Identification of targets genes of PRDM2 in G0 arrested myoblasts of C2C12
ABSTRACT: PRDM2 directly associates with the Myogenin promoter and repress its transcription. This led to the hypothesis that PRDM2 could potentially associate directly with other promoters and regulate their expression.To gain further insight into the pathways and genes controlled by PRDM2, ChIP-on-Chip analysis was performed using mouse 44K promoter array. Since PRDM2 represses transcription by H3K9 dimethylation, we were interested in determining which targets were occupied by PRDM2 as well as showed enrichment for H3K9me2. Hence ChIP-on-Chip analysis for H3K9me2 was performed to find the overlap between PRDM2 and H3K9me2 marked promoters. Agilent two-color ChIP-on-Chip experiment, Organism: Mus musculus ,Genotypic Technology designed Custom Mouse Promoter 244k ChIP-on-chip Array (AMADID-019046)
Project description:Knockdown of PRDM2 led to precocious differentiation. To understand the molecular basis for this phenotype, we performed microaary analysis of growing myoblasts. Genes differentially regulated by PRDM2 knock down were reveraled by microarray analysis using NIA15K mouse chips. Control and knock down cells were grown in proliferating conditions
Project description:Purpose: To identify the contribution of PRDM2 c.4459delA mutation to colorectal tumorigenesis Methods: We employed rAAV-mediated genome editing to correct somatic PRDM2 c.4459delA mutation in homozygously mutated cell line. Using next-generation sequencing we have compared transcriptional profile of parental and PRDM2-corrected cells. Results: RNA-seq profiling revealed that several hallmark cancer gene sets are affected by PRDM2 c.4459delA Overall design: To address if the PRDM2 wt cells were transcriptionally different at a global level compared to their parental cells that carry PRDM2 c.4459delA mutation, we performed RNA-sequencing of parental cells and three independently generated clones with corrected PRDM2. RNA-seq profile was generated for cells grown under normal culture conditions (10% FBS) as well as reduced serum conditions (0.5% FBS).
Project description:Knockdown of PRDM2 led to precocious differentiation. To understand the molecular basis for this phenotype, we performed microaary analysis of 28hr differentiated myoblasts. Genes differentially regulated by PRDM2 knock down were reveraled by microarray analysis using affymetrix mouse chips. Control and knock down cells were grown in differentiating conditions for 28hrs and total RNA was used to perform microarray analysis
Project description:Knockdown of PRDM2 led to precocious differentiation. To understand the molecular basis for this phenotype, we performed microaary analysis of quiescent myoblasts. Genes differentially regulated by PRDM2 knock down were reveraled by microarray analysis using affymetrix mouse chips. Control and knock down cells were synchronized at G0 by suspension culture and total RNA was used to perform microarray analysis
Project description:Increasing evidence supports a role for altered gene expression in mediating the lasting effects of cocaine on the brain, and recent work has demonstrated the involvement of chromatin modifications in these alterations. However, all such studies to date have been restricted by their reliance on microarray technologies which have intrinsic limitations. Here, we used advanced sequencing methods, RNA-seq and ChIP-seq, to obtain an unprecedented view of cocaine-induced changes in gene expression and associated adaptations in numerous modes of chromatin regulation in the nucleus accumbens, a key brain reward region. We identify unique combinations of chromatin changes, or signatures, that accompany cocaine’s regulation of gene expression, including the dramatic involvement of pre-mRNA alternative splicing in cocaine action. Together, this delineation of the cocaine-induced epigenome in the nucleus accumbens reveals several novel modes of drug regulation, thereby providing new insight into the biological basis of cocaine addiction. More broadly, the combinatorial chromatin and transcriptional approaches that we describe serve as an important resource for the field, as they can be applied to other systems to reveal novel transcriptional and epigenetic mechanisms of neuronal regulation. ChIP-seq of 6 marks (H3K27me3, H3K36me3, H3K4me1, H3K4me3, H3K9me2, RNApolII) were done on mouse nucleus accumbens 24 hr after 7 day daily cocaine ip injection with saline as control. Three replicates for each condition.
Project description:The H3K27me3 is a repressive histone mark associated with repressive chromatin and is important for X chromosome inactivation. ChIP-chip of H3K27me3 along the mouse X chromosome in male and female livers and p12.5 embryos demonstrated that H3K27me3 is absent at the genes that escape X inactivation. Comparison of H3K27me3 enrichment along the X chromosome in male and female adult livers and P12.5 embryos
Project description:We report widespread ChIP-seq bias at highly expressed genes in yeast that could lead to misinterpretation ChIP-seq for multiple transcription or chromatin-associated factors and negative controls
Project description:ChIP seq of Cfp-1 and H3K4me3 in C. elegans late embryos The coding region of F52B11.1a (cfp-1) was PCR amplified from N2 genomic DNA using Phusion polymerase (Finnzymes) and Gateway cloned into pDONR221. The cfp-1 coding region was then recombined into the MosSCI compatible vector pCFJ201 (which targets Mos site Mos1(cxTi10882) chrIV ) downstream of the dpy-30 promoter and upstream of gfp::tbb-2 3’UTR (Zeiser et al. 2011) to generae strain JA1597 expressing GFP tagged Cfp-1 protein. Late embryos were obtained by aging embryos collected by hypochlorite treatment 3.5 hrs prior to flash freezing in liquid nitrogen. Formaldehyde-fixed chromatin extracts and chromatin immunoprecipitations were as in (Kolasinska-Zwierz et al. 2009) except that DNA was sonicated to a size range of 200-400bp. ChIP assays were performed in 1 ml extract (1 mg protein) in FA buffer with 10 micrograms of anti-GFP rabbit serum (Abcam ab290) and anti-H3K4me3 (Abcam ab8580) individually. DNA sequencing libraries were constructed according using the Illumina Truseq sequencing kit and were sequenced on the Illumina platform.
Project description:Identification of target genes of PITX2 homeodomain transcription factor in ovary using human ovarian adenocarcinoma cells, SKOV-3 to determine the transcriptional network of PITX2. Genotypic Technology designed Custom Human Promoter 244k ChIP-on-chip Array (AMADID-019469)
Project description:ChIP sequencing for E2F1, E2F3A and E2F3B in mouse embryonic fibroblasts E2F1, E2F3A and E2F3B ChIPs were performed in mouse embryonic fibroblasts stably overexpressing the proteins. ChIP DNA was processed to generate libraries and then sequenced using the Illumina platform.