Unknown,Transcriptomics,Genomics,Proteomics

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Mecp2: an unexpected regulator of macrophage gene expression and function [ChIP-Seq]

ABSTRACT: Mutations in methyl-CpG-binding protein 2 (MeCP2), a major epigenetic regulator, are the predominant cause of Rett syndrome. We previously found that Mecp2-null microglia are deficient in phagocytic ability, and that engraftment of wild-type monocytes into the brain of Mecp2-deficient mice attenuates pathology. We have observed that Mecp2 deficiency is associated with increased levels of histone acetylation at the cis-regulatory regions of the Mecp2-regulated genes in macrophages. We hypothesized that Mecp2 recruits protein complexes containing histone deacetylases (HDACs) to repress the expression of its target genes. Our ChIP-Seq studies in bone-marrow derived macrophages revealed that Mecp2 co-localizes with Ncor2/Hdac3 protein complex at cis-regulatory regions of the target genes. These results suggest a role for Mecp2 in the recruitment and regulation of Ncor2/Hdac3 repressosome that plays a critical role in the regulation of inflammatory responses in macrophages. Examination of NCOR2 and HDAC3 genome-wide location in bone-marrow derived macrophages.

ORGANISM(S): Mus musculus

SUBMITTER: Stephen Turner

PROVIDER: E-GEOD-66501 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The conserved core domain of the TATA binding protein (TBP) interacts with multiple partners forming the complexes required for transcription by RNA Polymerases I, II and III. We use genetically modified mouse embryonic fibroblasts to show that many TBP core domain mutants complement loss of endogenous TBP, but this often results in a slow growth phenotype. Two TBP mutations, R188E and K243E, disrupt the TBP-BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not affect global genomic distribution of TBP, but positively or negatively affects TBP and/or RNA Polymerase II (Pol II) recruitment to a selected set of promoters. We identify a set of promoters where wild-type TBP assembles a partial inactive preinitiation complex lacking Pol II and TAF1. Our results suggest that an exchange of the B-TFIID complex in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters recruits Pol II to activate their expression. We also observe that both Wt and mutant TBP can occupy promoters without concurrent Pol II recruitment and active transcription. Our data reveal a novel regulatory mechanism involving the formation of a partial preinitiation complex that primes the promoter for productive preinitiation complex formation in mammalian cells. Using homologous recombination, we generated ES cells and mice harbouring a null allele in the Tbp gene by insertion of a hygromycin resistance cassette in exon III and a floxed allele, in which exon III is surrounded by LoxP sites. From these mice, we generated an embryonic fibroblast Tbplox/- cell line in which TBP expression can be inactivated by expression of the Cre recombinase leading to cell death. We adopted a two-step strategy to generate Tbp-/- cell lines expressing human (h)TBP. Cells were first infected with pBABE retrovirus vectors expressing Wt hTBP or a series of mutants in the TBP core region, all of which carry a Flag-HA tag on their N-terminus. Cells expressing endogenous mTBP and exogenous hTBP were then infected with a second retrovirus expressing the 4 hydroxy-tamoxifen (OHT) inducible Cre-ERT2. Subsequently, multiple clonal populations from the OHT treated cells were isolated and genotyped by PCR to identify the Tbp-/-clones. At least two independent clones where expression of the endogenous mTBP was lost and replaced by the exogenous hTBP were isolated.

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Mecp2: an unexpected regulator of macrophage gene expression and function [ChIP-Seq]

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