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

H Karimi Kinyamu,Pierre R Bushel,Trevor K Archer,Brian D Bennett

doi:10.1074/jbc.ra119.011174

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.

Highlights

Proteasome activity is required for diverse cellular processes, including transcriptional and epigenetic regulation
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
Commonlyenriched (Z score Ͼ1) Gene Ontology (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-␬B were enriched at 24 h (Fig. S1C)

Summary

Results

Proteasome inhibitors have emerged as powerful anti-cancer drugs, but downstream mechanisms of their antitumor effects are poorly understood. Analysis of the ChIP signal at differentially-expressed genes showed that proteasome inhibition enhanced H3K36me3 5Ј to 3Ј deposition at ϳ500 and 2600 genes that were induced during the 4- and 24-h treatment, respectively (Fig. 6, C–F) This effect was evident in metagene plots of ϳ500 and 2600 genes up-regulated after 4 and 24 h of treatment with proteasome inhibitor (Fig. 6, D and F). Proteasome inhibition causes global accumulation of the pSer-2 Pol II signal at all genes expressed in MCF-7 cells, at 24 h (Fig. 5, A and B), whereas H3K36me deposition correlates with time-dependent changes in transcriptional output. Inhibiting proteasome activity causes a distinct decoupling of pSer-2 Pol II and H3K36me upstream and downstream of TTS of induced (Fig. S6, B and C, UP) compared with repressed genes, and this effect is maximal in cells treated with inhibitor for 24 h (Fig. S6, D and E, DOWN). PSer-2 Poll II accumulation downstream of the TTS indicates proteasome activity is critical for RNA pol II transactions at the 3Ј end of genes and Pol II accumulation or stalling at the transcription termination sites during proteasome inhibition could result in defects in transcriptional termination

Discussion

Cell culture

Gene expression profiling and analysis

Preprocessing of the data

Statistical data analysis

Western blot analysis