promoters, with a major peak at ∼200-250 bp downstream from transcription start sites. Because ZFX binds within the transcribed region, we investigated whether it regulates transcriptional elongation. We used GRO-seq to show that loss or reduction of ZFX increased Pol2 pausing at ZFX-regulated promoters. To further investigate the mechanisms by which ZFX regulates transcription, we determined regions of the protein needed for transactivation and for recruitment to the chromatin. Interestingly, although ZFX has 13 grouped zinc fingers, deletion of the first 11 fingers produces a protein that can still bind to chromatin and activate transcription. We next used TurboID-MS to detect ZFX-interacting proteins, identifying ZNF593, as well as proteins that interact with the N-terminal transactivation domain (which included histone modifying proteins), and proteins that interact with ZFX when it is bound to the chromatin (which included TAFs and other histone modifying proteins). Our studies support a model in which ZFX enhances elongation at target promoters by recruiting H4 acetylation complexes and reducing pausing.

Selective regulation of lymphopoiesis and leukemogenesis by individual zinc fingers of Ikaros.

C2H2 zinc fingers are found in several key transcriptional regulators in the immune system. However, these proteins usually contain more fingers than are needed for sequence-specific DNA binding, which suggests that different fingers regulate different genes and functions. Here we found that mice lacking finger 1 or finger 4 of Ikaros exhibited distinct subsets of the hematological defects of Ikaros-null mice. Most notably, the two fingers controlled different stages of lymphopoiesis, and finger 4 was selectively required for tumor suppression. The distinct defects support the hypothesis that only a small number of genes that are targets of Ikaros are critical for each of its biological functions. The subcategorization of functions and target genes by mutagenesis of individual zinc fingers will facilitate efforts to understand how zinc-finger transcription factors regulate development, immunity and disease.

TENET 2.0: Identification of key transcriptional regulators and enhancers in lung adenocarcinoma.

Lung cancer is the leading cause of cancer-related death and lung adenocarcinoma is its most common subtype. Although genetic alterations have been identified as drivers in subsets of lung adenocarcinoma, they do not fully explain tumor development. Epigenetic alterations have been implicated in the pathogenesis of tumors. To identify epigenetic alterations driving lung adenocarcinoma, we used an improved version of the Tracing Enhancer Networks using Epigenetic Traits method (TENET 2.0) in primary normal lung and lung adenocarcinoma cells. We found over 32,000 enhancers that appear differentially activated between normal lung and lung adenocarcinoma. Among the identified transcriptional regulators inactivated in lung adenocarcinoma vs. normal lung, NKX2-1 was linked to a large number of silenced enhancers. Among the activated transcriptional regulators identified, CENPA, FOXM1, and MYBL2 were linked to numerous cancer-specific enhancers. High expression of CENPA, FOXM1, and MYBL2 is particularly observed in a subgroup of lung adenocarcinomas and is associated with poor patient survival. Notably, CENPA, FOXM1, and MYBL2 are also key regulators of cancer-specific enhancers in breast adenocarcinoma of the basal subtype, but they are associated with distinct sets of activated enhancers. We identified individual lung adenocarcinoma enhancers linked to CENPA, FOXM1, or MYBL2 that were associated with poor patient survival. Knockdown experiments of FOXM1 and MYBL2 suggest that these factors regulate genes involved in controlling cell cycle progression and cell division. For example, we found that expression of TK1, a potential target gene of a MYBL2-linked enhancer, is associated with poor patient survival. Identification and characterization of key transcriptional regulators and associated enhancers in lung adenocarcinoma provides important insights into the deregulation of lung adenocarcinoma epigenomes, highlighting novel potential targets for clinical intervention.

Characterization of the ZFX family of transcription factors that bind downstream of the start site of CpG island promoters.

Our study focuses on a family of ubiquitously expressed human C2H2 zinc finger proteins comprised of ZFX, ZFY and ZNF711. Although their protein structure suggests that ZFX, ZFY and ZNF711 are transcriptional regulators, the mechanisms by which they influence transcription have not yet been elucidated. We used CRISPR-mediated deletion to create bi-allelic knockouts of ZFX and/or ZNF711 in female HEK293T cells (which naturally lack ZFY). We found that loss of either ZFX or ZNF711 reduced cell growth and that the double knockout cells have major defects in proliferation. RNA-seq analysis revealed that thousands of genes showed altered expression in the double knockout clones, suggesting that these TFs are critical regulators of the transcriptome. To gain insight into how these TFs regulate transcription, we created mutant ZFX proteins and analyzed them for DNA binding and transactivation capability. We found that zinc fingers 11-13 are necessary and sufficient for DNA binding and, in combination with the N terminal region, constitute a functional transactivator. Our functional analyses of the ZFX family provides important new insights into transcriptional regulation in human cells by members of the large, but under-studied family of C2H2 zinc finger proteins.

ZFX acts as a transcriptional activator in multiple types of human tumors by binding downstream of transcription start sites at the majority of CpG island promoters.

High expression of the transcription factor ZFX is correlated with proliferation, tumorigenesis, and patient survival in multiple types of human cancers. However, the mechanism by which ZFX influences transcriptional regulation has not been determined. We performed ChIP-seq in four cancer cell lines (representing kidney, colon, prostate, and breast cancers) to identify ZFX binding sites throughout the human genome. We identified ~9,000 ZFX binding sites and found that the majority of the sites are in CpG island promoters. Moreover, genes with promoters bound by ZFX are expressed at higher levels than genes with promoters not bound by ZFX. To determine if ZFX contributes to regulation of the promoters to which it is bound, we performed RNA-seq analysis after knockdown of ZFX by siRNA in prostate and breast cancer cells. Many genes with promoters bound by ZFX were downregulated upon ZFX knockdown, supporting the hypothesis that ZFX acts as a transcriptional activator. Surprisingly, ZFX binds at +240 bp downstream of the TSS of the responsive promoters. Using Nucleosome Occupancy and Methylome Sequencing (NOMe-seq), we show that ZFX binds between the open chromatin region at the TSS and the first downstream nucleosome, suggesting that ZFX may play a critical role in promoter architecture. We have also shown that a closely related zinc finger protein ZNF711 has a similar binding pattern at CpG island promoters, but ZNF711 may play a subordinate role to ZFX. This functional characterization of ZFX provides important new insights into transcription, chromatin structure, and the regulation of the cancer transcriptome.

Three-dimensional analysis reveals altered chromatin interaction by enhancer inhibitors harbors TCF7L2-regulated cancer gene signature.

Distal regulatory elements influence the activity of gene promoters through chromatin looping. Chromosome conformation capture (3C) methods permit identification of chromatin contacts across different regions of the genome. However, due to limitations in the resolution of these methods, the detection of functional chromatin interactions remains a challenge. In the current study, we employ an integrated approach to define and characterize the functional chromatin contacts of human pancreatic cancer cells. We applied tethered chromatin capture to define classes of chromatin domains on a genome-wide scale. We identified three types of structural domains (topologically associated, boundary, and gap) and investigated the functional relationships of these domains with respect to chromatin state and gene expression. We uncovered six distinct sub-domains associated with epigenetic states. Interestingly, specific epigenetically active domains are sensitive to treatment with histone acetyltransferase (HAT) inhibitors and decrease in H3K27 acetylation levels. To examine whether the subdomains that change upon drug treatment are functionally linked to transcription factor regulation, we compared TCF7L2 chromatin binding and gene regulation to HAT inhibition. We identified a subset of coding RNA genes that together can stratify pancreatic cancer patients into distinct survival groups. Overall, this study describes a process to evaluate the functional features of chromosome architecture and reveals the impact of epigenetic inhibitors on chromosome architecture and identifies genes that may provide insight into disease outcome.

L3MBTL2 protein acts in concert with PcG protein-mediated monoubiquitination of H2A to establish a repressive chromatin structure.

We have identified human MBT domain-containing protein L3MBTL2 as an integral component of a protein complex that we termed Polycomb repressive complex 1 (PRC1)-like 4 (PRC1L4), given the copresence of PcG proteins RING1, RING2, and PCGF6/MBLR. PRC1L4 also contained E2F6 and CBX3/HP1γ, known to function in transcriptional repression. PRC1L4-mediated repression necessitated L3MBTL2 that compacted chromatin in a histone modification-independent manner. Genome-wide location analyses identified several hundred genes simultaneously bound by L3MBTL2 and E2F6, preferentially around transcriptional start sites that exhibited little overlap with those targeted by other E2Fs or by L3MBTL1, another MBT domain-containing protein that interacts with RB1. L3MBTL2-specific RNAi resulted in increased expression of target genes that exhibited a significant reduction in H2A lysine 119 monoubiquitination. Our findings highlight a PcG/MBT collaboration that attains repressive chromatin without entailing histone lysine methylation marks.

dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression.

Distinct epigenomic profiles of histone marks have been associated with gene expression, but questions regarding the causal relationship remain. Here we investigated the activity of a broad collection of genomically targeted epigenetic regulators that could write epigenetic marks associated with a repressed chromatin state (G9A, SUV39H1, Krüppel-associated box (KRAB), DNMT3A as well as the first targetable versions of Ezh2 and Friend of GATA-1 (FOG1)). dCas9 fusions produced target gene repression over a range of 0- to 10-fold that varied by locus and cell type. dCpf1 fusions were unable to repress gene expression. The most persistent gene repression required the action of several effector domains; however, KRAB-dCas9 did not contribute to persistence in contrast to previous reports. A 'direct tethering' strategy attaching the Ezh2 methyltransferase enzyme to dCas9, as well as a 'recruitment' strategy attaching the N-terminal 45 residues of FOG1 to dCas9 to recruit the endogenous nucleosome remodeling and deacetylase complex, were both successful in targeted deposition of H3K27me3. Surprisingly, however, repression was not correlated with deposition of either H3K9me3 or H3K27me3. Our results suggest that so-called repressive histone modifications are not sufficient for gene repression. The easily programmable dCas9 toolkit allowed precise control of epigenetic information and dissection of the relationship between the epigenome and gene regulation.