SPIDR: a highly multiplexed method for mapping RNA-protein interactions uncovers a potential mechanism for selective translational suppression upon cellular stress.

RNA binding proteins (RBPs) play crucial roles in regulating every stage of the mRNA life cycle and mediating non-coding RNA functions. Despite their importance, the specific roles of most RBPs remain unexplored because we do not know what specific RNAs most RBPs bind. Current methods, such as crosslinking and immunoprecipitation followed by sequencing (CLIP-seq), have expanded our knowledge of RBP-RNA interactions but are generally limited by their ability to map only one RBP at a time. To address this limitation, we developed SPIDR (Split and Pool Identification of RBP targets), a massively multiplexed method to simultaneously profile global RNA binding sites of dozens to hundreds of RBPs in a single experiment. SPIDR employs split-pool barcoding coupled with antibody-bead barcoding to increase the throughput of current CLIP methods by two orders of magnitude. SPIDR reliably identifies precise, single-nucleotide RNA binding sites for diverse classes of RBPs simultaneously. Using SPIDR, we explored changes in RBP binding upon mTOR inhibition and identified that 4EBP1 acts as a dynamic RBP that selectively binds to 5'-untranslated regions of specific translationally repressed mRNAs only upon mTOR inhibition. This observation provides a potential mechanism to explain the specificity of translational regulation controlled by mTOR signaling. SPIDR has the potential to revolutionize our understanding of RNA biology and both transcriptional and post-transcriptional gene regulation by enabling rapid, de novo discovery of RNA-protein interactions at an unprecedented scale.

ChIP-DIP: A multiplexed method for mapping hundreds of proteins to DNA uncovers diverse regulatory elements controlling gene expression.

Gene expression is controlled by the dynamic localization of thousands of distinct regulatory proteins to precise regions of DNA. Understanding this cell-type specific process has been a goal of molecular biology for decades yet remains challenging because most current DNA-protein mapping methods study one protein at a time. To overcome this, we developed ChIP-DIP (ChIP Done In Parallel), a split-pool based method that enables simultaneous, genome-wide mapping of hundreds of diverse regulatory proteins in a single experiment. We demonstrate that ChIP-DIP generates highly accurate maps for all classes of DNA-associated proteins, including histone modifications, chromatin regulators, transcription factors, and RNA Polymerases. Using these data, we explore quantitative combinations of protein localization on genomic DNA to define distinct classes of regulatory elements and their functional activity. Our data demonstrate that ChIP-DIP enables the generation of 'consortium level', context-specific protein localization maps within any molecular biology lab.

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.

A high-resolution 3D epigenomic map reveals insights into the creation of the prostate cancer transcriptome.

To better understand the impact of chromatin structure on regulation of the prostate cancer transcriptome, we develop high-resolution chromatin interaction maps in normal and prostate cancer cells using in situ Hi-C. By combining the in situ Hi-C data with active and repressive histone marks, CTCF binding sites, nucleosome-depleted regions, and transcriptome profiling, we identify topologically associating domains (TADs) that change in size and epigenetic states between normal and prostate cancer cells. Moreover, we identify normal and prostate cancer-specific enhancer-promoter loops and involved transcription factors. For example, we show that FOXA1 is enriched in prostate cancer-specific enhancer-promoter loop anchors. We also find that the chromatin structure surrounding the androgen receptor (AR) locus is altered in the prostate cancer cells with many cancer-specific enhancer-promoter loops. This creation of 3D epigenomic maps enables a better understanding of prostate cancer biology and mechanisms of gene regulation.

CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops.

BACKGROUND: Recent genome-wide association studies (GWAS) have identified more than 100 loci associated with increased risk of prostate cancer, most of which are in non-coding regions of the genome. Understanding the function of these non-coding risk loci is critical to elucidate the genetic susceptibility to prostate cancer. RESULTS: We generate genome-wide regulatory element maps and performed genome-wide chromosome confirmation capture assays (in situ Hi-C) in normal and tumorigenic prostate cells. Using this information, we annotate the regulatory potential of 2,181 fine-mapped prostate cancer risk-associated SNPs and predict a set of target genes that are regulated by prostate cancer risk-related H3K27Ac-mediated loops. We next identify prostate cancer risk-associated CTCF sites involved in long-range chromatin loops. We use CRISPR-mediated deletion to remove prostate cancer risk-associated CTCF anchor regions and the CTCF anchor regions looped to the prostate cancer risk-associated CTCF sites, and we observe up to 100-fold increases in expression of genes within the loops when the prostate cancer risk-associated CTCF anchor regions are deleted. CONCLUSIONS: We identify GWAS risk loci involved in long-range loops that function to repress gene expression within chromatin loops. Our studies provide new insights into the genetic susceptibility to prostate cancer.

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.

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.