Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcriptional profiling of PADI4 overexpression and inhibition in mouse embryonic stem cells

ABSTRACT: The enzymatic activity of PADI4 was investigated by overexpression and chemical inhibition to determine the effects of arginine citrullination on histone displacement, reprogramming efficiency and the maintenance of pluripotent stem cells.

ORGANISM(S): Mus musculus

SUBMITTER: Paul Bertone

PROVIDER: E-MTAB-1975 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Citrullination regulates pluripotency and histone H1 binding to chromatin.

Christophorou Maria A MA Castelo-Branco Gonçalo G Halley-Stott Richard P RP Oliveira Clara Slade CS Loos Remco R Loos Remco R Radzisheuskaya Aliaksandra A Mowen Kerri A KA Bertone Paul P Silva José C R JC Zernicka-Goetz Magdalena M Nielsen Michael L ML Gurdon John B JB Kouzarides Tony T

Nature 20140126 7490

Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thr ...[more]

PMID: 24463520

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Project description:Many transcription factors and DNA binding proteins play essential roles in the development of organs in which they are highly and/or specifically expressed. Embryonic stem cell (ESC) associated transcript (ECAT) 15-1 and ECAT15-2, also known as developmental pluripotency-associated (Dppa) 4 and Dppa2, are enriched in mouse ESCs and pre-implantation embryos, and encode homologous proteins with a common DNA binding domain known as the SAP motif. Previously, ECAT15-1 was shown to be important in the lung development, while it is dispensable in early development. In this study, we generated ECAT15-2 single and ECAT15-1/15-2 double knockout mice and found that almost all mutants, like ECAT15-1 mutants, died around birth with respiratory defects. Paradoxically, the expression of neither ECAT15-1 nor ECAT15-2 was detected in lung organogenesis. Several genes, such as Nkx2-5, Gata4, and Pitx2 were down-regulated in the ECAT15-2-null lung. On the other hand, genomic DNA of these genes showed inactive chromatin statuses in ECAT15-2-null ESCs, but not in wild-type ESCs. Chromatin immuno-precipitation (ChIP) assay revealed that ECAT15-2 binds to the regulatory region of Nkx2-5 in ESCs. These data suggest that ECAT15-2 have important roles in lung development where it is no longer expressed, by leaving epigenetic marks from earlier developmental stage. The ECAT15-2 mutant ESCs (15-1CT/CT, 15-2-/-) were established from E3.5 blasotcysts of 15-1+/CT, 15-2+/- intercross (CT allele; conditional targeted allele). Then the Flag-tagged ECAT15-2/Dppa2 transgenic vector based on piggyBac system were introduced. They were harvested on gelation coated dish with ES medium containing 15% FCS and LIF for three days. The total RNA from two wild type, two mock transduced ECAT15-2 deficient ESCs and two Flag-tagged ECAT15-2 transduced ECAT15-2 deficient ESCs were labeled with Cy3. Samples were hybridized with Whole Mouse Genome Microarray 4 x 44K (G4122F, Agilent), with the one color protocol. Data was analyzed by using GeneSpring GX11 software (Agilent).

Project description:Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10^6 reads corresponding to >10^4 insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors. Solid tumors in mice were generated by somatic transposon mutagenesis with a PiggyBac transposon system. Insertion sites of transposons in 11 tumors and 6 non-cancerous tail controls were determined by Illumina high-throughput sequencing. Insertions were determined both on 5' and 3' sides of the transposon (PB5 and PB3, respectively). Quantitative analysis of read numbers revealed enrichment of certain insertions in tumors, but not in controls, and these enriched insertions identify candidate cancer genes.

Dataset Information

Transcriptional profiling of PADI4 overexpression and inhibition in mouse embryonic stem cells

Publications

Citrullination regulates pluripotency and histone H1 binding to chromatin.

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