Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

ChIP-seq validation of recombinant antibodies to histone post-translational modifications

ABSTRACT: Recombinant antibodies to histone post-translational modifications (PTMs), with their essentially infinite renewability, could fundamentally eliminate a major source of low reproducibility in epigenetics research. Here, we report new recombinant antibodies to trimethylated Lys4 and Lys9, respectively, on histone H3. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications, including ChIP. These results demonstrate the feasibility of generating recombinant antibodies to a range of histone marks, which will accelerate epigenetics research. We characterized recombinant antibodies with native ChIP using HEK293 cells followed by deep sequencing.

ORGANISM(S): Homo sapiens

SUBMITTER: Takamitsu Hattori

PROVIDER: E-GEOD-66530 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation.

Hattori Takamitsu T Lai Darson D Dementieva Irina S IS Montaño Sherwin P SP Kurosawa Kohei K Zheng Yupeng Y Akin Louesa R LR Świst-Rosowska Kalina M KM Grzybowski Adrian T AT Koide Akiko A Krajewski Krzysztof K Strahl Brian D BD Kelleher Neil L NL Ruthenburg Alexander J AJ Koide Shohei S

Proceedings of the National Academy of Sciences of the United States of America 20160209 8

Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and hi ...[more]

PMID: 26862167

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Project description:We produced a genome-wide map of the distribution of macroH2A1 histone variants in mouse liver chromatin using high-throughput sequencing of DNA from macroH2A1 nucleosomes. Although macroH2A1 nucleosomes are widely distributed across the genome, their local concentration varies over a range of 100-fold or more. The transcribed regions of most active genes are depleted of macroH2A1, with many showing depletion of 3-fold or more. The inactive X chromosome appears to have relatively uniform macroH2A1 enrichment that averages approximately 3.9-fold, but most genes that are known to escape from X inactivation do not show this enrichment. We used macroH2A1 depletion to identify additional genes that escape X inactivation, but overall, our map supports the idea that relatively few genes escape in the mouse. The map also helped identify genes that appear to be direct targets of macroH2A1-mediated repression. MacroH2A1 nucleosomes were purified from female mouse liver chromatin by thiol-affinity chromatography (Mol. Cell Biol. 26, 4410 (2006)) and their distribution was compared to that of the bulk nucleosomes used as the starting material for the purification. Nucleosomal DNA from two independent preparations was pooled and mononucleosomal DNA was prepared from both macroH2A1 and starting material nucleosomes. Each preparation used 8 to 10 livers from 8 to 10 week old female mice. For the relative macroH2A1 content file (see supplementary file), we used tools in the rna-seq workflow of Partek Genomics Suite (version 6.5b, 6.09.0806, Partek Inc.) to calculate the relative macroH2A1 content of individual genes. This program counts and normalizes hits in predefined regions specified in a refFlat file. The gene list was the Mouse build 8 version of refFlat.txt obtained from the UCSC Genome site. All exon information was stripped from the refFlat.txt file, which left only the start and stop sites for the genes. Normalized RPKM (reads per kilobase per million reads) values were compared to calculate fold change and FDR-adjusted p-values for each transcript.

Dataset Information

ChIP-seq validation of recombinant antibodies to histone post-translational modifications

Publications

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation.

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