Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Analyses of cyclin D1 function using a "genetic-proteomic" approach

ABSTRACT: We examined the transcriptional function of cyclin D1 in mouse development using two approaches. First, we queried association of cyclin D1 with the genome of E14.5 mouse embryos using ChIP-on-chip approach. We observed binding of cyclin D1 to several promoter regions. Second, we compared gene expression profiles between wild-type and cyclin D1-null retinas. We observed several transcripts with altered levels in cyclin D1-null organs. Refer to individual Series

ORGANISM(S): Mus musculus

SUBMITTER: Frederic Bienvenu

PROVIDER: E-GEOD-13636 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transcriptional role of cyclin D1 in development revealed by a genetic-proteomic screen.

Bienvenu Frédéric F Jirawatnotai Siwanon S Elias Joshua E JE Meyer Clifford A CA Mizeracka Karolina K Marson Alexander A Frampton Garrett M GM Cole Megan F MF Odom Duncan T DT Odajima Junko J Geng Yan Y Zagozdzon Agnieszka A Jecrois Marie M Young Richard A RA Liu X Shirley XS Cepko Constance L CL Gygi Steven P SP Sicinski Piotr P

Nature 20100101 7279

Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. Here we developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in trans ...[more]

PMID: 20090754

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Project description:Cyclin D1 belongs to the core cell cycle machinery1, and it is frequently overexpressed in human cancers2. The full repertoire of cyclin D1 functions in normal development and in cancer cells is currently unknown. To address this question, here we introduce a novel approach that allows one to determine the set of cyclin D1-interacting proteins (D1 âinteractomeâ) and cyclin D1-bound genomic fragments (D1 âcistromeâ) in essentially any mouse organ, at any point of development or at any stage of cancer progression. Using this approach, we detected several novel tissue-specific interactors of cyclin D1. A significant number of these partners represent proteins involved in transcription. We show, using genome-wide location analysis3, that cyclin D1 occupies promoters of a very large number of genes in the developing mouse, where it binds in close proximity to transcription start sites. Bioinformatics analyses of cyclin D1-bound genomic segments in the developing embryo revealed DNA recognition sequences for several transcription factors. By querying SAGE libraries4, promoter CpG content5 and gene expression profiles of cyclin D1-null organs, we demonstrate that cyclin D1 binds promoters of highly expressed genes, and that it functions to activate or to repress gene expression in vivo. Analyses of cyclin D1 transcriptional targets reveal that cyclin D1 contributes to cell proliferation by upregulating genes required for S-phase entry and progression. Hence, cyclin D1 plays a broad transcriptional regulatory function in vivo during normal mouse development. We hypothesized that cyclin D1 may play role in transcription by interacting with transcriptional machinery at the promoters of target genes. To test this possibility, we employed chromatin immunoprecipitation coupled to DNA microarray analysis (genome-wide location analysis or ChIP-chip) to study association of cyclin D1 with genomic DNA sequences. In this procedure, a protein of interest is crosslinked to DNA, immunoprecipitated, and the bound DNA is hybridized to an array containing probes that span the genome. Since anti-FLAG antibodies have been successfully used for ChIP-chip in several different systems including murine cells, knock-in mice expressing tagged cyclin D1 provided us with a novel tool to query the association of cyclin D1 with the genome in vivo, in a living mouse.

Project description:The zebrafish u-boot (ubo) gene encodes the transcription factor Prdm1, which is essential for the specification of the primary slow twitch muscle fibres that derive from adaxial cells. Here we show that Prdm1 executes this function by acting as a transcriptional repressor. Expression of the slow twitch isoform of the myosin heavy chain in adaxial cells is achieved by Prdm1 mediated repression of the transcriptional repressor Sox6. Using Chromatin immuno precipitation (ChIP) we found that genes encoding fast specific isoforms of sarcomeric proteins are direct targets of Prdm1. These genes are ectopically expressed in the adaxial cells of ubotp39 mutant embryos, suggesting that Prdm1 promotes slow twitch fibre differentiation by acting both as a global repressor of fast fibre specific genes as well as of a repressor of slow specific genes. Keywords: ChIP-chip Genomic array design. Microarrays were designed as described below and manufactured by Agilent Technologies (www.agilent.com). We have previously described the ChIP-chip technique in zebrafish and the design of promoter microarrays which cover a 2kb region around the transcription start sites (TSSs) of 11,512 zebrafish genes based on Zv4 (Wardle et al, 2006. Genome Biology 7:R71). In order to capture additional regulatory information further from the basal promoter region we also designed an expanded set of 60mer probes that cover 9kb upstream and 3kb downstream of the TSS of these genes using the same parameters as for the 2kb microarrays. These probe sequences were re-mapped to Zv6 over the course of the project and the coordinates given are for Zv6. We also incorporated several sets of control probes, both positive and negative. On each array there are 769 negative probes designed against zebrafish gene desert regions and Arabidopsis thaliana genes. In addition we included duplicates of promoter probes for several genes that we anticipated may be bound by factors of interest for this and other studies (tnnt3a, mylz2, myhz2, wnt11, flh, vent, msgn1, myod, fgf8, pcdh8) and these probes are arrayed on both slides slide. Finally on each array there are 1804 controls added by Agilent. The final design contained 352,490 probes divided between two microarray slides. Further information on design and manufacture of the microarrays can be found at the Agilent Technologies website.

Dataset Information

Analyses of cyclin D1 function using a "genetic-proteomic" approach

Publications

Transcriptional role of cyclin D1 in development revealed by a genetic-proteomic screen.

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