Proteomic Analyses Identify a Novel Role for EZH2 in the Initiation of Cancer Cell Drug Tolerance

Acquisition of drug resistance remains a chief impediment to successful cancer therapy, and we previously described a transient drug-tolerant cancer cell population (DTPs) whose survival is in part dependent on the activities of the histone methyltransferases G9a/EHMT2 and EZH2, the latter being the catalytic component of the polycomb repressive complex 2 (PRC2). Here, we apply multiple proteomic techniques to better understand the role of these histone methyltransferases (HMTs) in the establishment of the DTP state. Proteome-wide comparisons of lysine methylation patterns reveal that DTPs display an increase in methylation on K116 of PRC member Jarid2, an event that helps stabilize and recruit PRC2 to chromatin. We also find that EZH2, in addition to methylating histone H3K27, also can methylate G9a at K185, and that methylated G9a better recruits repressive complexes to chromatin. These complexes are similar to complexes recruited by histone H3 methylated at K9. Finally, a detailed histone post-translational modification (PTM) analysis shows that EZH2, either directly or through its ability to methylate G9a, alters H3K9 methylation in the context of H3 serine 10 phosphorylation, primarily in a cancer cell subpopulation that serves as DTP precursors. We also show that combinations of histone PTMs recruit a different set of complexes to chromatin, shedding light on the temporal mechanisms that contribute to drug tolerance

Spike-in normalization reveals the expected H3K27me3 decrease following EZH2 inhibition.

(A) IGV browser image of spike-in normalized H3K27me3 ChIP-seq data from cells treated for 4 and 8 days with CPI-360. (B) Browser image of spike-in normalized H3K27me3 ChIP-seq data from cells treated for 5 days with GSK126. (C) Scatter plots representing the correlation of all H3K27me3 ChIP-seq peaks before and after CPI-360 treatment. Spike-in normalization dramatically decreases H3K27me3 signal in cells treated for 4 days and 8 days. (D) Scatter plots representing the correlation of all H3K9me3 ChIP-seq peaks before and after CPI-360 treatment. Spike-in normalization does not significantly affect H3K9me3 signal in cells treated with CPI-360 for 4 day and 8 days. (E) Scatter plots representing the correlation of all H3K27me3 and H3K4me3 ChIP-seq peaks before and after GSK126 treatment. Spike-in normalization dramatically decreases H3K27me3 signal in inhibitor treated cells but does not significantly affect H3K4me3 signal. (F) Box plots representing H3K27me3 ChIP-seq data sets from DMSO, 4 day and 8 day CPI-360 treated cells. (G) Box plots representing H3K9me3 ChIP-seq data sets from DMSO, 4 day and 8 day CPI-360 treated cells. (H) Box plots representing H3K27me3 and H3K4me3 ChIP-seq data sets from DMSO and GSK126 treated cells.</p

An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition

<div><p>Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) has been instrumental in inferring the roles of histone post-translational modifications in the regulation of transcription, chromatin compaction and other cellular processes that require modulation of chromatin structure. However, analysis of ChIP-seq data is challenging when the manipulation of a chromatin-modifying enzyme significantly affects global levels of histone post-translational modifications. For example, small molecule inhibition of the methyltransferase EZH2 reduces global levels of histone H3 lysine 27 trimethylation (H3K27me3). However, standard ChIP-seq normalization and analysis methods fail to detect a decrease upon EZH2 inhibitor treatment. We overcome this challenge by employing an alternative normalization approach that is based on the addition of <i>Drosophila melanogaster</i> chromatin and a <i>D</i>. <i>melanogaster-</i>specific antibody into standard ChIP reactions. Specifically, the use of an antibody that exclusively recognizes the <i>D</i>. <i>melanogaster</i> histone variant H2Av enables precipitation of <i>D</i>. <i>melanogaster</i> chromatin as a minor fraction of the total ChIP DNA. The <i>D</i>. <i>melanogaster</i> ChIP-seq tags are used to normalize the human ChIP-seq data from DMSO and EZH2 inhibitor-treated samples. Employing this strategy, a substantial reduction in H3K27me3 signal is now observed in ChIP-seq data from EZH2 inhibitor treated samples.</p></div

EZH2 inhibition reduces global H3K27me3 levels, however standard ChIP-seq methods do not reveal the reduction.

<p><b>(A)</b> Western blot showing reduced global H3K27me3 levels in KARPAS-422 cells treated with 1.5 μM CPI-360 for 4 and 8 days. Whole cell extracts were resolved by SDS page and immuno-blotted with anti-H3K27me3. Anti-H3 immuno-blots show equal levels of total H3. <b>(B)</b> Western blot showing reduced global H3K27me3 levels in PC9 cells treated with 1 μM of GSK126 for 5 days. Whole cell extracts were resolved by SDS page and immuno-blotted with anti-H3K27me3. Anti-H3 immuno-blots show equal levels of total H3. <b>(C, D)</b> Representation of H3K27me3 ChIP-seq data using IGV. No obvious differences are detected in CPI-360 (C) and GSK126 (D) treated KARPAS-422 and PC9 cells when compared to vehicle-treated controls. <b>(E, F)</b> Genome-wide data from H3K27me3 ChIP-seq experiments under different treatment conditions are represented as scatter plots.</p

<i>D</i>. <i>melanogaster</i> tag counts from H3K27me3 ChIP-seq reactions are elevated in EZH2 inhibitor treated samples.

<p>H2Av bound regions of the <i>D</i>. <i>melanogaster</i> genome were determined using the H2Av antibody in ChIP-seq reactions containing <i>D</i>. <i>melanogaster</i> S2 or OSS chromatin. <i>D</i>. <i>melanogaster</i> tags from ChIP-seq spike-in reactions were mapped only to these pre-defined H2Av regions. <b>(A)</b> H3K27me3 ChIP-seq reactions with <i>D</i>. <i>melanogaster</i> spike-in in KARPAS-422 cells have a substantial increase in <i>D</i>. <i>melanogaster</i> tags in spike-in libraries prepared from CPI-360 treated cells both at 4 days and 8 days after treatment. <b>(B)</b> The increase was not observed in the control H3K9me3 reactions. <b>(C)</b> H3K27me3 ChIP-seq reactions with <i>D</i>. <i>melanogaster</i> spike-in in PC9 cells have a substantial increase in <i>D</i>. <i>melanogaster</i> tags in spike-in libraries prepared from GSK126 treated cells. <b>(D)</b> The substantial increase in tags was not observed in the control H3K4me3 ChIP-seq spike-in reactions.</p

Reduced H3K27me3 binding is detected by ChIP-qPCR.

<p><b>(A)</b> ChIP was performed using chromatin from KARPAS-422 cells treated with the EZH2 inhibitor CPI-360. qPCR using the positive control primer <i>MYT1</i> showed reduced H3K27me3 occupancy in the presence of the inhibitor. <b>(B)</b> ChIP was performed using chromatin from PC9 cells treated with the EZH2 inhibitor GSK126. qPCR using the positive control primer <i>MYT1</i> showed reduced H3K27me3 occupancy in cells treated with the inhibitor. (<b>C</b>) Libraries were generated from KARPAS-422 cells using 15 cycles of PCR amplification. Library DNA was diluted and qPCR was performed using positive control primers for <i>MYT1</i> and <i>CCND2</i>. (<b>D</b>) Libraries were generated from PC9 cells as described in (C) and library DNA was used for qPCR using positive control primers for <i>MYT1</i> and <i>CCND2</i>. All experiments are represented as the mean of two independent experiments with qPCRs performed in triplicate ±SD. The <i>ACTB</i> promoter served as a negative control for all experiments.</p

Schematic representation of the ChIP-seq spike-in protocol.

<p>ChIP-seq spike-in reactions are set up by adding the test chromatin of interest (human or other), the target antibody of interest, a small portion of <i>D</i>. <i>melanogaster</i> chromatin and the <i>D</i>. <i>melanogaster-</i>H2Av-specific antibody. The <i>D</i>. <i>melanogaster</i> spike-in chromatin is added in equal amounts and the H2Av antibody functions to pull down a small portion of the <i>D</i>. <i>melanogaster</i> chromatin in each reaction. After sequencing, tags are mapped to the genome corresponding to the test chromatin as well as to the <i>D</i>. <i>melanogaster</i> genome. The total number of tags uniquely mapping to the <i>D</i>. <i>melanogaster</i> genome are counted for each sample and used to generate correction factors (DMSO tags/inhibitor tags). The test chromatin tag counts are then normalized using the correction factors.</p