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

ABSTRACT: 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.

ORGANISM(S): Neurospora crassa

PROVIDER: GSE81129 | GEO | 2017/01/01

SECONDARY ACCESSION(S): PRJNA320685

REPOSITORIES: GEO

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Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track was produced as part of the ENCODE project. It reports the percentage of DNA molecules that exhibit cytosine methylation. In general, DNA methylation within a gene's promoter is associated with gene silencing, and DNA methylation within the exons and introns of a gene is associated with gene expression. Proper regulation of DNA methylation is essential during development and aberrant DNA methylation is a hallmark of cancer. DNA methylation status was assayed with Whole Genome Bisulfite Sequencing (WGBS). Genomic DNA was sheared by sonication, end-repaired and then ligated to methylated sequencing adapters. The library fragments were treated with sodium bisulfite and amplified by PCR to convert every unmethylated cytosine to a thymine while leaving methylated cytosines intact. The sequenced fragments were aligned to a bisulfite-converted reference genome. For each assayed cytosine, the number of sequencing reads covering that C and the percentage of those reads that were methylated were reported. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf DNA methylation at cytosines across the genome was assayed with Whole Genome Bisulfite Sequencing (WGBS). WGBS was performed on cell lines grown by ENCODE production groups. WGBS was carried out by the Myers production group at the HudsonAlpha Institute for Biotechnology. Isolation of Genomic DNA: Genomic DNA was isolated from each cell line using the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer. DNA concentrations for each genomic DNA preparation were determined using fluorescent DNA-binding dye and a fluorometer (Invitrogen Quant-iT dsDNA High Sensitivity Kit and Qubit Fluorometer). Typically, 2 µg of genomic DNA is used to make WGBS libraries. WGBS Library Construction and Sequencing: WGBS library construction started with sonication of genomic DNA on a Covaris S2 instrument. Sheared ends were then repaired and blunted with DNA polymerase I, T4 DNA polymerase and T4 polynucleotide kinase in the presence of dATP, dGTP and dTTP. After end repair, Klenow exo- DNA Polymerase was used to add an adenosine as a 3' overhang. Next, a methylated version of the Illumina paired-end adapters was ligated onto the DNA. Adapter-ligated 400 bp genomic DNA fragments were selected using a 2% agarose SizeSelect E-gel. The selected adapter-ligated fragments were treated with sodium bisulfite using the Zymo Research EZ DNA Methylation Gold Kit, which converts unmethylated cytosines to uracils and leaves methylated cytosines unchanged. Bisulfite-treated DNA was amplified in a final PCR reaction which was optimized to uniformly amplify diverse fragment sizes and sequence contexts in the same reaction. During this final PCR reaction, uracils were copied as thymines, resulting in a thymine in the PCR products wherever an unmethylated cytosine existed in the genomic DNA. These libraries were then sequenced with an Illumina HiSeq 2000 according to the manufacturer's recommendations as paired-end 50 bp reads. Libraries were sequenced to a depth of 600 million aligned reads. Data Analysis: To analyze the sequence data, Bismark (Krueger and Andrews, 2011) was used to align sequences reads. Generally, each read went through a conversion of Cs to Ts and was then aligned to fully converted plus and minus strands of the hg19 build of the human genome. A few custom refinements were made to the Bismark program. Since these libraries were made in a directional orientation with the first read always being C-poor, we skipped unnecessary alignments to impossible orientations. We also implemented a more stringent uniqueness filter, only allowing reads that have one acceptable alignment (based on default Bowtie parameters) across both strands. Once reads were aligned, the percent methylation was calculated for each cytosine using the original sequence reads. The percent methylation and number of reads is reported for each CpG in the wgEncodeHaibMethylWgbsXXXXCpg.bigBed file and for each non CpG cytosine in the wgEncodeHaibMethylWgbsXXXXNoncpg.bigBed file.

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

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