Epigenome microarray platform for proteome-wide dissection of chromatin-signaling networks - PubMed (original) (raw)

. 2009 Aug 26;4(8):e6789.

doi: 10.1371/journal.pone.0006789.

Alex J Kuo, Peggie Cheung, Chih Long Liu, Valentina Migliori, Alexsandra Espejo, Fabio Casadio, Christian Bassi, Bruno Amati, Mark T Bedford, Ernesto Guccione, Or Gozani

Affiliations

Epigenome microarray platform for proteome-wide dissection of chromatin-signaling networks

Dennis J Bua et al. PLoS One. 2009.

Abstract

Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Key steps in the human epigenome peptide microarray (HEMP) procedure.

Figure 2

Figure 2. HEMP slides as a diagnostic tool for testing antibody specificity.

(a) Array images for antibodies: i) anti-H3K9me1, ii) anti-H3R2me2 (asymmetric), iii) anti-H3K18ac, iv) anti-γH2AX, and v) anti-H3K20me3 with schematic of array layout and key. (b) Heatmap representation of antibody HEMP slide data (See Table S2 for additional antibody details). The epitope(s) that the antibody was generated against is/are highlighted with a white border. See “Heatmap PTM key” for details about peptides with post-translational modifications (PTMs). Note, that di-methyl arginine residues with blue circles are symmetrically di-methylated. SNR RN, signal-to-noise ratio range-normalized. n.t., not tested.

Figure 3

Figure 3. Detection of known chromatin effector-histone PTM interactions using HEMP slides.

(a) The chromodomain of Drosophila melanogaster heterochromatin protein 1 alpha (dsHP1CD), (b) the plant homeodomain of human inhibitor of growth 3 (ING3PHD), and (c) the double chromodomains of human chromodomain-helicase-DNA-binding protein 1 (CHD1CD), all recognize, as indicated, their cognate histone ligand on the peptide array. All these protein domains are expressed as GST-fusions and an array probed with GST alone (d) serves as a negative control. For order of peptide spotting, see schematic in Figure 2a.

Figure 4

Figure 4. Identification of three novel methyl-histone binding modules.

(a) Table summarizing the number of domains tested in this study and the number of interactions detected. CD, chromodomain. TD, tudor domain. (b) Array images for: i) MPP8CD, ii) TDRD7TD, and iii) JMJ2CTD. Peptide/s detected in each experiment is indicated. See Figure 2a for array schematic. (c) Validation of array results in peptide-binding assays. Biotinylated peptide pull-down assay using peptides detected in (b) and the indicated GST-fusion proteins. (d) Co-localization of CDYL1 with H3K9me3. Representative immunofluorescence images of U2OS cells transfected with His-tagged CDYL1 and co-stained with the indicated antibodies. K9 = H3K9me3, K4 = H3K4me3. (e) Validation of array results in bulk-histone binding assays. Calf-thymus histone pull-down with the indicated proteins: MPP8CD, TDRD7TD, and JMJ2CTD. In each case the domain was pulled-down and the pellet was probed with the indicated antibodies. (f) MPP8CD binds to HeLa-purified nucleosomes enriched for H3K9me3 but not H3K4me3. Pull-downs of GST or GST-MPP8CD protein after incubation with HeLa nucleosomes were probed with the antibodies indicated (see Fig. S1 for quantitation).

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