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Proteogenomic characterization and mapping of nucleosomes decoded by Brd and HP1 proteins

Gary LeRoy1, Iouri Chepelev2, Peter A DiMaggio1, Mario A Blanco1, Barry M Zee13, Keji Zhao2 and Benjamin A Garcia1345*

Author Affiliations

1 Department of Molecular Biology, Princeton University, 415 Schultz Laboratory, Princeton NJ 08544, USA

2 Systems Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA

3 Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 1009C Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, PA19104, USA

4 Department of Chemistry, Princeton University, Princeton NJ 08544, USA

5 Quantitative and Computational Biology Program, Princeton University, Princeton, NJ 08544, USA

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Genome Biology 2012, 13:R68  doi:10.1186/gb-2012-13-8-r68

Published: 16 August 2012

Additional files

Additional file 1:

Western blots of whole cell extracts from cell lines expressing FLAG-Brd4, FLAG-HP1β and control cell line (empty vector). Blots were probed with anti-Brd4 (mAb Epitomics, 5716 Burlingame, CA, USA), anti-HP1β (pAb Cell Signaling Technology 2613 Danvers, MA, USA) and β-actin control (mAb Santa Cruz, sc-81178 Santa Cruz, CA, USA).

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Additional file 2:

Table of relative PTM abundances determined by quantitative mass spectrometry on histones H3 and H4 averaged from three independent ChIP experiments with each Brd and HP1 protein and data from three experiments with HEK293 genomic chromatin.

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Additional file 3:

Table of relative PTM abundances determined by quantitative mass spectrometry on the histone H4 peptide (amino acids 4 to 17) averaged from three independent ChIP experiments with each Brd and HP1 protein and data from three experiments with HEK293 genomic chromatin.

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Additional file 4:

P-values from t-tests performed on the fold changes (ChIP/Genomic) from the histone H4 data presented in Additional file 3. t-Tests were performed with data from three independent ChIP experiments for each Brd and HP1 protein and data from three experiments with HEK293 genomic chromatin. P-values were adjusted using the Benjamini-Hochberg correction method to control the false discovery rate (FDR).

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Additional file 5:

P-values from t-tests performed on the fold changes (ChIP/Genomic) from the histone data presented in Additional file 2. t-Tests were performed with data from three independent ChIP experiments for each Brd and HP1 protein and data from three experiments with HEK293 genomic chromatin. P-values were adjusted using the Benjamini-Hochberg correction method to control the false discovery rate (FDR).

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Additional file 6:

Table of relative combinatorial PTM abundances determined by quantitative mass spectrometry on the histone H3 peptides (amino acids 9 to 17), (amino acids 18 to 26) and (amino acids 27 to 40) averaged from three independent ChIP experiments with each Brd and HP1 protein and data from three experiments with HEK293 genomic chromatin.

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Additional file 7:

P-values from t-tests performed on the fold changes (ChIP/Genomic) from the histone H3 data presented in Additional file 6. t-Tests were performed with data from three independent ChIP experiments for each Brd and HP1 protein and data from three experiments with HEK293 genomic chromatin. P-values were adjusted using the Benjamini-Hochberg correction method to control the false discovery rate (FDR).

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Additional file 8:

Spreadsheets of all promoters bound by the Brd and HP1 proteins. Promoters are ranked by P-values.

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Additional file 9:

Heatmap of motifs enriched in the HP1 and Brd ChIPs. Lists of consensus sequences (motifs) are found in the matrix used to create the heatmap (Additional file 10)

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Additional file 10:

Spreadsheets containing matrix used to create the heatmap of motifs enriched in Brd and HP1 ChIPs (Additional file 9).

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Additional file 11:

Spreadsheets containing Gene Ontology terms enriched in Brd and HP1 ChIPs. Gene Ontology terms are ranked by false discovery rates (FDRs).

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Additional file 12:

Products from PCR reactions were run on 2% agarose gels stained with ethidium bromide and visualized on a Gel Doc XR system (BioRad® Hercules, CA, USA). One half of each PCR reaction was loaded. Gel is labeled corresponding to the templates used for the PCR reactions: control ChIP (beads alone), Brd4 ChIP and ChIP input DNA.

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Additional file 13:

Western blots of whole cell extracts from Brd4 shRNA knockdown, HP1β shRNA knockdown and control shRNA knockdown cell lines. Blots were probed with anti-Brd4 (mAb Epitomics, 5716), anti-HP1β (pAb Cell Signaling Technology 2613) and β-actin control (mAb Santa Cruz, sc-81178).

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Additional file 14:

Supplemental Materials and methods.

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