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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.

Nuclear receptors such as the glucocorticoid receptor (GR) are ligand-dependent transcription factors that mediate transcription of target genes by recruiting factors that modulate chromatin structure. In this study, curcumin, a compound known to inhibit GR-mediated transcription, was used to examine the different mechanisms by which GR regulates transcription. The mechanisms of transcription regulation of metallothioneine-2A (MT2A) and solute carrier family 19 member 2 (SLC19A2), two GR target genes where the hormone-dependent gene activation is inhibited or unaffected by curcumin treatment, respectively, were analyzed by chromatin immunoprecipitation and RT-PCR experiments. The data suggest that the loss of hormone-dependent MT2A gene expression is due to the inhibition of continued transcription activity after initial assembly of the transcription machinery. In contrast, the hormone-dependent SLC19A2 gene expression is maintained because the continued transcription output after assembly of transcription machinery is unaffected by curcumin. These results suggest that the two GR target genes use alternate mechanisms to regulate expression levels at the level of continued transcription output after transcription machinery assembly.

Reporter enzymes are commonly used in cell biology to study transcriptional activity of genes. Recently, reporter enzymes in combination with compounds that inhibit proteasome function have been used to study the effect of blocking transcription factor degradation on gene activation. While investigating the effect of proteasome inhibition on steroid receptor activation of the mouse mammary tumor virus (MMTV) promoter, we found that treatment with proteasome inhibitors enhanced glucocorticoid activation of the promoter attached to a chloramphenicol acetyltransferase (CAT) reporter, but inhibited activation of MMTV attached to a firefly luciferase or beta-galactosidase reporter. MMTV RNA levels under these conditions correlated with the promoter activity observed using the CAT reporter, suggesting that proteasome inhibitor treatment interfered with luciferase or beta-galactosidase reporter assays. Washout experiments demonstrated that the majority of luciferase activity was lost if the proteasome inhibitor was added at the same time luciferase was produced, not once the functional protein was made, suggesting that proteasome inhibition interferes with production of luciferase protein. Indeed, we found that proteasome inhibitor treatment dramatically reduced the levels of luciferase and beta-galactosidase protein produced, as determined by Western blot. Thus, treatment with proteasome inhibitors interferes with luciferase and beta-galactosidase reporter assays, possibly by inhibiting production of a functional reporter protein.

We have previously shown a connection between histone H1 phosphorylation and the transcriptional competence of the hormone inducible mouse mammary tumor virus (MMTV) promoter. Prolonged exposure of mouse cells to dexamethasone concurrently dephosphorylated histone H1 and rendered the MMTV promoter refractory to hormonal stimulation and, therefore, transcriptionally unresponsive. Using electrospray mass spectrometry, we demonstrate here that prolonged dexamethasone treatment differentially effects a subset of the six somatic H1 isoforms in mouse cells. H1 isoforms H1.0, H1.1, and H1.2 are non-responsive to hormone whereas prolonged dexamethasone treatment effectively dephosphorylated the H1.3, H1.4, and H1.5 isoforms. The protein kinase inhibitor staurosporine, shown to dephosphorylate histone H1 and down-regulate MMTV in cultured cells, appears only to completely dephosphorylate the H1.3 isoform. These results suggest that dephosphorylation of specific histone H1 isoforms may contribute to the previously observed decrease in transcriptional competence of the MMTV promoter through the modulation of chromatin structure. In a broader sense, this work advances the hypothesis that post-translational modifications of individual histone H1 isoforms directly influence the transcriptional activation/repression of specific genes.

We examined the mechanism(s) by which the progesterone receptor (PR) is able to inhibit glucocorticoid receptor (GR) activation from the mouse mammary tumor virus (MMTV) promoter in vivo. Using specific hormone antagonists, we demonstrate that the PR complexed with an type II antiprogestin blocks glucocorticoid-induced activation of the MMTV promoter. However, when complexed with a type I antiprogestin the PR is unable to block glucocorticoid-induced activation. PR repression of GR activity results from the inhibition of the ability of the GR to remodel chromatin such that the antiprogestin-occupied/PR prevents the glucocorticoid induced assembly of a preinitiation complex at MMTV promoter. These experiments suggest that the specific chromatin organization of the MMTV promoter provides a mechanism for regulating cross-talk between the GR and PR in vivo.

We have demonstrated that physiological concentrations of 17 beta-estradiol increase nuclear estrogen-specific binding sites in the human hepatoma cell line HepG2 7- to 10-fold and the rate of accumulation of secreted apolipoprotein C-II (apo-C-II), 2.5-fold (Tam, S-P., Archer, T. K., and Deeley, R. G. (1985) J. Biol. Chem. 260, 1670-1675). Apo-C-II is the major activator of lipoprotein lipase, an enzyme which plays a key role in lipoprotein catabolism. In order to define more precisely the mechanism by which estrogen influences apo-C-II production, we have synthesized a triacontanucleotide DNA probe that is complementary to apo-C-II mRNA. We have used the probe both in Northern hybridization experiments and in DNA excess titrations to quantify apo-C-II mRNA in hormonally treated HepG2 cells and various primate tissues. These studies revealed that: 1) the concentration of apo-C-II mRNA in HepG2 cells is comparable with that present in human liver; 2) treatment of the cells with low levels of estrogen results in a doubling of the apo-C-II mRNA concentration; 3) the apo-C-II mRNA concentration in monkey liver is 60- to 70-fold greater than in the intestine and 2.5-fold higher than in human liver.

We have examined the effect of estrogen on the rate of accumulation of apolipoproteins secreted by the human hepatocarcinoma cell line, HepG2. Prior to exposure to hormone, we detected less than 300 high-affinity, nuclear, estrogen-binding sites/cell. Within 48 h of growth in the presence of 20 nM 17 beta-estradiol this number rose to 3000-3500 sites/cell. Rates of accumulation of two of the major apolipoproteins, apo-C-II and apo-A-I increased 2.5- and 2.0-fold, respectively, in response to estrogen treatment. Other major apolipoproteins were not affected at this concentration of hormone. Induction of both proteins was completely antagonized by 20 nM testosterone. The density distribution of apolipoproteins secreted by the hepatocytes was similar to that reported using perfused liver systems. The consequences of estrogen treatment were to increase the apo-C-II/apo-C-III ratio in very low density lipoproteins as well as to decrease the overall very low density lipoprotein:high density lipoprotein ratio.

Apolipoprotein (apo) A-I is the principal protein component of high density lipoproteins and the major in vivo activator of lecithin-cholesterol acyltransferase. We have used the human hepatoma cell line, HepG2, as a model to examine the ability of estrogen to modulate hepatic synthesis of apo-A-I. Primer extension studies have demonstrated that the major transcriptional initiation site of the apo-A-I gene utilized in the hepatoma cells is the same as that used in human liver. The kinetics of induction of high- and low-affinity estrogen-binding sites, rates of secretion of apolipoproteins, and apo-A-I mRNA levels were examined following treatment of the cells with estrogen. Initial concentrations of 20 nM 17 beta-estradiol resulted in a 14-15-fold increase in the levels of high-affinity nuclear estrogen-binding sites within 8 h, while the level of low-affinity sites increased by only 10%. During the same period, the levels of apo-A-I mRNA and the rate of accumulation of the secreted protein increased by 55 and 50%, respectively. New steady state levels of apo-A-I mRNA and rates of accumulation of protein, approximately twice those in control cultures, were established within 24-48 h of exposure to hormone. Experiments with a 50-fold higher concentration of estrogen resulted in only an additional 10% increase in mRNA levels. The increase in mRNA levels following estrogen treatment was adequate to account for 85-90% of the elevation observed in the rate of accumulation of secreted apo-A-I. Comparison of the apo-A-I mRNA levels in HepG2 cells with those present in human liver revealed that the concentration of the mRNA was approximately 3-fold lower than that found in vivo.