Project description:The Nrd1-Nab3-Sen1 (NNS) complex plays a pivotal role in the control of pervasive transcription and the generation of sn- and snoRNAs in S. cerevisiae. The NNS-complex terminates transcription of non-coding RNA genes and promotes processing/degradation of transcripts by the nuclear exosome. To assess the role of the Nrd1p CTD-interacting domain (CID) in the function of the NNS-complex, we re-examined whether this domain is required for efficient transcription termination by the NNS pathway. We compared the RNA polymerase II distribution by ChIP and tiling arrays (ChIP-chip) in wild type and nrd1 deltaCID cells. Moreover, we compared the genome-wide chromatin distribution of Nrd1p in the presence and the absence of the CID by ChIP-chip analysis. ChIP of RNA polymerase II was performed using an anti-Rpb3 antibody (1Y26, Neoclone). ChIP of Nrd1 was performed using TAP-tagged S. cerevisiae strains. For details see protocols. To download the wild-type Pol II and Nrd1 data go to E-MTAB-1626 and E-MTAB-1060, respectively.
Project description:Sir2 and the homologous proteins, Hst1, Hst2, Hst3, and Hst4 from Saccharomyces cerevisiae are NAD+-dependent histone deacetylases of the sirtuin protein family. Sir2 functions in transcriptional silencing at the silent mating-type loci, telomeres, and rDNA locus, but also promotes replicative lifespan. To gain a better understanding of the chromatin-regulatory roles carried out by Sir2 and the Hst proteins, we performed ChIP-sequencing analysis on all five sirtuins and Sum1, the DNA binding partner for Hst1. Sir2, Hst1, and Sum1 were abundantly, and functionally co-enriched at several major targets, including the telomeric repeats, where they were required for maintaining proper telomere repeat length. At tRNA target genes they were required for efficient cohesin and condensin deposition. Across the open reading frames of glycolytic and ribosomal protein genes, Sir2 and Hst1 functioned in NAD+-dependent transcriptional repression at the diauxic shift, directly linking Sir2 to glucose metabolism, which could have implications for longevity. Six factors and Input ChIP-seq samples were analyzed in Saccharomyces eerevisiae.
Project description:To obtain rates of mRNA synthesis and decay in yeast, we established dynamic transcriptome analysis (DTA). DTA combines non-perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA was used to monitor the cellular response to osmotic stress in comparison to the wild type. Genomic occupancy profiling of RNA polymerase (Pol) II was used to predict changes in mRNA synthesis rates.
Project description:ChIP-chip was performed to identify the genomic binding locations for the termination factors RNA14 and RNA15, and for Rpb3 in wildtype and mutant backgrounds.
Project description:Cell-specific transcriptional regulations exerted by the estrogen (E2) receptor alpha (ER) heavily rely upon timely and spatially coordinated processes. We engaged a comparative analysis of such dynamic molecular events at the TFF locus harbouring a cluster of genes co-regulated by E2, in two distinct breast cancer cell lines. Using a combination of methods, we show that the recruitment of ER on cell-specific sites triggers dynamic local modifications of chromatin, which are coordinated in time all along the locus. DNA-FISH experiments further demonstrate that these changes are associated with an E2-dependent reduction in plasticity of this genomic region and are dependent upon cohesin. Importantly, 3C/4C experiments and the use of triplex forming oligonucleotides (TFOs) allowed us to precisely map the three-dimensional network of regulatory events that permits the estrogenic response of this genomic region. These data also evidenced an unexpected functional redundancy of enhancers. Independent duplicate array series, using on one array pooled ChIP triplicates prepared from separate MDA::ER or MCF-7 cell cultures treated with estradiol for 50 minutes.
Project description:CENP-A, a variant of histone H3, is incorporated into centromeric chromatin and plays a role during kinetochore establishment. In fission yeast, the localization of CENP-A is limited to a region spanning 10 to 20 kb of the core domain of the centromere. Here, we report a mutant (rpt3-1) in which this region is expanded to 40 to 70 kb. Likely due to abnormal distribution of CENP-A, this mutant exhibits chromosome instability and enhanced gene silencing. Interestingly, the rpt3+ gene encodes a subunit of the 19S proteasome, which localizes to the nuclear membrane. While Rpt3 associates with centromeric chromatin, the mutant protein has lost this localization. A loss of the cut8+ gene encoding an anchor of the proteasome to the nuclear membrane causes similar phenotypes as observed in the rpt3-1 mutant. Thus, we propose that the proteasome (or its subcomplex) associates with centromeric chromatin and regulates distribution of CENP-A. Chromosomal distributions of differentially expressed centromere protein A in wild-type and a proteasome mutant.
Project description:The goal of this study was to assay the extent of variation in chromatin organization between 3 ant castes (major and minor female workers and males) in one colony of Camponotus floridanus carpenter ant using ChIPseq. 45 samples total: 30 ChIP samples and 3 inputs for total histone H3, 7 histone H3 PTMs and RNA Pol II in major, minor, and male ants; CBP in major and minor ants; the major H3K27ac sample was replicated. 4 ChIP samples for H3 and H3K27ac in brains of majors and minors, and 2 inputs. 2 RNAseq samples for major and minor ants head+thorax; 4 RNAseq samples for brain (majors and minors with 2 replicates each).
Project description:To investigate the chromatin transcription cycle, we determined genome-wide occupancy profiles for RNA polymerase (Pol) II, its phosphorylated forms, and transcription factors in growing yeast. ChIP-chip was performed to identify the genomic binding locations for Rpb3, TFIIB, Tfg1, Kin28, Cet1, Spt4, Spt5, Spt6, Elf1, Spn1, Bur1, Ctk1, Paf1, Spt16, Pcf11, and Rpb1 phosphorylated at serine 2, 5, and 7 residues of the CTD, respectively.
Project description:Although the majority of genomic binding sites for the insulator protein CTCF are constitutively occupied, a subset show variable occupancy. Such variable sites provide an opportunity to assess context-specific CTCF functions in gene regulation. Here we have identified a variably occupied CTCF site in the Ultrabithorax (Ubx) gene in Drosophila. This site is occupied in tissues where Ubx is active (third thoracic imaginal leg disc) but is not bound in tissues where the Ubx gene is repressed (first thoracic imaginal leg disc). Comparison of CTCF binding in T1 leg disc vs T3 leg disc in from 3rd instar larva