Project description:We performed a CRISPR-based functional genetic screen targeting CTCF binding elements (CBEs) located in the vicinity of ERα-bound enhancers. The screen identified four functional CBEs whose targeting resulted in a marked negative effect on the proliferation of MCF-7 cells (ERα-positive cells) but no significant effect on MDA-MB-231 (ERα-negative cells) cells. We then carried out RNA-seq analysis on MCF-7 cells induced with sgRNA vectors targeting these four CBEs: sgRNA_1118, sgRNA_1659, sgRNA_680 and sgRNA_810, as well as non-targeting sgRNA as control.
Project description:We performed a CRISPR-based functional genetic screen targeting CTCF binding elements (CBEs) located in the vicinity of ERα-bound enhancers. The screen identified four functional CBEs whose targeting resulted in a marked negative effect on the proliferation of MCF-7 cells (ERα-positive cells) but no significant effect on MDA-MB-231 (ERα-negative cells) cells. Here, we carried out GRO-seq analysis on MCF-7 cells induced with an sgRNA vector targeting one of the hits detected by the screen: sgRNA_1118, as well as non-targeting sgRNA and sg1830 as control.
Project description:We report the high-throughput profiling of histone modifications, CTCF and HP1a binding sites in MCF7 breast cancer cells. ChIP-chip experiments were performed using the Agilent Human Genome CGH Microarray 1x1M. Regulatory markers H3K4Me1, H3K4Me3, H3K4Ac, H3K9Ac, CTCF are known to be positively correlated with gene expression, and H3K9Me2, H3K27Me3 and HP1a are negative markers. Together with MCF7 methylation data, we showed hypomethylated promoters are significantly enriched with positive regulatory elements, and lacks repressive markers.
Project description:ChIP-Seq study in human MCF7 and HEPG2 cells using antibodies against CTCF (Millipore, 07-729), STAG1 (abcam, ab4457), RAD21 (abcam, ab992), ERa (santa cruz, sc-543), CEBPa (santa cruz, sc-9314)
Project description:We performed a CRISPR-based functional genetic screen targeting TEAD4 binding motif located within the YAP-bound enhancers. The screen identified seven functional enhancers whose targeting resulted in a marked negative effect on the proliferation of MCF10A-YAP-5SA cells (overexpressed constrictively activated YAP) but no significant effect on MCF10A (parental control) cells. We then carried out RNA-seq analysis on MCF10A-YAP-5SA cells transduced with sgRNA vectors targeting these seven enhancers (enhancers A, B, C, D, E, F, G) as well as the non-targeting sgRNA as control (NT).
Project description:Analysis of the expression profiles of MCF7 cells transduced with a control shRNA and an TSC2-targeted shRNA (leading to tuberin depletion). MCF7 cells were cultured in standard conditions, transduced, selected with puromycin (pLKO.1 shRNA vectors) and processed for expression profiling.
Project description:The transcription factor CTCF appears indispensable in defining topologically associated domain boundaries and maintaining chromatin loop structures within these domains, supported by numerous functional studies. However, acute depletion of CTCF globally reduces chromatin interactions but does not significantly alter transcription. Here we systematically integrated multi-omics data including ATAC-seq, RNA-seq, WGBS, Hi-C, Cut&Run, CRISPR-Cas9 survival dropout screening, time-solved deep proteomic and phosphoproteomic analyses in cells carrying auxin-induced degron at endogenous CTCF locus. Acute CTCF protein degradation markedly rewired genome-wide chromatin accessibility. Increased accessible chromatin regions were largely located adjacent to CTCF-binding sites at promoter regions and insulator sites and were associated with enhanced transcription of nearby genes. In addition, we used CTCF-associated multi-omics data to establish a combinatorial data analysis pipeline to discover CTCF co-regulatory partners in regulating downstream gene expression. We successfully identified 40 candidates, including multiple established partners (i.e., MYC) supported by all layers of evidence. Interestingly, many CTCF co-regulators (e.g., YY1, ZBTB7A) that have evident alterations of respective downstream gene expression do not show changes at their expression levels across the multi-omics measurements upon acute CTCF loss, highlighting the strength of our system to discover hidden co-regulatory partners associated with CTCF-mediated transcription. This study highlights CTCF loss rewires genome-wide chromatin accessibility, which plays a critical role in transcriptional regulation