Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Abstract

Proteasome activity is required for diverse cellular processes, including transcriptional and epigenetic regulation. However, inhibiting proteasome activity can lead to an increase in transcriptional output that is correlated with enriched levels of trimethyl H3K4 and phosphorylated forms of RNA polymerase (Pol) II at the promoter and gene body. Here, we perform gene expression analysis and ChIP followed by sequencing (ChIP-seq) in MCF-7 breast cancer cells treated with the proteasome inhibitor MG132, and we further explore genome-wide effects of proteasome inhibition on the chromatin state and RNA Pol II transcription. Analysis of gene expression programs and chromatin architecture reveals that chemically inhibiting proteasome activity creates a distinct chromatin state, defined by spreading of the H3K4me3 mark into the gene bodies of differentially-expressed genes. The distinct H3K4me3 chromatin profile and hyperacetylated nucleosomes at transcription start sites establish a chromatin landscape that facilitates recruitment of Ser-5- and Ser-2–phosphorylated RNA Pol II. Subsequent transcriptional events result in diverse gene expression changes. Alterations of H3K36me3 levels in the gene body reflect productive RNA Pol II elongation of transcripts of genes that are induced, underscoring the requirement for proteasome activity at multiple phases of the transcriptional cycle. Finally, by integrating genomics data and pathway analysis, we find that the differential effects of proteasome inhibition on the chromatin state modulate genes that are fundamental for cancer cell survival. Together, our results uncover underappreciated downstream effects of proteasome inhibitors that may underlie targeting of distinct chromatin states and key steps of RNA Pol II–mediated transcription in cancer cells. Proteasome activity is required for diverse cellular processes, including transcriptional and epigenetic regulation. However, inhibiting proteasome activity can lead to an increase in transcriptional output that is correlated with enriched levels of trimethyl H3K4 and phosphorylated forms of RNA polymerase (Pol) II at the promoter and gene body. Here, we perform gene expression analysis and ChIP followed by sequencing (ChIP-seq) in MCF-7 breast cancer cells treated with the proteasome inhibitor MG132, and we further explore genome-wide effects of proteasome inhibition on the chromatin state and RNA Pol II transcription. Analysis of gene expression programs and chromatin architecture reveals that chemically inhibiting proteasome activity creates a distinct chromatin state, defined by spreading of the H3K4me3 mark into the gene bodies of differentially-expressed genes. The distinct H3K4me3 chromatin profile and hyperacetylated nucleosomes at transcription start sites establish a chromatin landscape that facilitates recruitment of Ser-5- and Ser-2–phosphorylated RNA Pol II. Subsequent transcriptional events result in diverse gene expression changes. Alterations of H3K36me3 levels in the gene body reflect productive RNA Pol II elongation of transcripts of genes that are induced, underscoring the requirement for proteasome activity at multiple phases of the transcriptional cycle. Finally, by integrating genomics data and pathway analysis, we find that the differential effects of proteasome inhibition on the chromatin state modulate genes that are fundamental for cancer cell survival. Together, our results uncover underappreciated downstream effects of proteasome inhibitors that may underlie targeting of distinct chromatin states and key steps of RNA Pol II–mediated transcription in cancer cells. The 26S proteasome is a large multiprotease component of the ubiquitin proteasome system (UPS) 2The abbreviations used are: UPSubiquitin proteasome systemDEGdifferentially expressed geneGSEAGene Set Enrichment AnalysisTSStranscription start sitePolpolymeraseCTDC-terminal domainTTStranscription termination sitePICpreinitiation complexPTMposttranslational modificationFDRfalse discovery rateGOGene OntologyERestrogen receptorDIFFdifferenceChIP-seqChIP sequencingMNasemicrococcal nucleaseMEMmodified Eagle's mediumUNTRuntreated. that recognizes and destroys ubiquitylated and misfolded proteins (1Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67 (9759494): 425-47910.1146/annurev.biochem.67.1.425Crossref PubMed Scopus (6878) Google Scholar). Proteasome activity is required for multiple DNA transactions, and there is increasing evidence the 26S proteasome regulates transcription, chromatin organization, and ultimately the expression of genetic information that governs gene networks critical for cellular homeostasis. Dysfunction of the proteolytic activity of the proteasome disrupts many cellular processes that are important in health and disease (2Schmidt M. Finley D. Regulation of proteasome activity in health and disease.Biochim. Biophys. Acta. 2014; 1843 (23994620): 13-2510.1016/j.bbamcr.2013.08.012Crossref PubMed Scopus (309) Google Scholar). ubiquitin proteasome system differentially expressed gene Gene Set Enrichment Analysis transcription start site polymerase C-terminal domain transcription termination site preinitiation complex posttranslational modification false discovery rate Gene Ontology estrogen receptor difference ChIP sequencing micrococcal nuclease modified Eagle's medium untreated. 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Acta. 2011; 1809 (20728592): 109-11810.1016/j.bbagrm.2010.08.005Crossref PubMed Scopus (11) Google Scholar, 32McCann T.S. Tansey W.P. Functions of the proteasome on chromatin.Biomolecules. 2014; 4 (25422899): 1026-104410.3390/biom4041026Crossref PubMed Scopus (20) Google Scholar), the effects of proteasome inhibition on chromatin modifications and RNA Pol II transcription have not been extensively examined genome-wide in cancer cells. Exploring how histone modifications and proteasome activity cross-talk to regulate gene transcription may help to better understand how the proteasome pathway impacts cellular processes critical for cell survival. In this study, we exposed MCF-7 breast cancer cells to the proteasome inhibitor MG132 to better understand how blocking protein turnover impacts chromatin state and subsequent gene-expression programs. We show that proteasome inhibition establishes a distinct hyperacetylated chromatin landscape characterized by the spreading of the H3K4me3 mark into the gene body. This chromatin environment facilitates the recruitment and processivity of RNA Pol II leading to the expression of genes whose functions are relevant to breast cancer pathology. Proteasome inhibitors have emerged as powerful anti-cancer drugs, but downstream mechanisms of their antitumor effects are poorly understood. To begin to address mechanisms of proteasome inhibitors in tumor cells, we monitored the changes in gene expression and chromatin state in MCF-7 breast cancer cells exposed to MG132, a drug that effectively blocks the activity of the 26S proteasome complex. We performed microarray analysis of MCF-7 cells treated with vehicle (DMSO) or 1 μm MG132 for 4 h (MG4H) and 24 h (MG24H) and found profound time-dependent changes in gene expression, with ∼700 (519 up; 194 down) and ∼5000 (2637 up; 2434 down) genes being significantly changed (false discovery rate (FDR) <0.05 and fold change >|1.5|) at the 4- and 24-h time points, respectively (Fig. 1, A and B). A majority of genes (∼90%) changed at 4 h were also changed at 24 h, as indicated by the overlap on the Venn diagram (Fig. 1B). Genes shared between 4 and 24 h included induced genes PMAIP1 and GABARAPL1 and down-regulated genes CYP26A1 and METTL7A, which were also validated by quantitative PCR (Fig. S1A). To confirm the effectiveness of the proteasome inhibition, we first examined changes in the expression of proteasome subunits. As expected, genes encoding proteasomal subunits are up-regulated by MG132 treatment, demonstrating proteasome inhibition was effective in our experimental system (Fig. S1B). Conversely, treatment for 24 h elicits expression of an expanded set of genes, unique to the 24-h treatment (Fig. 1B). Examples of genes changed at 24 h include KLF6, IL6, ESR1, and E2F2 (Fig. S1A). Gene Ontology (GO) analysis of the differentially changed genes revealed significantly-enriched molecular terms that were time-dependent (Fig. S1C). As expected, GO terms representing the proteasome ubiquitin pathway and unfolded protein response were highly enriched in proteasome-inhibited cells (Table S1). Commonly-enriched (Z score >1) GO terms of genes up-regulated by MG132 at 4 and 24 h included NRF2-mediated oxidative stress response, hypoxia signaling in the cardiovascular system, death receptor signaling, and PI3K/AKT signaling (Fig. S1C). p53 signaling was an enriched term at 4 h, whereas downstream signaling pathways like IL-6, nerve growth factor (NGF), and NF-κB were enriched at 24 h (Fig. S1C). Down-regulated genes were predominantly enriched for terms representing cell cycle and DNA damage, including Wnt/β-catenin signaling, role of BRCA1 in DNA damage response, estrogen-mediated S-phase entry, and aryl hydrocarbon receptor signaling (Fig. S1C). Ingenuity pathway analysis (IPA) upstream regulator analysis was done to predict relevant transcriptional regulators that could play a role in the observed gene expression changes. The analysis revealed several regulators of genes in the enriched GO pathways. Upstream regulators of genes differentially changed at the 4-h time point predominantly modulate cell cycle and oxidative stress (Fig. 1C, left panel). Upstream regulators of up-regulated genes include transcription factors, for example TP53, whose activity is most increased and nuclear factor erythroid 2 like 2 (NFE2L2, NRF2), a positive regulator of NRF2-mediated oxidative stress. The loss in activity of aurora kinase (AURK) and anillin actin-binding protein (ANLN) leads to up-regulation of genes that control cell cycle, cell proliferation, and migration (Fig. 1C, left panel, bold). The activation of the chromatin modifier, nuclear protein 1, transcriptional regulator (NUPR1), and miRNAs together with inhibition of TNF and ERBB2 activity in cells treated with MG132 for 4 h suggests gene repression may be mediated by these factors (Fig. 1C, left panel). Consistent with proteasome inhibition, the activity of the 26S proteasome is decreased (negative Z score) in cells treated for 24 h (Fig. 1C, right panel, bold), and this reduced activity results in up-regulation of a subset of genes, including those encoding a majority of proteasome subunits (Fig. S1B). Other upstream regulators of 24-h–induced DEGs are factors involved in cell proliferation and migration, including Erb-B2 receptor tyrosine kinase 2 (ERBB2, HER2/neu), Kirsten rat sarcoma (KRAS), and epidermal growth factor (EGF) (Fig. 1C, right panel, bold). Finally, gene repression at 24 h may be partly mediated by NUPR1 and transcription factors, melanocyte-inducing transcription factor, ERBB2, and FOXM1, whose activities primarily promote cell differentiation, proliferation, and survival, and hence their activity is decreased by MG132 treatment (Fig. 1C, right panel). Intriguingly, the latter transcription factors largely regulate the activity of the estrogen receptor α (ESR1), whose activity is also decreased by proteasome inhibition (Fig. 1C, right panel). Consistent with the finding that p53 and estrogen receptor (ERα) signaling pathways were activated and inhibited at 4 and 24 h, respectively (Fig. 1C and Fig. S1C), gene set enrichment analysis indicated that genes induced at 4 h were enriched in p53 targets, and genes repressed at 24 h were enriched in ER targets (Fig. 1D). Notably, in agreement with the gene expression data, p53 and ERα protein levels increased and decreased at 4 and 24 h, respectively (Fig. S1D). These data support the hypothesis that proteasome inhibition leads to significant changes in expression of multiple genes, including genes important for distinct pathways whose predicted upstream regulators are key drivers of biological processes relevant to breast cancer. 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We first analyzed chromatin features of genes that were differentially expressed upon MG132 treatment, focusing on genomic regions surrounding the transcription start sites (TSS). H3K4me3 signal centered around the TSS (±2 kb) of all expressed (∼13,500) and differentially-expressed genes (up- and down-regulated) in MCF-7 cells were similar between vehicle (UNTR) and MG132-treated cells (Fig. S2A). However, in contrast to regions in close proximity to the TSS (∼+750 bp), a subset of genes up-regulated upon MG132 treatment showed a tendency of increased H3K4me3 signal downstream of TSS starting (∼+1 kb) and extending into the gene body, as is evident in the heatmaps showing the difference in H3K4me3 signal (±5 kb) of the up-regulated genes (Fig. 2A and Fig. S2B). We observed spreading of the H3K4me3 mark into the gene body of the up-regulated but not the down-regulated genes on the metagene plots representing the difference between H3K4me3 signal in control and cells treated for 4 and 24 h (Fig. 2B, DIFF 4H and DIFF 24H). Interestingly, close examination of the metagene plots reveal a loss in H3K4me3 (−1)-modified nucleosome signal and what seems to be a shift in the +1 H3K4me3-modified nucleosome into the gene body at 24 h (DIFF 24 h). The differences in the distribution of the H3K4me3 at induced compared with repressed genes can be observed on examples of UCSC genome browser tracks showing the spreading of H3K4me3 signal into the gene body of GABARAPL1, which is the induced and not the repressed CYP26A1 (Fig. 2C and Fig. S1A). 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