Genome-wide recruitment profile of Tat protein on cellular genes in HIV-1 infected T-cells
ABSTRACT: HIV-1 Tat is a multifunctional protein, that in addition to its primary function of transactivating viral transcription also tends to modulate cellular gene expression, the molecular mechanism of which remains to be clearly elucidated. We have reported earlier NFκB enhancer binding activity of Tat and proposed its potential role in regulation of cellular gene expression. In the present study, we have analysed genome wide occupancy of Tat protein in HIV-1 infected cells. Our results identify an entire spectrum of binding sites of Tat protein on chromatin and also reveal that Tat is recruited majorly on gene promoters indicating its possible involvement in the regulation of cellular gene expression. Agilent two-color ChIP-on-Chip experiment, Organism: Homo sapiens ,Genotypic Technology designed Custom Human Promoter 244k ChIP-on-chip Array (AMADID-019469)
Project description:The discovery of TET proteins, enzymes that oxidize 5-methylcytosine (5mC) in DNA, has revealed novel mechanisms for the regulation of DNA methylation. We have mapped 5-hydroxymethylcytosine (5hmC) at different stages of T cell development in the thymus and T cell differentiation in the periphery. We show that 5hmC is enriched in the gene body of highly expressed genes at all developmental stages, and that its presence correlates positively with gene expression. Further emphasizing the connection with gene expression, we find that 5hmC is enriched in active thymus-specific enhancers, and that genes encoding key transcriptional regulators display high intragenic 5hmC levels in precursor cells at those developmental stages where they exert a positive effect. Our data constitute a valuable resource that will facilitate detailed analysis of the role of 5hmC in T cell development and differentiation. Examine the distribution of the H3K27Ac in DP T cells. The presence of H3K27Ac in enhancers enable us to distinguish poised(H3Kme1 enriched, but devoid of H3K27Ac) versus active enhancers (enriched for H3K4me1 and H3K27Ac).
Project description:Transcription factors mediate precise regulation of gene expression programs to modulate key biological processes. In addition to controlling HIV transcription, Tat appears to modulate cellular transcription to alter the biology of target immune cells and generate a permissive state for HIV infection. However, the molecular mechanisms of transcriptional control have remained elusive. Here, we identified the direct target genes of Tat using genomics and defined mechanisms by which Tat selectively rewires cellular transcriptional programs. Interestingly, Tat functions as both transcriptional activator and repressor of a defined set of genes sharing functional annotations and regulated by master transcriptional regulators such as T-cell identity factors. Tat is recruited to precise genomic domains (promoters and intragenic enhancers) through interaction with master transcriptional regulators to control both the transcription initiation and elongation steps. Tat mediates transcription initiation by modulating Pol II recruitment to promoters and intragenic enhancers and fine-tuning chromatin looping. Tat stimulates or blocks RNA Polymerase (Pol) II recruitment or promoter-proximal pause release thereby promoting gene activation or repression, respectively. Global analysis of chromatin signatures revealed correlation of transcription activity marks with Pol II recruitment or pause release status at gene promoters and enhancers, and transcription elongation at coding units. We propose that Tat has evolved these unique properties to hijack precise genomic domains to control cellular transcription using unexpected regulatory mechanisms, which showed marked differences to the regulation of the HIV genome. Our studies also reveal that Tat can be used as a molecular probe to decode general principles of transcriptional regulation. ChIP of various DNA binding proteins
Project description:Turnover and exchange of nucleosomal histones and their variants, a process long believed to be static in post-replicative cells, remains largely unexplored in brain. Here, we describe a novel mechanistic role for HIRA (histone cell cycle regulator) and proteasomal degradation associated histone dynamics in the regulation of activity-dependent transcription, synaptic connectivity and behavior. We uncover a dramatic developmental profile of nucleosome occupancy across the lifespan of both rodents and humans, with the histone variant H3.3 accumulating to near saturating levels throughout the neuronal genome by mid-adolescence. Despite such accumulation, H3.3 containing nucleosomes remain highly dynamic–in a modification independent manner–to control neuronal- and glial- specific gene expression patterns throughout life. Manipulating H3.3 dynamics in both embryonic and adult neurons confirmed its essential role in neuronal plasticity and cognition. Our findings establish histone turnover as a critical, and previously undocumented, regulator of cell-type specific transcription and plasticity in mammalian brain. All ChIP-seq samples were generated to test the impact of neuronal activity/adult physiological plasticity on histone turnover in the central nervous system. This was tested in cultured neurons and astrocytes, FACS purified neurons or FACS purified Glia.
Project description:HIV-1 Tat can modulate the expression of both HIV and cellular genes. In antigen-presenting cells Tat induces the expression of a subset of interferon-stimulated genes (ISGs) in the absence of interferons (IFNs). We investigated the genome-wide Tat association with cellular promoters in immature dendritic cells (iDC) and in monocyte derived macrophages (MDM). Chromatin immunoprecipitation (ChIP) of Tat together with chromatin profiling by ChIP-on-chip analysis demonstrated that Tat associates with the MAP2K6 and MAP2K3 promoters and mediates its increase, which also affects the induction of some ISGs. comparison of KG-1 cells expressing tTA control vs Tat or mutant TatSF2G48R57A
Project description:The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in the M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host. ChiP-Seq: M. tuberculosis H37Rv (GC1237) cultures grown in vitro to exponential phase. Two wild type samples plus one isogenic phoP mutant as control
Project description:To investigate how dSETDB1 regulated the genome-wide distribution of HP1 proteins, we performed ChIP-chip assay on the third instar larval lysate from the dSETDB1null mutants in comparison to the wild type. Third instar larvae from the wild type and dSETDB1null mutants were collected. Three independent biological replicates of ChIP with anti-HP1 were performed.
Project description:We characterize the acetylation of H3K122 for the first time. Towards this we mapped the genomic distribution of H3K122Ac, identified the enzyme introducing H3K122Ac, and addressed the functional contribution H3K122Ac to transcription. We found that H3K122Ac is associated with chromatin marks and genomic regions associated with active transcription and is catalysed by p300/CBP and can be regulated by estrogen signaling in MCF-7. Moreover H3K122Ac stimulates transcription as dermined by in vitro transcription assays ChIP seq study
Project description:Whole-exome sequencing studies have implicated chromatin modifiers and transcriptional regulators in autism spectrum disorder (ASD) through the identification of de novo loss of function mutations in affected individuals. Many of these genes are co-expressed in mid-fetal human cortex, suggesting ASD risk genes converge in regulatory networks that are perturbed in ASD during neurodevelopment. To elucidate such networks we mapped promoters and enhancers bound by the chromodomain helicase CHD8, which is strongly enriched in ASD-associated de novo loss of function mutations, using ChIP-seq in mid-fetal human brain, human neural stem cells (hNSCs), and embryonic mouse cortex. We find that CHD8 targets are strongly enriched for ASD risk genes that converge in ASD-associated co-expression networks in human midfetal cortex. CHD8 knockdown in hNSCs results in significant dysregulation of ASD risk genes targeted by CHD8, as well as additional genes important for neurodevelopment, including members of the Wnt/β-catenin signaling pathway. Integration of CHD8 binding data with genetic and gene co-expression data in ASD risk models provides support for additional ASD risk genes. Together, our results suggest that loss of CHD8 function contributes to ASD through regulatory perturbation of other ASD risk genes during human cortical development. Two biological replicates for each ChIP with appropriate Input control Four biological replicates for each condition in knockdown experiments (Ctrl construct, Chd8 target C, and Chd8 target G)
Project description:SAGA member Ada2 is required for the majority of H3K9 acetylation in C. neoformans. To identify specific genomic loci that exhibit Ada2-dependent H3K9 acetylation, we performed ChIP-Seq against H3K9ac in wildtype and ada2Δ cells. ChIP-Seq was performed using antibodies for H3K9ac in KN99 wildtype cells and ada2Δ cells. Input and IPed DNA was collected in triplicate from each strain and sequenced on an Illumnina HiSeq 2000 flow cell producing 84 million reads. Due to the lack of quality scores, raw reads are omitted from the submission.
Project description:Bone marrow-derived multipotent stromal cells (BM-MSCs) exhibit therapuetically valuable properties, including the capacity to differentiate into skeletal tissues and modulate immune system activity. These properties depend on proper regulation of dynamic gene expression in response to environmental and developmental stimuli. This study used chromatin immunoprecipitation (ChIP) coupled with human promoter tiling microarray analysis (ChIP-on-chip) to profile histones H3K4me3 and H3K27me3 at promoters genome-wide. The goal of the study was to identify gene promoters marked by H3K27me3 and H3K4me3 in BM-MSCs. ChIP-on-chip performed with antibodies to H3K4me3 and H3K27me3 on BM-MSCs from 3 different donors (labeled 1632, 167696, and 8F3560) and with technical replicates.