Unknown,Transcriptomics,Genomics,Proteomics

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4C-seq from viewpoint at MYC promoter (VP-MYC1 and VP-MYC2), in wild type (Hap) and variously modified alleles around the locus in human iPS cells.

ABSTRACT: We reconstituted arrays of CTCF binding sites (L1, L2, L3, L4, R1, R2 and R3) and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. We inserted STITCH into five different positions of the remaining allele of the locus: \\"STITCH+30kb\\", \\"STITCH+440kb\\", \\"STITCH+1760kb\\" and \\"STITCH+1790kb\\" have the STITCH insertions away from the MYC promoter for the indicated distances to the telomeric side of the p arm of the chromosome. \\"STITCH-30kb\\", at the 30-kb upstream from the MYC. We also made a deletion clone of the enhancer region, termed del(30-440). We made deletion of each CTCF array, L (delL) and R (delR), inversion of R (invR), deletion of the middle five binding sites from L2 to R2 (del(L2-R2)), and deletion of the six sites but for R3 (del(L1-R2)) in STITCH+30kb. We also obtained deletion and inversion of the whole of STITCH (del(L1-R3) and inv(L1-R3)). We integrated a transgene consisting of tetR-KRAB followed by DNA encoding the 2A peptide and the puromycin resistant gene with piggyBac transposition into the genome in the STITCH+30kb clone (STITCH/KRAB). We performed 4C-seq (Circular chromatin conformation capture assay followed by deep-sequencing) from the MYC promoter as a viewpoint to see how STITCH impacts on the chromatin conformation.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Taro Tsujimura

PROVIDER: E-MTAB-7668 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Polyamines are aliphatic polycations that have emerged as important determinants of cell growth and viability in rapidly proliferating cells, including in the pathogenic protozoan parasite Leishmania donovani. In L. donovani, the polyamine spermidine is synthesized by the successive conversion of ornithine into putrescine (catalyzed by ornithine decarboxylase or ODC) and putrescine into spermidine (catalyzed by spermidine synthase or SPDSYN). Deletion of either ODC (del-odc) or SPDSYN (del-spdsyn) from the L. donovani genome renders these parasites auxotrophic for polyamines and these mutants are impaired in their ability to survive both in culture and within the mammalian host without the addition of exogenous polyamine supplementation. Significantly, del-odc parasites immediately cease proliferation after putrescine is removed from the culture media and perish within two weeks, while spermidine starved del-spdsyn mutants, which retain intracellular putrescine pools, show a slow-growth phenotype, and persist for several weeks in culture. To elucidate the key differences within the proteome of putrescine-starved del-odc cells and spermidine-starved del-spdsyn parasites, a shotgun quantitative proteomics approach was undertaken using TMT labeling and LC-MS/MS analysis. Briefly, three biological replicates each for mid-log phase del-odc and del-spdsyn promastigotes grown in the presence of exogenous putrescine (for del-odc) or spermidine (for del-spdsyn) supplementation were washed to remove the exogenous polyamine supplementation and incubated in polyamine-free media. At 24 and 48 h, cells from each biological replicate were isolated and prepared for tandem mass tag (TMT) labeling and downstream LC-MS/MS analyses. Peptides were identified using a database generated from the reference genome of L. donovani BPK282A1 strain. Changes in relative protein abundance for the polyamine-starved del-odc and del-spdsyn cell lines at 24 and 48 h were calculated by comparing aggregate total reporter ion intensities for each protein to that of the corresponding polyamine-supplemented 0-h timepoint.

Dataset Information

4C-seq from viewpoint at MYC promoter (VP-MYC1 and VP-MYC2), in wild type (Hap) and variously modified alleles around the locus in human iPS cells.

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