Project description:Changes in histone post-translational modifications are associated with aging through poorly defined mechanisms. Histone 3 lysine 4 (H3K4) methylation at promoters is deposited by SET1 family methyltransferases acting within conserved multiprotein complexes known as COMPASS. Using co-immunopurification coupled to mass spectrometry-based proteomics, we characterized complex members and binding partners in various genetic contexts, using WDR-5 or CFP-1 proteins as baits.
Project description:The SIN3 transcriptional coregulator influences gene expression through multiple interactions that include histone deacetylases (HDACs). Haploinsufficiency and mutations in SIN3 are the underlying cause of Witteveen-Kolk syndrome and related intellectual disability (ID)/autism syndromes, emphasizing its key role in development. However, little is known about the diversity of its interactions and functions in developmental processes. To gain insight in the repertoire of proteins that interact with SIN-3, we performed IP-MS on mCherry::SIN-3 expressing embryos. Furthermore, we analyzed the partnership of ARID-1::GFP, ARID-1 being an already described subunit of the SIN-3L complex.
Project description:Small non-coding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of Metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that trans-generationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the H3K9me3 mark on genomic piRNA cluster sequences. The HP1 homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that trans-generationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels, by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors. ChIPseq of Rhino and Cutoff in Drosophila melanogaster ovaries The Rhino-BioTAP flies were made by fusing the BioTAP tag (Alekseyenko et al. 2014 )to the C-terminal region of the Rhino gene under the UASp promoter. The Cutoff-EGFP fly line (Nanos-GAL4/UASp-Cutoff-GFP), Cuff^wm25 and Cuff^qq37 were a generous gift from T. Schupbach.
Project description:In this project, we identified a novel RNA-binding protein, MHZ9. And we analyzed the potential proteins interacted with MHZ9 through immunoprecipitation-mass spectrometry (IP-MS). The N-terminal domain of MHZ9 (MHZ9-N) contains a putative RNA splicing and modification domain PRP4. To identify RNA binding sites in the MHZ9-N. We performed XRNAX-IP-MS assay.
Project description:Piwi interacting (pi)RNAs repress diverse transposable elements in the germ cells of metazoans and are essential for fertility in both invertebrates and vertebrates. The precursors of piRNAs are transcribed from distinct genomic regions, the so-called piRNA clusters; however, how piRNA clusters are differentiated from the rest of the genome is not known. To address this question, we studied piRNA biogenesis in two Drosophila virilis strains that show differential ability to generate piRNAs from several genomic regions. We found that active piRNA biogenesis correlates with high levels of histone 3 lysine 9 trimethylation (H3K9me3) over genomic regions that give rise to piRNAs. Furthermore, piRNA biogenesis in the progeny requires the trans-generational inheritance of an epigenetic signal, presumably in form of homologous piRNAs that are generated in the maternal germline and deposited into the oocyte. The inherited piRNAs enhance piRNA biogenesis by installment of H3K9me3 mark on piRNA clusters and by promoting ping-pong processing of homologous transcripts into mature piRNAs. We submitted the resequencing data together with the functional genomic datasets because it was generated with the sole purpose of supporting those. The SRA accession numbers are SRR1536176 and SRR1536175. ChIP-seq against H3K9me3 and Pol2, Total RNA-seq, in Drosophila virilis Strain9 and Strain160 as well as crosses between them
Project description:ChIP-chip study using Saos-2 cell line infected with adenoviruses encoding GFP (Mock) or GFP together with the p53 H1 helix mutants EL or RE. DNA-protein-complexes were precipitated with monoclonal p53-antibody (clone DO-1). Mock-chromatin was immunoprecipitated in the absence of antibody.<br>Three completely independent biological replicates were performed for each antibody, obtaining the corresponding input as total genomic DNA reference. Hybridizations were performed using Affymetrix GeneChip Human Tiling 2.0R Array set (7 arrays set).<br><br> Affymetrix .BAR files and an additional processed data file containing the coordinates of the identified binding sites can be found in the FTP directory for this experiment. <A HREF="ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-1748">FTP directory</A>
Project description:To identify those proteins interacting with the cytoplasmic dynein-2 using mammalian cell lines stably expressing GFP-tagged intermediate chain subunits, WDR34 (DYNC2I2) or WDR60 (DYNC2I1).
Project description:To identify those proteins interacting with the cytoplasmic dynein-2 using mammalian cell lines stably expressing GFP-tagged intermediate chain subunits, WDR34 (DYNC2I2) or WDR60 (DYNC2I1).