Unknown,Transcriptomics,Genomics,Proteomics

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ChIP-seq analysis of Guanylate Cyclase Soluble Subunit Beta-3 (GUCY1B3 or sGCÎ²1) binding to chromatin DNA

ABSTRACT: To identify sGCÎ²1 binding sites to chromatin DNA, we performed ChIP analysis by using anti-sGCÎ²1 antibody (Sigma G4405). We have successfully identified 4169 intervals. In this study, sGCÎ²1 was found to bind to many genes' promoter region. We used U-87 MG (ATCCÂ® HTB-14) glioma cells which overexpress sGCÎ²1 (generated by us) to perform ChIP assay by using anti-sGCÎ²1 antibody (Sigma G4405) and genome-wide sGCÎ²1 binding was determined with sequencing. The ChIP DNA was processed into a standard Illumina ChIP-Seq library and sequence. The 75-nt sequence reads generated by Illumina sequencing (using NextSeq 500) are mapped to the hg19 genome using the BWA algorithm with default settings. Only reads that pass Illuminaâs purity filter, align with no more than 2 mismatches, and map uniquely to the genome are used in the subsequent analysis. Intervals (= Peaks) were determined using the MACS peak finding algorithm.

ORGANISM(S): Homo sapiens

SUBMITTER: haijie xiao

PROVIDER: E-GEOD-83419 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Eukaryotic nitric oxide (NO) signaling involves modulation of cyclic GMP (cGMP) levels through activation of the soluble isoform of guanylate cyclase (sGC). sGC is a heterodimeric hemoprotein that contains a Heme-Nitric oxide and OXygen binding (H-NOX) domain, a Per/ARNT/Sim (PAS) domain, a coiled-coil (CC) domain, and a catalytic domain. To evaluate the role of these domains in regulating the ligand binding properties of the heme cofactor of NO-sensitive sGC, we constructed chimeras by swapping the rat ?1 H-NOX domain with the homologous region of H-NOX domain-containing proteins from Thermoanaerobacter tengcongensis, Vibrio cholerae, and Caenorhabditis elegans (TtTar4H, VCA0720, and Gcy-33, respectively). Characterization of ligand binding by electronic absorption and resonance Raman spectroscopy indicates that the other rat sGC domains influence the bacterial and worm H-NOX domains. Analysis of cGMP production in these proteins reveals that the chimeras containing bacterial H-NOX domains exhibit guanylate cyclase activity, but this activity is not influenced by gaseous ligand binding to the heme cofactor. The rat-worm chimera containing the atypical sGC Gcy-33 H-NOX domain was weakly activated by NO, CO, and O(2), suggesting that atypical guanylate cyclases and NO-sensitive guanylate cyclases have a common molecular mechanism for enzyme activation. To probe the influence of the other sGC domains on the mammalian sGC heme environment, we generated heme pocket mutants (Pro118Ala and Ile145Tyr) in the ?1 H-NOX construct (residues 1-194), the ?1 H-NOX-PAS-CC construct (residues 1-385), and the full-length ?1?1 sGC heterodimer (?1 residues 1-619). Spectroscopic characterization of these proteins shows that interdomain communication modulates the coordination state of the heme-NO complex and the heme oxidation rate. Taken together, these findings have important implications for the allosteric mechanism of regulation within H-NOX domain-containing proteins.

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ChIP-seq analysis of Guanylate Cyclase Soluble Subunit Beta-3 (GUCY1B3 or sGCÎ²1) binding to chromatin DNA

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