Transcription_in_Mecp2_null_and_Cfp1_null_cells. Transcription_in_Mecp2_null_and_Cfp1_null_cells

The dinucleotide sequence 5’CG acts as a signalling module that can influence the epigenome and modulate chromosome function. CG exists in three chemically distinct forms that differ due to the modification status of the cytosine base: unmethylated, methylated or hydroxymethylated. The Cfp1 protein binds to unmethylated CG clusters in vivo and is required for the coincident trimethylation of lysine 4 in histone H3 by the Setd1 complex. On the other hand, the methyl-CG binding protein MeCP2 can direct deacetylation of histones. These findings align with evidence that nonmethylated CpG islands mark promoters, whereas CG methylation leads to gene silencing. Interfering with Cfp1 or Mecp2 function will therefore impact chromatin structure, possibly resulting in alteration of transcription. Protocol:Isolated nuclei were submitted to nuclear run-on in the presence of Br-UTP. Bulk RNA were then isolated, fragmented and Br-UTP-incorporated run-on transcripts were selectively enriched using anti-Br-dUTP antibody. Cells were crosslinked using 1% formaldehyde and chromatin sheared using sonication. Protein-DNA complexes were immunoprecipitated with the appropriate antibody and DNA was subsequently extracted.Adaptors were ligated and affinity-enriched RNA was reverse transcribed then amplified by PCR. The purified cDNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols Sequencing was performed at the Wellcome Trust Sanger Institute; Hinxton, Cambridge, UK This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/