Dual chromatin recognition by the histone deacetylase complex HCHC is required for proper DNA methylation in Neurospora crassa

Whole-Genome Bisulfite Sequencing of HCHC mutants To extend our understanding of the role of the HCHC complex in Neurospora, we carried out whole-genome bisulfite sequencing (WGBS) of cdp-2, chap, and hda-1 mutants.  Consistent with prior analyses, the WGBS revealed both hypomethylated and hypermethylated regions in the three HCHC mutants while the ; sequences with a lower Combined RIP Index (CRI) tend to show reduced methylation in the mutants, while sequences with higher CRI scores show increased methylation. Analysis of the WGBS data also demonstrated that shorter methylated regions, regardless of their degree of methylation in wild type, commonly have reduced methylation in the HCHC mutants, while longer methylated sequences tend to have elevated methylation.  In addition, the borders of normally methylated regions typically lose methylation and show a contraction of boundary methylation in the HCHC mutants.  Sequences near telomeres that are normally methylated were found to lose methylation in the mutants, while most of the increased DNA methylation of the three HCHC mutants is found at centromeres. CHAP in vitro DNA-affinity purification HT-seq and CHAP DamID-Seq To test whether the CHAP AT-hook motifs bind AT-rich RIP’d DNA, we performed in vitro DNA-affinity purification with the recombinant CHAP-N-terminus (1-274 residues) containing the two AT-hook motifs and analyzed the purified DNA with high throughput sequencing (HT-Seq). To complement this approach, we also assessed binding of CHAP in vivo with DamID-seq using the CHAP-Dam strain. Together, these techniques gave us a detailed genomic view of the specific localization and binding of CHAP to AT-rich RIP’d DNA, which is nearly coincident with methylated DNA regions. Whole Genome Bisulfite-Seq and analysis of Neurospora crassa's four HCHC mutants (hda-1, cdp-2, chap, and hpo) and a wild type control strain. In vitro DNA-affinity purification using the amino terminus of the CHAP protein followed by high-throughput sequencing. DamID-seq analysis of CHAP-Dam in vivo binding. The CHAP AT-hook motifs specifically bind highly RIP?d/AT-rich DNA