DamID (LmnB1/Dam) Log2 ratios of C57Bl/6 fibroblasts and RAG2-/- pro-B cells.
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Karen Reddy
PROVIDER: E-GEOD-56990 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:How gene positioning to the nuclear periphery regulates transcription remains largely unclear. We have previously observed the differential compartmentalization of transcription factors and histone modifications at the nuclear periphery in mouse C2C12 myoblasts. Here, we have integrated high throughput DNA sequencing into the DNA adenine methyltransferase identification (DamID) assay, and have identified ~15, 000 sequencing-based Lamina-Associated Domains (sLADs) in mouse 3T3 fibroblasts and C2C12 myoblasts. These genomic regions range from a few kb to over 1 Mb and cover ~30% of the genome, and are spatially proximal to the nuclear lamina (NL). Active histone modifications such as H3K4me2, H3K9Ac, H3K36me3 and H3K79me2 are all localized away from the nuclear periphery microscopically, and distributed predominantly out of sLADs genome-wide. Therefore, the spatial compartmentalization of active histone modifications likely characterizes a major portion of chromatin at the nuclear periphery in mammalian cells. Genomic regions around transcription start sites of expressed sLAD genes display reduced associations with the NL and possess active histone modifications; in contrast, gene bodies of expressed sLAD genes possess very low levels of active histone modifications. Our genome-wide analyses of NL-associated chromatin have enabled functional and mechanistic dissections of gene positioning on transcription regulation. generate DamID maps of genome-NL interaction for mouse 3T3 fibroblasts and C2C12 myoblasts
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Project description:Secondary diversification of antibodies through somatic hypermutation (SHM) and class switch recombination (CSR) is a critical component of the immune response. Activation-induced deaminase (AID) initiates both processes by deaminating cytosine residues in immunoglobulin genes. The resulting U:G mismatch can be processed by alternative pathways to give rise to a mutation (SHM) or a DNA double-strand break (CSR). Central to this processing is the activity of uracil-N-glycosylase (UNG), an enzyme normally involved in error-free base excision repair. We used next generation sequencing to analyze the contribution of UNG to the resolution of AID-induced lesions. Loss- and gain-of-function experiments showed that UNG activity can promote both error-prone and high fidelity repair of U:G lesions. Unexpectedly, the balance between these alternative outcomes was influenced by the sequence context of the deaminated cytosine, with individual hotspots exhibiting higher susceptibility to UNG-triggered error-free or error-prone resolution. These results reveal UNG as a new molecular layer that shapes the specificity of AID-induced mutations and may provide new insights into the role of AID in cancer development. Next Generation Sequencing analysis of mutations introduced by AID in non B cells. NIH-3T3 cells were co-transduced with mOrangeSTOP and AID-ERM-bM-^@M-^Sexpressing vectors, together with Ugi (UNG inhibitor), UNG, or empty vector as control (n=3). Transduced cells were cultured in the presence of OHT during 11 d. AID-E58Q-ER vector (catalytically inactive form of AID) was used as a negative control in combination with the previously described constructions (n=3).
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Project description:The nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, so-called lamin associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions. In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2β to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina as well as identified putative LAD proximal proteins. Our analysis identifies many heterochromatin related proteins related to H3K9 methylation processes as well as many proteins related to cell cycle regulation identifying important proteins essential for LAD function.
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Project description:Following androgen ablation treatment for advanced prostate cancer, almost all men relapse after a period of initial response to therapy, which eventually is life threatening. We have previously found that purine-rich element binding protein, PUR alpha, was significantly repressed in androgen-independent prostate cancer cell lines in comparison to an androgen-dependent line. Moreover, over-expressing PURa in androgen-independent prostate cancer cells attenuated their cell proliferation. The aim of the studies described here was to uncover some of the mechanisms by which over-expression of PURa attenuates cell proliferation. A set of common genes induced by over-expressing PURa both in PC3 and LNCaP cells was analyzed by DNA microarray. The results were then validated utilizing quantitative reverse transcription-PCR. Using a 5.3-kb region of the PSA promoter containing androgen response elements, the participation of PURa in androgen regulated gene expression was determined. Genes involved in stress response and cell differentiation were induced in cells over-expressing PURa. Some of the genes that are targets of androgen regulation are also induced. Most strikingly, ectopic expression of PURa induced transcriptional activity of the 5.3-kb PSA promoter containing androgen response elements, without androgen stimulation. Based upon the consideration that some of the genes involved in cell stress and differentiation are also regulated by androgens our data suggest that PURa shares some common pathway regulated by the androgen receptor. These findings suggest that regulation of PURa expression in prostate cancer cells may serve as a therapeutic target for hormone refractory prostate cancer. Experiment Overall Design: PC3 cells (low in PURa expression) was stably over-expressed PURa. Total RNA of the cells was collected simultaneously with that of PC3 transfected with mock vector. Experiment Overall Design: LNCaP cells (Express PURa more than PC3 cells) was PURa-over-expressed with retrovirus vector (MSCV-PIG) and selected with puromycin for 72hr. Total RNA of the cells was collected simultaneously with that of LNCaP cells infected with mock vector. Experiment Overall Design: One set of RNA derived from PC3 cells and two independent RNA derived from LNCaP cells retrovirus infection were used for DNA microarray. Quality and concentration of total RNA was verified using Agilent Bioanalyzer and Nanodrop Spectrophotometer, respectively. T7 oligo(dT) primer (Sigma-Proligo, Boulder, CO) and 5ug total RNA are combined for first strand cDNA synthesis. Following second strand cDNA synthesis and cDNA cleanup (Phase Lock Gel Light, Eppendorf, Hamburg, Germany) an in vitro transcription reaction was performed overnight using T7 RNA transcript labeling kit provided by Enzo Life Sciences, Inc. (Farmingdale, NY). IVT reactions were cleaned using RNeasy Mini Kit (Qiagen) and concentration determined by Nanodrop Spectrophotometer. 15ug cRNA was fragmented using 5X fragmentation buffer (made in-house). Hybridization cocktail was made in accordance with Affymetrix eukaryotic expression array protocol (Affymetrix, Santa Clara, CA) and combined with fragmented cRNA. 10ug cRNA was loaded onto Human U133Plus 2.0 genome arrays (Affymetrix, Santa Clara, CA) and hybridized overnight for 16 hours. After hybridization, staining and washing of the arrays was performed following the Affymetrix eukaryotic expression array protocol, including staining with streptavidin-phycoerythrin, antibody stain, and a second streptavidin-phycoerythrin stain. 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This probe level data processing includes a normalization procedure utilizing the quantile normalization method (Bolstad BM et al. 2003) to reduce the obscuring variation between microarrays, which might be introduced during the processes of sample preparation, manufacture, fluorescence labeling, hybridization and/or scanning. Exploratory data analysis (EDA) was performed with the preprocessed data above. Between-treatment and between-replicate variations were examined with the pair-wise MvA plots, in which the base 2 log ratios (M) between two samples are plotted against their averaged base 2 log signals (A). With the signal estimates, Principal Component analysis (PCA) was also performed to assess sample variability. 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