Project description:ETS1 and RAS/ERK regulate a common gene expression program in establishing enviroment suitable for prostate cancer cell migration. ChIP-sequencing of various transcription factors
Project description:Knockdowns of c-JUN and JUND had opposite effects on PC3 prostate cell migration. We predicted that c-JUN and JUND control the same set of cell migration genes, but in opposite directions. To test this hypothesis, mRNA with expression changes in c-JUN and JUND knockdown PC3 cell lines were compared to mRNA levels in control (luciferase knockdown) PC3 cells by RNA-seq. mRNA profiles of luciferase knockdown (WT), c-Jun knockdown, and Jun-D knockdown in PC3 cells were generated using deep sequencing, in triplicate, using Illumina HiSeq. Knockdowns were stable shRNA expression from a lentiviral construct selected with puromycin.
Project description:ETS1 and RAS/ERK regulate a common gene expression program in establishing enviroment suitable for prostate cancer cell migration. mRNA profiles of luciferase knockdown (WT), ETS1 knockdown, and U0126 treated DU145 cells were generated using deep sequencing, in triplicate, using Illumina HiSeq. Knockdowns were stable shRNA expression from a lentiviral construct selected with puromycin.
Project description:Derailed gene expression programs within the developing nervous system, encompassing both transcriptional and posttranscriptional processes, can cause diverse neurodevelopmental diseases (NDD). The NDD FOXG1-syndrome lacks full understanding of the mechanistic role of its eponymous gene product. While it is known that FOXG1 acts in part at the chromatin by binding to regulative regions, it is unclear what factors control its presence at specific sites. Long non-coding RNAs (lncRNAs) can mediate site-directed transcription factor binding, but their potential role in FOXG1-syndrome has not been described. Here, we show that FOXG1 localisation is regulated at selected loci through the lncRNA Pantr1. We identified FOXG1 as an upstream transcriptional activator of Pantr1 in human and mice. Further, we discovered that FOXG1 has the ability to associate with RNAs. Both, transcriptional regulation of Pantr1 by FOXG1 and association of both partners, build up a regulative network that impacts the localisation of FOXG1 at selected genomic loci. Specifically, Pantr1 facilitates cooperative presence of FOXG1/NEUROD1 at specific sites, and Pantr1 reduction leads to redistribution of FOXG1 to comparably more generic binding sites. The rescue of impaired dendritic outgrowth upon FOXG1 reduction by simultaneous overexpression of Pantr1 underlines the importance of the FOXG1/Pantr1 regulative network.
Project description:Genome-wide maps of nucleosome organization in human CD4+ T-cells, CD8+ T-cells, granulocytes, and from in vitro reconstitution. 8 samples include: nucleosome-bound DNA isolated from primary human CD4+ T-cells, nucleosome bound-DNA isolated from primary human CD8+ T-cells, nucleosome-bound DNA isolated from primary human granulocytes, nucleosome-bound DNA isoalted from in vitro reconstitution of nucleosomes with human genomic DNA, control DNA generated by micrococcal nuclease treatment of human genomic DNA, RNA-seq from human CD4+ T-cells, RNA-seq from human CD8+ T-cells, RNA-seq from human Granulocytes.
Project description:A Parallel Analysis of six mouse tissues to investigate cleavage products and degradome Survey of polyA transcripts having 5' monophosphates (a characteristic of RNA cleavage events) in six mouse tissues.
Project description:Histone methyltransferases (HMTases), as chromatin modifiers, regulate the transcriptomic landscape in normal development as well in diseases such as cancer. Here, we molecularly order two HMTases, EZH2 and MMSET that have established genetic links to oncogenesis. EZH2, which mediates histone H3K27 trimethylation and is associated with gene silencing, was shown to be coordinately expressed and function upstream of MMSET, which mediates H3K36 dimethylation and is associated with active transcription. We found that the EZH2-MMSET HMTase axis is coordinated by a microRNA network and that the oncogenic functions of EZH2 require MMSET activity. Together, these results suggest that the EZH2-MMSET HMTase axis coordinately functions as a master regulator of transcriptional repression, activation, and oncogenesis and may represent an attractive therapeutic target in cancer. Examination of H3K36me2 mark in control and stable EZH2 knockdown cells
Project description:Background: Adenosine deaminases that act on RNA (ADARs) bind to double-stranded and structured RNAs and deaminate adenosines to inosines. This A to I editing is widespread and required for normal life and development. Besides mRNAs and repetitive elements, ADARs can target miRNA precursors. Editing of miRNA precursors can affect processing efficiency and alter target specificity. Interestingly, ADARs can also influence miRNA abundance independent of RNA-editing. In mouse embryos where editing levels are low, ADAR2 was found to be the major ADAR protein that affects miRNA abundance. Here we extend our analysis to adult mouse brains where high editing levels are observed. Results: Using Illumina deep sequencing we compare the abundances of mature miRNAs and editing events within them, between wild-type and ADAR2 knockout mice in the adult mouse brain. Reproducible changes in abundance of specific miRNAs are observed in ADAR2 deficient mice. Most of these quantitative changes seem unrelated to A to I editing events. However, many A to G transitions in cDNAs prepared from mature miRNA sequences, reflecting A to I editing events in the RNA, are observed with frequencies reaching up to 80%. About half of these editing events are primarily caused by ADAR2 while a few miRNAs show increased editing in the absence of ADAR2, suggesting preferential editing by ADAR1. Moreover, novel, previously unknown editing events were identified in several miRNAs. In general 64% of all editing events are located within the seed region of mature miRNAs. In one of these cases retargeting of the edited miRNA could be verified in reporter assays. Also, altered processing efficiency upon editing near a processing site could be experimentally verified. Conclusions: ADAR2 can significantly influence the abundance of certain miRNAs in the brain. Only in a few cases changes in miRNA abundance can be explained by miRNA editing. Thus, ADAR2 binding to miRNA precursors, without editing them, may influence their processing and thereby abundance. ADAR1 and ADAR2 have both overlapping and distinct specificities for editing of miRNA editing sites. Over 60% of editing occurs in the seed region possibly changing target specificities for many edited miRNAs. Examination of the effect of ADAR2 on mature miRNA abundance and sequence in adult mouse brain.
Project description:Polycomb Repressive Complexes 1 and 2 (PRC1 and 2) play a critical role in the epigenetic regulation of transcription during cellular differentiation, stem cell pluripotency, and neoplastic progression1-3. Here we show that the Polycomb group protein EED, a core component of PRC2, physically interacts with and functions as part of the PRC1 complex. Components of PRC1 and PRC2 compete for EED binding. EED functions to recruit PRC1 to H3K27me3 loci and enhances PRC1 mediated H2A ubiquitin E3 ligase activity. Taken together, we uncover the integral role of EED as an epigenetic exchange factor coordinating the activities of PRC1 and 2. EED, uH2A, RING1A, RING1B, BMI1 and H3K27Me3 ChIP-seq in EED stable knockdown and control Scramble DU145 prostate cancer cell line
Project description:Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single cell Hi-C, combined with genome-wide statistical analysis and structural modeling of single copy X chromosomes, to show that individual chromosomes maintain domain organisation at the megabase scale, but show variable cell-to-cell chromosome territory structures at larger scales. Despite this structural stochasticity, localisation of active gene domains to boundaries of territories is a hallmark of chromosomal conformation, affecting most domains in most nuclei. Single cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organisation underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns. Mouse Th1 single-cell Hi-C maps were produced and paired-end sequenced. 10 single-cell samples and a multi-sample pool together with a population Hi-C sample are included.