Abstract Background DNA methylation and repressive histone modifications cooperate to silence promoters. One mechanism by which regions of methylated DNA could acquire repressive histone modifications is via methyl DNA-binding transcription factors. The zinc finger protein ZBTB33 (also known as Kaiso) has been shown in vitro to bind preferentially to methylated DNA and to interact with the SMRT/NCoR histone deacetylase complexes. We have performed bioinformatic analyses of Kaiso ChIP-seq and DNA methylation datasets to test a model whereby binding of Kaiso to methylated CpGs leads to loss of acetylated histones at target promoters. Results Our results suggest that, contrary to expectations, Kaiso does not bind to methylated DNA in vivo but instead binds to highly active promoters that are marked with high levels of acetylated histones. In addition, our studies suggest that DNA methylation and nucleosome occupancy patterns restrict access of Kaiso to potential binding sites and influence cell type-specific binding. Conclusions We propose a new model for the genome-wide binding and function of Kaiso whereby Kaiso binds to unmethylated regulatory regions and contributes to the active state of target promoters.

It has been proposed that ZNF217, which is amplified at 20q13 in various tumors, plays a key role during neoplastic transformation. ZNF217 has been purified in complexes that contain repressor proteins such as CtBP2, suggesting that it acts as a transcriptional repressor. However, the function of ZNF217 has not been well characterized due to a lack of known target genes. Using a global chromatin immunoprecipitation (ChIP)-chip approach, we identified thousands of ZNF217 binding sites in three tumor cell lines (MCF7, SW480, and Ntera2). Further analysis of ZNF217 in Ntera2 cells showed that many promoters are bound by ZNF217 and CtBP2 and that a subset of these promoters are activated upon removal of ZNF217. Thus, our in vivo studies corroborate the in vitro biochemical analyses of ZNF217-containing complexes and support the hypothesis that ZNF217 functions as a transcriptional repressor. Gene ontology analysis showed that ZNF217 targets in Ntera2 cells are involved in organ development, suggesting that one function of ZNF217 may be to repress differentiation. Accordingly we show that differentiation of Ntera2 cells with retinoic acid led to down-regulation of ZNF217. Our identification of thousands of ZNF217 target genes will enable further studies of the consequences of aberrant expression of ZNF217 during neoplastic transformation.

Background

The ZNF217 gene, encoding a C2H2 zinc finger protein, is located at 20q13 and found amplified and overexpressed in greater than 20% of breast tumors. Current studies indicate ZNF217 drives tumorigenesis, yet the regulatory mechanisms of ZNF217 are largely unknown. Because ZNF217 associates with chromatin modifying enzymes, we postulate that ZNF217 functions to regulate specific gene signaling networks. Here, we present a large-scale functional genomic analysis of ZNF217, which provides insights into the regulatory role of ZNF217 in MCF7 breast cancer cells.

Results

ChIP-seq analysis reveals that the majority of ZNF217 binding sites are located at distal regulatory regions associated with the chromatin marks H3K27ac and H3K4me1. Analysis of ChIP-seq transcription factor binding sites shows clustering of ZNF217 with FOXA1, GATA3 and ERalpha binding sites, supported by the enrichment of corresponding motifs for the ERalpha-associated cis-regulatory sequences. ERalpha expression highly correlates with ZNF217 in lysates from breast tumors (n = 15), and ERalpha co-precipitates ZNF217 and its binding partner CtBP2 from nuclear extracts. Transcriptome profiling following ZNF217 depletion identifies differentially expressed genes co-bound by ZNF217 and ERalpha; gene ontology suggests a role for ZNF217-ERalpha in expression programs associated with ER+ breast cancer studies found in the Molecular Signature Database. Data-mining of expression data from breast cancer patients correlates ZNF217 with reduced overall survival.

Conclusions

Our genome-wide ZNF217 data suggests a functional role for ZNF217 at ERalpha target genes. Future studies will investigate whether ZNF217 expression contributes to aberrant ERalpha regulatory events in ER+ breast cancer and hormone resistance.

Abstract Background The orphan nuclear receptor TR4 (human testicular receptor 4 or NR2C2) plays a pivotal role in a variety of biological and metabolic processes. With no known ligand and few known target genes, the mode of TR4 function was unclear. Results We report the first genome-wide identification and characterization of TR4 in vivo binding. Using chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq), we identified TR4 binding sites in 4 different human cell types and found that the majority of target genes were shared among different cells. TR4 target genes are involved in fundamental biological processes such as RNA metabolism and protein translation. In addition, we found that a subset of TR4 target genes exerts cell-type specific functions. Analysis of the TR4 binding sites revealed that less than 30% of the peaks from any of the cell types contained the DR1 motif previously derived from in vitro studies, suggesting that TR4 may be recruited to the genome via interaction with other proteins. A bioinformatics analysis of the TR4 binding sites predicted a cis regulatory module involving TR4 and ETS transcription factors. To test this prediction, we performed ChIP-seq for the ETS factor ELK4 and found that 30% of TR4 binding sites were also bound by ELK4. Motif analysis of the sites bound by both factors revealed a lack of the DR1 element, suggesting that TR4 binding at a subset of sites is facilitated through the ETS transcription factor ELK4. Further studies will be required to investigate the functional interdependence of these two factors. Conclusions Our data suggest that TR4 plays a pivotal role in fundamental biological processes across different cell types. In addition, the identification of cell type specific TR4 binding sites enables future studies of the pathways underlying TR4 action and its possible role in metabolic diseases.

Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with increased risk for colorectal cancer (CRC). A molecular understanding of the functional consequences of this genetic variation is complicated because most GWAS SNPs are located in non-coding regions. We used epigenomic information to identify H3K27Ac peaks in HCT116 colon cancer cells that harbor SNPs associated with an increased risk for CRC. Employing CRISPR/Cas9 nucleases, we deleted a CRC risk-associated H3K27Ac peak from HCT116 cells and observed large-scale changes in gene expression, resulting in decreased expression of many nearby genes. As a comparison, we showed that deletion of a robust H3K27Ac peak not associated with CRC had minimal effects on the transcriptome. Interestingly, although there is no H3K27Ac peak in HEK293 cells in the E7 region, deletion of this region in HEK293 cells decreased expression of several of the same genes that were downregulated in HCT116 cells, including the MYC oncogene. Accordingly, deletion of E7 causes changes in cell culture assays in HCT116 and HEK293 cells. In summary, we show that effects on the transcriptome upon deletion of a distal regulatory element cannot be predicted by the size or presence of an H3K27Ac peak.

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.

Biologists possess the detailed knowledge critical for extracting biological insight from genome-wide data resources, and yet they are increasingly faced with nontrivial computational analysis challenges posed by genome-scale methodologies. To lower this computational barrier, particularly in the early data exploration phases, we have developed an interactive pattern discovery and visualization approach, Spark, designed with epigenomic data in mind. Here we demonstrate Spark's ability to reveal both known and novel epigenetic signatures, including a previously unappreciated binding association between the YY1 transcription factor and the corepressor CTBP2 in human embryonic stem cells.

Next-generation sequencing is revolutionizing the identification of transcription factor binding sites throughout the human genome. However, the bioinformatics analysis of large datasets collected using chromatin immunoprecipitation and high-throughput sequencing is often a roadblock that impedes researchers in their attempts to gain biological insights from their experiments. We have developed integrated peak-calling and analysis software (Sole-Search) which is available through a user-friendly interface and (i) converts raw data into a format for visualization on a genome browser, (ii) outputs ranked peak locations using a statistically based method that overcomes the significant problem of false positives, (iii) identifies the gene nearest to each peak, (iv) classifies the location of each peak relative to gene structure, (v) provides information such as the number of binding sites per chromosome and per gene and (vi) allows the user to determine overlap between two different experiments. In addition, the program performs an analysis of amplified and deleted regions of the input genome. This software is web-based and automated, allowing easy and immediate access to all investigators. We demonstrate the utility of our software by collecting, analyzing and comparing ChIP-seq data for six different human transcription factors/cell line combinations.

Background

Rewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems.

Results

Here, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9.

Conclusions

These data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type.

The H3K9me3 histone modification is often found at promoter regions, where it functions to repress transcription. However, we have previously shown that 3' exons of zinc finger genes (ZNFs) are marked by high levels of H3K9me3. We have now further investigated this unusual location for H3K9me3 in ZNF genes. Neither bioinformatic nor experimental approaches support the hypothesis that the 3' exons of ZNFs are promoters. We further characterized the histone modifications at the 3' ZNF exons and found that these regions also contain H3K36me3, a mark of transcriptional elongation. A genome-wide analysis of ChIP-seq data revealed that ZNFs constitute the majority of genes that have high levels of both H3K9me3 and H3K36me3. These results suggested the possibility that the ZNF genes may be imprinted, with one allele transcribed and one allele repressed. To test the hypothesis that the contradictory modifications are due to imprinting, we used a SNP analysis of RNA-seq data to demonstrate that both alleles of certain ZNF genes having H3K9me3 and H3K36me3 are transcribed. We next analyzed isolated ZNF 3' exons using stably integrated episomes. We found that although the H3K36me3 mark was lost when the 3' ZNF exon was removed from its natural genomic location, the isolated ZNF 3' exons retained the H3K9me3 mark. Thus, the H3K9me3 mark at ZNF 3' exons does not impede transcription and it is regulated independently of the H3K36me3 mark. Finally, we demonstrate a strong relationship between the number of tandemly repeated domains in the 3' exons and the H3K9me3 mark. We suggest that the H3K9me3 at ZNF 3' exons may function to protect the genome from inappropriate recombination rather than to regulate transcription.