Figure 1. Distinct chromatin regions isolated by the N-ChroP strategy

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Figure 1. Distinct chromatin regions isolated by the N-ChroP strategy Figure 1. Distinct chromatin regions isolated by the N-ChroP strategy. (A) Scheme of the N-ChroP experimental approach. (B) Heatmap visualisation of histone PTM relative enrichment (log<sub>2</sub>) in H3K4me1, H3K4me3 and H3K9me3-immunopurified nucleosomes; modifications are clustered in peptides belonging to histone H3 (up) and H4 (down). Data from three biological independent replicates (I, II and III) are shown. (C) Volcano plots of the histone PTMs that were identified and quantified. P-values and t-test differences were plotted against each other in the volcano plot, comparing ChIPs of H3K4me1 (left) and H3K4me3 (right) with that of H3K9me3. Histone PTMs with P ≤ 0.05 were selected and highlighted as significantly enriched, using two-side t-test and Bonferroni correction. (D) Western blot validation of H3K36me2 (left) and H3K79me2 (right) enrichment in H3K4me1 ChIPs, with intensity signals quantified by ImageJ. The enrichment for each modification in the different ChIPs was calculated as follows: the signal of each PTM in the ChIPs and in the input was divided by the signal of the corresponding unmodified H3, after which the obtained ratio in each ChIP was normalized to the input value. From: Chromatin proteomics reveals novel combinatorial histone modification signatures that mark distinct subpopulations of macrophage enhancers Nucleic Acids Res. 2017;45(21):12195-12213. doi:10.1093/nar/gkx821 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 2. ChIP-seq analysis validates the MS-based histone PTM profiling. (A) Global genomic distribution of peaks for H3K36me2, H3K36me3, H3K79me2 and histone PTMs used as baits in N-ChroP, at the basal state. TSS are defined as regions ±1 kb around known transcription starting sites in the RefSeq database. (B) Heatmap representing the chromatin state model generated by ChromHMM for histone PTMs at the basal state. (C) Relative quantification of the overlap between H43K4me1 and other histone modifications based on chromatin state identification. Overlaps between H3K4me1 and H3K36me2 (26% of H3K4me1-containing chromatin states), H3K36me3 (10%) or H3K79me2 (24%) are highlighted. From: Chromatin proteomics reveals novel combinatorial histone modification signatures that mark distinct subpopulations of macrophage enhancers Nucleic Acids Res. 2017;45(21):12195-12213. doi:10.1093/nar/gkx821 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 3. Genome-wide distribution of H3K36me2, H3K36me3 and H3K79me2 around Pu.1. (A) Reads distribution around Pu.1 binding sites that overlap with enhancers marked by H3K36me2, H3K36me3 or H3K79me2. Chromatin states #2, #11 and #5 indicate regions in which H3K4me1 co-localized with H3K36me2, H3K36me3 or H3K79me2, respectively. (B) Representative ChIP-seq signal snapshots showing the distribution of H3K36me2, H3K36me3 and H3K79me2 relative to the H3K4me1 and Pu.1 profiles. From: Chromatin proteomics reveals novel combinatorial histone modification signatures that mark distinct subpopulations of macrophage enhancers Nucleic Acids Res. 2017;45(21):12195-12213. doi:10.1093/nar/gkx821 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 4. Dynamic profiling of histone PTMs at enhancers during the inflammatory response. (A) Experimental design for the N-ChroP time-course using H3K4me1 as bait. UT, untreated cells; 1h or 4h, cells treated with lipopolysaccharide (LPS) for 1 or 4 h, respectively. (B) Heatmap visualization of the dynamic relative enrichment of histone PTMs during LPS treatment (log<sub>2</sub>). Data are shown as a mean of three independent biological replicates (n = 3). (C) Line graphs for a subset of histone PTMs, representing the fold enrichment over time. UT, 1 h and 4 h (x-axis) correspond to cells untreated or treated with LPS for 1 h or 4 h, respectively. (D) Left histograms represent the log<sub>2</sub> relative enrichment of peptides containing K36me2 and K36me3 with K27me in the H3 27–40 aa peptide. The right histogram indicates the relative enrichment of K79 modifications in the H3 73–83 aa peptide. Values are expressed as mean ± s.e. (standard error) from three independent biological experiments (n = 3), before and after LPS treatment. (E) Global genomic distribution of peaks for H3K36me2, H3K36me3 and H3K79me2 after 1 h or 4 h (upper and lower panels, respectively) of LPS treatment. TSS are defined as regions ±1 kb around known TSS in the RefSeq database. (F) Representative ChIP-Seq signal snapshots showing the distribution of H3K4me1, H3K36me2, H3K36me3 and H3K79me2, before and after LPS treatment. From: Chromatin proteomics reveals novel combinatorial histone modification signatures that mark distinct subpopulations of macrophage enhancers Nucleic Acids Res. 2017;45(21):12195-12213. doi:10.1093/nar/gkx821 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 5. Characterization of different intronic enhancer subpopulations. (A) Distribution and enrichment of chromatin states in a 10-kb window surrounding TSS. Chromatin states #5 and #6 correspond to regions in which H3K4me1 co-localized with H3K79me2 alone or with H3K4me3, respectively. Chromatin state #7 represents regions in which H3K79me2 was co-enriched with H3K4me3. The chromatin state #11 represents regions in which H3K4me1 and H3K36me3 co-localized. (B) Global distribution of chromatin states #5, #6, #7 and #11. (C) Heatmap representing the chromatin state model at basal level and after 1 h or 4 h of LPS treatment, including ChIP-seq profiles for H3K27ac and H3K27me3. (D) Distribution of RNA quantification values, either from total (left and central panel) or nascent (right panel) RNA analysis, associated with different sets of chromatin states. Chromatin states #7, #8, #14 and #17 represent regions in which H3K4me1 co-localized with K79me2, K27ac/K79me2, K27ac/K36me3 or K27ac, respectively. Chromatin states #16 and #18 represents instead regions in which H3K4me1 is present alone or in combination with H3K27me3. (E) TF binding motifs enriched for enhancers marked by H3K4me1/K36me3 and H3K4me1/K79me2. From: Chromatin proteomics reveals novel combinatorial histone modification signatures that mark distinct subpopulations of macrophage enhancers Nucleic Acids Res. 2017;45(21):12195-12213. doi:10.1093/nar/gkx821 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 6. Functional analysis of genes associated with different populations of intronic enhancers. (A) Gene ontology (GO) analysis and enriched pathways in genes hosting K4me1-, K4me1/K79me2- or K4me1/K36me3-marked active enhancers, at basal state or after LPS stimulus. Significantly enriched GO terms are indicated both by colour code and the P-value shown within each cell. (B) Conversion of intronic enhancers after LPS induction: schematic representation of class II enhancers (K4me1/K27me3) that convert to class I enhancers (K4me1/K27ac) after LPS stimulus. GO enrichment of the novel class I enhancers marked by H3K4me1/K27ac and H3K4me1/K27ac/K36me3 is shown. From: Chromatin proteomics reveals novel combinatorial histone modification signatures that mark distinct subpopulations of macrophage enhancers Nucleic Acids Res. 2017;45(21):12195-12213. doi:10.1093/nar/gkx821 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 7. The combinatorial mark H3K36me2/K4me1 associates with transcribed enhancers. (A) Comparison between the number of intergenic enhancers marked by the H3K4me1/K36me2 combination (states #2 and #3) or by H3K4me1 alone (states #16 and #17), as identified by the 27-state ChromHMM model. Within these classes, active or non-active enhancers were also divided based on the presence or absence of H3K27ac, and the distribution of transcript levels associated with the two groups is shown. (B) Examples of representative genomic regions displaying H3K4me1, H3K36me2, H3K27ac and nascent RNA enrichment. (C) Distribution of transcribed extragenic enhancers belonging to the K4me1 and K4me1/K36me2 groups, as compared to annotated genomic features; the enrichment of active and super-enhancers in the H3K4me1/K36me2 group is highlighted. (D) Distribution of H3K4me1 (left) and H3K36me2 (right) normalized read counts in H3K4me1/K36me2-marked enhancers in cells untreated or LPS-stimulated, with or without flavopiridol (FP) pretreatment. (E) mRNA levels of NSD1 and NSD2 measured by RT-qPCR analyses in RAW 264.7 cells expressing the scrambled (shScramble) and NSD1- and NSD2-specific shRNAs (shNSD1 and shNSD2). Gene expression is normalized to TBP mRNA level. Values are expressed as mean ± s.e. (standard error) of two biological replicates (n = 2). (F) NSD1 protein level in cells expressing scrambled (shScramble) and NSD1-specific shRNA (upper panel). Protein LFQ ratio distribution from label-free quantitative (LFQ) MS-analysis of whole extracts from cells expressing NSD2-specific shRNAs (shNSD2) versus shScramble cells. NSD2 LFQ ratio value is indicated with the green dot and equals log<sub>2</sub> = –1.4, confirming its down-regulation. (G) Western blot analysis of H3K36me2 from protein extracts from cells expressing scrambled (shScramble) and NSD1- and NSD2-specific shRNAs (shNSD1 and shNSD2). Vinculin and Ponceau staining are used as loading control. (H) qPCR of extragenic regions marked by H3K4me1/K36me2 after H3K36me2 ChIP in cells expressing scrambled (shScramble) or NSD1- and NSD2-specific shRNAs (shNSD1 and shNSD2), at 4 h after LPS treatment. Data are expressed as percent of the input. (I) Quantitative RT-qPCR using 4sU-labeled RNAs on extragenic regions marked by H3K4me1/K36me2 in cells expressing scrambled (shScramble) and NSD1- and NSD2-specific shRNAs (shNSD1 and shNSD2), at 4 h after LPS treatment. Values are expressed as mean ± s.e. (standard error) of two biological replicates (n = 2). From: Chromatin proteomics reveals novel combinatorial histone modification signatures that mark distinct subpopulations of macrophage enhancers Nucleic Acids Res. 2017;45(21):12195-12213. doi:10.1093/nar/gkx821 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com