Project description:This experiment is a part of global location analysis of the human DREAM complex including subunits: E2F4, RBL2/p130, LIN9 and LIN54. Specifically, the regions in Human Promoter Array 1.0R (GPL5082) bound by: E2F4 in G0-arrested T98G cells E2F4 in S-phase synchronized T98G cells LIN54 in G0-arrested T98G cells LIN54 in S-phase synchronized T98G cells LIN9 in G0-arrested T98G cells LIN9 in S-phase synchronized T98G cells p130 in G0-arrested T98G cells p130 in S-phase synchronized T98G cells were compared with input chromatin. MAT (Model-based Analysis for Tiling Arrays) .bar and .bed output files provided as supplementary files. Keywords: CHiP-chip genomic DNA
Project description:The mammalian Retinoblastoma (RB) family including pRB, p107, and p130 represses E2F target genes through mechanisms that are not fully understood. In D. melanogaster, RB-dependent repression is mediated in part by the multisubunit protein complex Drosophila RBF, E2F, and Myb (dREAM) that contains homologs of the C. elegans synthetic multivulva class B (synMuvB) gene products. Using an integrated approach combining proteomics, genomics, and bioinformatic analyses, we identified a p130 complex termed DP, RB-like, E2F, and MuvB (DREAM) that contains mammalian homologs of synMuvB proteins LIN-9, LIN-37, LIN-52, LIN-54, and LIN-53/RBBP4. DREAM bound to more than 800 human promoters in G0 and was required for repression of E2F target genes. In S phase, MuvB proteins dissociated from p130 and formed a distinct submodule that bound MYB. This work reveals an evolutionarily conserved multisubunit protein complex that contains p130 and E2F4, but not pRB, and mediates the repression of cell cycle-dependent genes in quiescence. Experiment Overall Design: Gene expression during the cell cycle in T98G cells. Cells were serum starved for 72 hours to induce G0 and then restimuated to enter cell cycle by serum addition. BrdU and FACS was done simultaneously to confirm their cell cycle phase.
Project description:The mammalian Retinoblastoma (RB) family including pRB, p107, and p130 represses E2F target genes through mechanisms that are not fully understood. In D. melanogaster, RB-dependent repression is mediated in part by the multisubunit protein complex Drosophila RBF, E2F, and Myb (dREAM) that contains homologs of the C. elegans synthetic multivulva class B (synMuvB) gene products. Using an integrated approach combining proteomics, genomics, and bioinformatic analyses, we identified a p130 complex termed DP, RB-like, E2F, and MuvB (DREAM) that contains mammalian homologs of synMuvB proteins LIN-9, LIN-37, LIN-52, LIN-54, and LIN-53/RBBP4. DREAM bound to more than 800 human promoters in G0 and was required for repression of E2F target genes. In S phase, MuvB proteins dissociated from p130 and formed a distinct submodule that bound MYB. This work reveals an evolutionarily conserved multisubunit protein complex that contains p130 and E2F4, but not pRB, and mediates the repression of cell cycle-dependent genes in quiescence. Keywords: Gene expression analysis during cell cycle
Project description:genomic localization of the budding yeast cohesin complex was mapped in mitotically arrested cells by Mcd1 ChIP followed by hybridization to high-density tiled microarrays Cells were synchronized first in G1 and then released into media containing the microtubule poison nocodazole. Cells were fixed and processed for ChIP once arrested as large-budded cells. Immunoprecipitated DNA and total genomic DNA not subjected to immunoprecipitation were competitively hybridized to Nimblegen whole genome arrays.
Project description:The master transcriptional repressor DREAM (dimerization partner, RB-like, E2F and multi-vulval class B) complex regulates cell cycle in eukaryotes, but much remains unknown about how it transmits repressive signals on chromatin to the primary transcriptional machinery (e.g., Pol II). Through a genetic screen, we identified BTE1 (barrier of transcription elongation 1), a key component in the plant DREAM complex. The subsequent characterization demonstrated that the DREAM complex represses Pol II elongation. The core DREAM subunits E2F transcription factors recruit BTE1 to accumulate at the promoter-proximal regions of target genes. DREAM target genes exhibit characteristic enrichment of H2A.Z and H3K4me2 modification on chromatin. The DREAM complex BTE1 subunit interacts with WDR5a, repressing WDR5a chromatin binding and the productive elongation of transcription on BTE1-targeted genomic loci. Thus, we demonstrate a major role of DREAM complex in the control of transition from transcription initiation to productive elongation around TSSs on the open chromatin via inhibiting WDR5a function.
Project description:The master transcriptional repressor DREAM (dimerization partner, RB-like, E2F and multi-vulval class B) complex regulates cell cycle in eukaryotes, but much remains unknown about how it transmits repressive signals on chromatin to the primary transcriptional machinery (e.g., Pol II). Through a genetic screen, we identified BTE1 (barrier of transcription elongation 1), a key component in the plant DREAM complex. The subsequent characterization demonstrated that the DREAM complex represses Pol II elongation. The core DREAM subunits E2F transcription factors recruit BTE1 to accumulate at the promoter-proximal regions of target genes. DREAM target genes exhibit characteristic enrichment of H2A.Z and H3K4me2 modification on chromatin. The DREAM complex BTE1 subunit interacts with WDR5a, repressing WDR5a chromatin binding and the productive elongation of transcription on BTE1-targeted genomic loci. Thus, we demonstrate a major role of DREAM complex in the control of transition from transcription initiation to productive elongation around TSSs on the open chromatin via inhibiting WDR5a function.
Project description:The master transcriptional repressor DREAM (dimerization partner, RB-like, E2F and multi-vulval class B) complex regulates cell cycle in eukaryotes, but much remains unknown about how it transmits repressive signals on chromatin to the primary transcriptional machinery (e.g., Pol II). Through a genetic screen, we identified BTE1 (barrier of transcription elongation 1), a key component in the plant DREAM complex. The subsequent characterization demonstrated that the DREAM complex represses Pol II elongation. The core DREAM subunits E2F transcription factors recruit BTE1 to accumulate at the promoter-proximal regions of target genes. DREAM target genes exhibit characteristic enrichment of H2A.Z and H3K4me2 modification on chromatin. The DREAM complex BTE1 subunit interacts with WDR5a, repressing WDR5a chromatin binding and the productive elongation of transcription on BTE1-targeted genomic loci. Thus, we demonstrate a major role of DREAM complex in the control of transition from transcription initiation to productive elongation around TSSs on the open chromatin via inhibiting WDR5a function.
Project description:The Retinoblastoma-like pocket proteins p130 and p107 act as gatekeepers of the cell cycle through their activity within the DREAM (Dp/Rb-like/E2F/MuvB) transcriptional repressor complex. The goal of this study was to address how the pocket protein contributes to DREAM complex assembly and function on chromatin by utilizing a protein null mutant of the only C. elegans pocket protein LIN-35. We performed ChIP-seq of C. elegans DRM subunits in wild-type and lin-35 null late embryos to assess the effect on their chromatin localization following loss of LIN-35.
Project description:Knockdown of MLL5 led to deregulation of S phase. To understand the molecular basis for this phenotype, we performed microarray analysis of S phase synchronized myoblasts. Genes differentially regulated by MLL5 knock down were revealed by microarray analysis using NIA15K mouse chips. Control and knock down cells were synchronized at G0 by suspension culture and reactivated to enter S phase by replating for 24hrs in growth medium.
Project description:DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs must be repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in cycling cells at the G1 or G2 phase cells was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.