DNA methylation at transcription start sites blocks TBP binding and transcription onset by RNApolII [ChIPbisSeq]
ABSTRACT: We report the application of ChIP bisulfite sequencing (ChIPbisSeq) to establish the methylation state of DNA bound to RNApol2 phosphorylated in Ser5 (RNAPol2Ser5) in the mouse cortex. We first profiled the RNAPol2Ser5 binding using regular ChIPSeq from 45 million raw read pairs (31 million unique pairs were aligned to the genome). Then we used bisulfite converted DNA immunoprecipitated with an antibody against RNAPol2Ser5 (87 million raw read pairs, 38 million unique read pairs aligned to the genome) to map RNAPol2Ser5 sites and to establish the methylation state at these sites. Examination of methylation state at RNAPol2Ser5 binding sites in the mouse cortex.
Project description:Reward-related memory is an important factor in cocaine seeking. One necessary signaling mechanism for long-term memory formation is the activation of poly(ADP-ribose) polymerase-1 (PARP-1), via poly(ADP-ribosyl)ation. We demonstrate herein that auto-poly(ADP-ribosyl)ation of activated PARP-1 was significantly pronounced during retrieval of cocaine-associated contextual memory, in the central amygdala (CeA) of rats expressing cocaine-conditioned place preference (CPP). Intra-CeA pharmacological and shRNA depletion of PARP-1 activity during cocaine-associated memory retrieval abolished CPP. In contrast, PARP-1 inhibition after memory retrieval did not affect CPP reconsolidation process and subsequent retrievals. Chromatin Immuoprecipitation (ChIP) sequencing revealed that PARP-1 binding in the CeA is highly enriched in genes involved in neuronal signaling. We identified amongst PARP targets in CeA a single gene, yet uncharacterized and encoding a putative transposase inhibitor, at which PARP-1 enrichment dramatically increases during cocaine-associated memory retrieval and positively correlates with CPP. Our findings have important implications for understanding drug-related behaviors, and suggest possible future therapeutic targets for drug abuse. 4 samples, each is pooled central amygdalae tissues collected from 2 rats. Rats were trained for cocaine-conditioned place-preference (CPP), tissues were harvested immediately following cocaine-CPP retrieval. Three groups of rats were used: high cocaine CPP, low cocaine CPP and control saline only trained rats.
Project description:In order to support our research of bladder cancer in human genome, we conducted massively parallel pyrosequencing of mRNAs (RNA-Seq) using normal, paracancerouse and cancerous human bladder tissues. We obtained a total of 30.0 million read pairs from normal, 33.1 million read pairs from paracancerous and 36.5 million read pairs from cancerous. The RNA-Seq data derived from the sample illustrated the differencially expression genes among normal, paracancerous and cancerous bladder tissues of human. 3 samples examined: normal tissue, paracancerous tissue, cancerous tissue.
Project description:ChIP-Seq Analysis of H3K9Ac in pairs of mouse and human samples carrying either the Gfi136S or the GFi136N variants. The objective of the study was to identify the changes in H3K9 acetylation at gene promoters that occur in samples expressing the 36N variant of the Gfi1 gene. 3 pairs of bone-marrow AML samples were obtained from mice where 1 mouse in each pair was homozygous for Gfi136S and 1 heterozygous for Gfi136N, or homozygous for 36N in one case. 2 pairs of AML samples were obtained from human patients were 1 patient was homozygous for Gfi1 36S and one was heterozygous for Gfi1 36N. H3 and H3K9Ac ChIP-Seq was carried out on each sample.
Project description:We report the application of ChIP bisulfite sequencing (ChIPbisSeq) to establish the methylation state of DNA bound to RNApol2 phosphorylated in Ser5 (RNAPol2Ser5) in the mouse cortex. We first profiled the RNAPol2Ser5 binding using regular ChIPSeq from 45 million raw read pairs (31 million unique pairs were aligned to the genome). Then we used bisulfite converted DNA immunoprecipitated with an antibody against RNAPol2Ser5 (87 million raw read pairs, 38 million unique read pairs aligned to the genome) to map RNAPol2Ser5 sites and to establish the methylation state at these sites. Overall design: Examination of methylation state at RNAPol2Ser5 binding sites in the mouse cortex.
Project description:To estimate the reproducibility of standard and micro-scaled H3K4me2 ChIP-Seq assay we performed the genome-wide correlation analysis of H3K4me2 enrichment patterns from 7 independent standard ChIP-Seq assays (2 million D10 cells), and micro-scaled ChIP-Seq 100,000 cells sample –(N=12), 10,000 cells sample–(N=3), and 1000 cell sample – (N=4) Examination of H3K4me2 histone modifications in mouse derived lymphoblastic cell line (D10.G4.1 cells, ATCC).
Project description:Capture Hi-C (CHi-C) is a state-of-the art method for profiling chromosomal interactions involving targeted regions of interest (such as gene promoters) globally and at high resolution. Signal detection in CHi-C data involves a number of statistical challenges that are not observed when using other Hi-C-like techniques. We present a background model, and algorithms for normalisation and multiple testing that are specifically adapted to CHi-C experiments, in which many spatially dispersed regions are captured, such as in Promoter CHi-C. We implement these procedures in CHiCAGO (http://regulatorygenomicsgroup.org/chicago), an open-source package for robust interaction detection in CHi-C. We validate CHiCAGO by showing that promoter-interacting regions detected with this method are enriched for regulatory features and disease-associated SNPs. Three human CHi-C biological replicates were generated (comprising 1, 2and 3 technical replicates). Two mouse CHi-C biological replicates were generated (both comprising three technical replicates) and a mouse Hi-C dataset. The publicly available HiCUP pipeline (doi: 10.12688/f1000research.7334.1) was used to process the raw sequencing reads. This pipeline was used to map the read pairs against the mouse (mm9) and human (hg19) genomes, to filter experimental artefacts (such as circularized reads and re-ligations), and to remove duplicate reads. For the CHi-C data, the resulting BAM files were processed into CHiCAGO input files, retaining only those read pairs that mapped, at least on one end, to a captured bait. CHiCAGO then identified Hi-C restriction fragments interacting, with statistical significant, to captured baits.
Project description:In order to support our research of chronic myeloid leukemia in human genome, we conducted massively parallel pyrosequencing of mRNAs (RNA-seq) using chronic myeloid leukemia blood in early disease. We obtained a total of 17.74 million read pairs from blood in early disease.The RNA-seq data derived from the sample illustrated the expreesion genes in chronic myeloid leukemia blood in early disease of human. 1 sample examined: blood in early disease.
Project description:Latency-associated nuclear antigen (LANA), a multifunctional protein expressed by the Kaposi sarcoma-associated herpesvirus (KSHV) in latently-infected cells, is required for stable maintenance of the viral episome. This is mediated by two interactions: LANA binds to specific sequences (LBS1 and 2) on viral DNA, and also engages host histones, tethering the viral genome to host chromosomes in mitosis. LANA has also been suggested to affect host gene expression, but both the mechanism(s) and role of this dysregulation in KSHV biology remain unclear. Here we have examined LANA interactions with host chromatin on a genome-wide scale using ChIP-seq, and show that LANA predominantly targets human genes near their transcriptional start sites (TSSs). These host LANA-binding sites are generally found within transcriptionally active promoters and display striking overrepresentation of a consensus DNA sequence virtually identical to the LBS1 motif in KSHV DNA. Comparison of the ChIP-seq profile with whole transcriptome (RNA-seq) data reveals that few of the genes that are differentially regulated in latent infection are occupied by LANA at their promoters. This suggests that direct LANA binding to promoters is not the prime determinant of altered host transcription in KSHV-infected cells. Most surprisingly, the association of LANA to both host and viral DNA is strongly disrupted during the lytic cycle of KSHV. This disruption can be prevented by the inhibition of viral DNA synthesis, suggesting the existence of novel and potent regulatory mechanisms linked to either viral DNA replication or late gene expression. Profiling of KSHV LANA positioning on the host genome and examination of gene expression from promoters bound by KSHV LANA.
Project description:ChIP-chip was performed using chromatin isolated from formaldehyde crosslinked, stage 5 embryos, and 8WG16 monoclonal antibody against RNA polymerase II(Covance Inc.). The chromatin immunoprecitation and hybridization for each antibody were carried out in duplicates, and in each chromatin immunoprecitation and hybridization series, two mock IP controls and two input DNA controls were also generated. The microarray used in this study is the Affymetrix Drosophila genomic DNA tiling array, which has about 3 million oligo pairs (perfect match-mismatch), covering the whole euchromatic sequences of the fly genome at a resolution of about 35 bp.
Project description:This data set is part of a study where the genome of Malassezia sympodialis (strain ATCC 42132) was sequenced using long-read technology and annotated using RNA-seq and proteogenomics. RNA was extracted at two different culture times (2 and 4 days). Seven RNA-seq libraries were prepared from independent samples. Two samples (P2 and P3) were enriched for protein-coding RNA using poly(A)-selection. The remaining five samples were processed with RiboMinus to deplete ribosomal RNA, and thus retain both mRNA and non-ribosomal noncoding RNA for sequencing. In total, we obtained 71 million RNA-seq read pairs mapping to genomic regions other than the highly expressed ribosomal loci.