Nuclear RNA-seq in WT, Ring1a KO and Ring1a/b dKO mouse ES cells
ABSTRACT: Nuclear RNA was isolated from all three cell types to enable differential expression analysis of both coding and non-coding RNA shortly after tamoxifen treatment, that resulted in conditional knock-out of Ring1b in the Ring1a -/- background.
Project description:H3K4me1 (ab8895 Abcam) and H3K27ac (ab4729 Abcam) antibodies were used for ChIP-seq in Ring1a-/- mouse ES cells and after 48h tamoxifen treatment in conditional knock-out of Ring1b in the Ring1a -/- background.
Project description:Genome organization influences transcriptional regulation by facilitating interactions between gene promoters and distal regulatory elements. To analyse distal promoter contacts mediated by the PRC1 complex we used Capture Hi-C (CHi-C) to enrich for promoter-interactions in a HiC library in Ring1a KO and Ring1a/b dKO mouse ES cells.
Project description:QuantSeq-Rev method to generate highly strand-specific next-generation sequencing (NGS) libraries enabling transcript quantification and identification of the 3'end of polyadenylated RNAs
Project description:In this experiment, we sought to analyze how the transcriptome of WT, Δ5|6, and Δ5|6:7|9 cells vary during differentiation of ESCs into cervical motor neurons 3 lines (WT, Δ5|6, Δ5|6:7|9)
Project description:Polycomb protein complexes are critical for stem cell renewal and tumorigenesis. Some components of Polycomb repressive complex 2 (PRC2) have been frequently found to be overexpressed in a variety of human cancers and contribute to tumor initiation and development partially by transcriptional silencing of tumor suppressor genes. However, the exact underlying mechanisms still remain elusive. To further characterize PRC2-directed transcriptome and explore its implication in tumorigenesis, here we performed a genome-wide profiling of EZH2-associated, a key component of PRC2, transcriptome in human gastric cancer cell lines by RNA immunoprecipitation sequencing (RIP-seq). We observed that the EZH2-interacting transcripts are distributed in protein-coding, intergenic, intronic and promoter regions and transcribed from both strands. Many transcripts are originated from cancer-related loci and transcribed from active promoters. Particularly, we demonstrated that EZH2-associated lncRNA MALAT1 tethers EZH2 to PCDH10 promoter for its transcriptional repression and contributes to gastric cellular invasion and migration.
Project description:Long noncoding RNAs (lncRNAs) are important regulators of cell fate, and their mis-expression has been implicated in many diseases. While distinct polymerases generate messenger vs. noncoding ribosomal or tRNAs3, little is known about distinct mechanisms controlling lncRNA expression. Here we show that transcription of lncRNAs is quantitatively different from that of messenger RNAs (mRNAs)--as revealed by deficiency of Dicer (Dcr), a key ribonuclease that generates microRNAs (miRNAs). Loss of Dcr in mouse embryonic stem cells (mESCs) led surprisingly to decreased level of the majority of lncRNAs. The canonical Dgcr8-Dcr-miRNA pathway is required for robust lncRNA expression, at the level of transcriptional initiation and elongation of lncRNA genes rather than at the level of their stability. cMyc, an oncogenic transcription factor, whose expression is indirectly regulated by Dcr-miRNA in mESCs, is partly responsible for lncRNA transcription. Loss of cMyc led to a more dramatic decrease of lncRNAs than mRNAs, and cMyc overexpression rescues lncRNA expression in Dcr KO cells. A quantitative metric of “mRNA-lncRNA decoupling” revealed that Dcr and cMyc differentially regulate lncRNAs vs. mRNAs in diverse cell types and in vivo, as evidenced by hundreds of microarray experiments. Thus, Dcr and cMyc may allow numerous lncRNAs to be activated or deactivated as a class, implicating lncRNAs to potential regulatory roles in development and disease states where Dcr and cMyc have been associated with. RNA was sequenced from WT and Dcr KO mESCs and the expression of lncRNAs and mRNAs are compared between WT and Dcr KO mESCs.
Project description:The histone variant macroH2A1 and the poly(ADP-ribose) polymerase PARP-1 both regulate gene transcription by modulating chromatin structure and function. Of the two macroH2A1 splice variants, macroH2A1.1 and macroH2A1.2, the former is often suppressed in cancer and has the unique ability to interact with poly(ADP-ribose). Using ChIP-seq in primary lung fibroblasts, we demonstrate that macroH2A1 is incorporated into either of two spatially and functionally distinct types of chromatin; the first is marked by H3 K27 trimethylation, while the second contains a set of nine histone acetylations. MacroH2A1-regulated genes are involved in cancer progression are specifically found in macroH2A1-containing acetylated chromatin. Through the recruitment of PARP-1, macroH2A1.1 promotes the acetylation of H2B K12 and K120 which plays a key role in the regulation of macroH2A1 target genes in primary cells. The macroH2A1/PARP-1 pathway regulating H2B K12 and K120 acetylation is disrupted in cancer cells, in part, explaining macroH2A1’s role in cancer suppression. Three biological replicates of RNA-seq from cells expressing shRNA directed against macroH2A1 or luciferase as a control
Project description:The chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on the prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to the recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. Genetic ablation of catalytic subunit of the PRC1 complex (RINGA/B) and ChIP-seq analysis of PRC1 and PRC2 components confirmed genome-wide decreases in PRC2 occupancy and H3K27me3 levels at PRC target sites. This activity is restricted to variant PRC1 complexes and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for polycomb domain formation and normal development. Together these observations provide a surprising new PRC1-dependent logic for PRC2 occupancy and polycomb domain formation. RING1A-/-;RING1Bfl/fl ES cells were treated with 800µM tamoxifen for 48hours and compared to untreated control cells by ChIP-seq for RING1B, SUZ12, EZH2 and H3K27me3.