Volume 61, Issue 1, Pages 170-180 (January 2016) A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes  Peter van Galen, Aaron D. Viny, Oren Ram, Russell J.H. Ryan, Matthew J. Cotton, Laura Donohue, Cem Sievers, Yotam Drier, Brian B. Liau, Shawn M. Gillespie, Kaitlin M. Carroll, Michael B. Cross, Ross L. Levine, Bradley E. Bernstein  Molecular Cell  Volume 61, Issue 1, Pages 170-180 (January 2016) DOI: 10.1016/j.molcel.2015.11.003 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 61, 170-180DOI: (10.1016/j.molcel.2015.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 A Multiplexed, Quantitative, and Low-Input Assay for Profiling Chromatin States (A) Overview of Mint-ChIP protocol. Following (1) lysis and MNase digestion, (2) a ligation mix inactivates MNase, repairs DNA ends, and ligates barcoded T7-adapters to nucleosomes (index #1). (3) The indexed samples are pooled and then split for parallel ChIP assays. (4) The DNA is isolated and amplified by in vitro transcription, yielding RNA, which is then (5) reverse transcribed. (6) PCR amplification yields (7) an Illumina seq library (index #2). (8) The seq data are de-multiplexed in silico based on their barcodes, yielding profiles for each sample (index #1) and each mark (index #2). (B) Plot depicts proportions of Mint-ChIP reads that align to the human or mouse genomes. The x axis indicates the relative ratios of T7-adapter-ligated chromatin (human) to carrier chromatin (mouse). The mouse carrier lacks T7-adapters and is not amplified or sequenced. The data are shown as mean ± SD of 4 ChIP assays × 5 MNase concentrations. (C) Pie charts indicate T7-adapter barcode representations in Mint-ChIP seq data for total H3. These data validate the Mint-ChIP procedures for indexing and pooling chromatin and in silico de-multiplexing. (D) Four human samples (K562, T7-adapter barcode 1–4) and two mouse samples (YAC-1, T7-adapter barcode 5–6) were indexed, pooled, and split for three parallel Mint-ChIP assays. The plot depicts the proportions of reads for each barcode that align to the human or mouse genomes. The data are shown as mean ± SD of three ChIP assays. See also Figure S1. Molecular Cell 2016 61, 170-180DOI: (10.1016/j.molcel.2015.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Validation of Chromatin Data and Sensitivity to Low-Input Samples (A and B) Data tracks show (A) H3K4me3 and (B) H3K27me3 profiles derived by Mint-ChIP using indicated starting cell numbers. For comparison, ENCODE data generated by conventional ChIP-seq are also shown. (C) Density plots compare Mint-ChIP and ENCODE data for K562 cells. The datapoints compare the number of reads in Mint-ChIP (x axis) versus ENCODE (y axis) for all promoter intervals (H3K4me3), H3K27ac peaks called from ENCODE data (H3K27ac), or all annotated transcripts (H3K27me3). The R indicates Pearson correlation. (D) Workflow for hematopoietic stem cell analysis. The CD34+CD38−CD45RA− cells were isolated from human bone marrow by flow cytometry. Mint-ChIP was used to analyze histone modifications. (E) Data tracks show H3K27ac, H3K27me3, and H3K36me3 profiles of hematopoietic stem cells at the HOXA locus. (F) Density plots depict correlation between methylation within genes (H3K36me3 and H3K27me3) and mRNA expression in human hematopoietic stem cells. Each data point corresponds to a single gene; some genes are highlighted as examples. See also Figure S1. Molecular Cell 2016 61, 170-180DOI: (10.1016/j.molcel.2015.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Mint-ChIP Quantitative Normalization Clarifies Global Differences in Histone Modification Levels (A) Graphic for Mint-ChIP quantitative normalization. The control or drug treated cells are indexed, pooled, and then split for parallel ChIP assays. The ratio between H3K27me3 reads and H3 reads is used to compare global H3K27me3 levels between samples and normalize corresponding profiles. (B) Western blot shows H3K27ac and H3K27me3 levels in K562 cells following treatment with the p300 inhibitor C646 or the EZH2 inhibitor GSK126 (compared to DMSO control). The total H3 is shown as a loading control. (C) Bar plots show global modification levels inferred from western blot (top) or Mint-ChIP (bottom). The respective methods were applied in parallel to the same sample of K562 cells treated for 48 hr with the indicated inhibitors. The data are shown as mean ± SD of n = 3 independent experiments (symbols indicate values from independent experiments). (D) Diagram explains difference between normalization methods. The global differences in histone modification levels (e.g., by demethylase inhibition) may be masked by conventional ChIP-seq signal normalization (RPM). In contrast, quantitative normalization enables direct peak height comparisons between the samples. (E) Western blot shows increased H3K4me3 levels in K562 cells following treatment with the demethylase inhibitor KDM5-C70. The total H3 is shown as a loading control and n = 3 experiments are shown. (F) Bar plots show global H3K4me3 levels inferred from Mint-ChIP. The data are shown as mean ± SD of four replicates and n = 3 experiments are shown. (G) Data tracks show H3K4me3 profiles, scaled by conventional or quantitative normalization. (H) Composite plot depicts average H3K4me3 signals in K562 cells treated with DMSO or KDM5-C70. There are 10 kb regions surrounding the centers of 36,875 peaks that are shown. (I) Bar plot shows the fraction of peaks within size windows. The peaks of >10 kb were classified as 10 kb such that the total area is one. (J) Venn diagram shows the number of peaks detected in K562 cells treated with DMSO or KDM5-C70. See also Figure S2. Molecular Cell 2016 61, 170-180DOI: (10.1016/j.molcel.2015.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Quantitative Normalization Resolves Distinct Chromatin Landscapes Resulting from Cancer Mutations and Drug Treatment (A and B) Heatmaps compare H3K27me3 or H3K27ac levels in different cell lines treated with GSK126, as quantified by Mint-ChIP. These experiments were performed using two different MNase concentrations, which are typically averaged. (C) Bar plot shows mass spectrometry quantification of H3K27ac in Pfeiffer, SKM-1, and Toledo (Jaffe et al., 2013). The mass spectrometry data match the normalized Mint-ChIP data. (D) Composite plots depict average H3K27ac signals over 20 kb regions surrounding the centers of 23,176 peaks. The values were computed by conventional normalization, wherein signal is relative to total read numbers (RPM, left) or by the quantitative normalization afforded by Mint-ChIP (right). (E) Bar plots depict viable cell counts following 72 hr GSK126 treatment of Pfeiffer, SKM-1, and Toledo. The data are shown as mean ± SD of technical triplicates × n = 2 independent experiments (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001). (F) Composite plots depict average H3K27me3 signals over 40 kb regions surrounding the centers of 2,052 peaks. Together, these data demonstrate the unique capacity of Mint-ChIP to quantitatively map and compare chromatin landscapes and modification levels between cell types and epigenetic inhibitor treatments. See also Figures S3 and S4. Molecular Cell 2016 61, 170-180DOI: (10.1016/j.molcel.2015.11.003) Copyright © 2016 Elsevier Inc. Terms and Conditions