The 26S proteasome complex tightly regulates receptor turnover and transcription of many steroid hormone receptors, including the GR (36
). Previous work showed that proteasome inhibition increases glucocorticoid receptor-mediated transcriptional activity from the MMTV promoter (8
). Here we have extended the study to understand the mechanism by which proteasome inhibition influences GR-mediated gene transcription. Although GR levels are increased in the cell, ChIP analysis monitoring GR occupancy revealed a decrease in GR loading on the MMTV promoter after proteasome inhibition (Fig. ). This is consistent with the previous observation that proteasome inhibition decreases the mobility of the receptor within the nucleus, and this could lead to less GR on the promoter (8
). Our observation is further supported by fluorescence recovery after photobleaching experiments showing no correlation between GR protein levels and transcriptional changes at the MMTV array in cells treated with proteasome inhibitor (49
). Additionally, we show that neither the BRG-1 complex nor p300 are increased at the promoter after proteasome inhibition, suggesting that hormone-dependent promoter chromatin hypersensitivity after proteasome inhibition does not account for the increase in MMTV expression. These initial observations led us to propose that apart from receptor turnover, the proteasome can regulate components of the transcriptional machinery and chromatin structure modifications that modulate the hormone response.
The role of the 26S proteasome in recycling of receptor/transcriptional complexes has been suggested as the main mechanism involved in controlling gene expression (36
). For the GR, proteasome inhibition results in increased gene expression, suggesting that proteolytically linked recycling of the receptor is not a key mechanism for the observed increase in gene expression. Nonproteolytic activities of the proteasome such as coactivator recruitment have been proposed to be important for transcriptional regulation. As such, the differential occupancy by the 19S and 20S proteasome subunits on the promoter and the transcribed region of the gene has significant regulatory potential. One way the proteasome subunits can act at the DNA template is by facilitating activator-coactivator interactions required for the assembly of the transcription complex and activation of productive transcription. An interesting possibility is that the 19S ATPase complex can facilitate chromatin-modifying machines, allowing alteration in chromatin structure and transcription to occur as demonstrated for the SAGA complex (26
). However, at the MMTV locus, chromatin remodeling is highly dependent on the BRG-1 but not the p300 hypoxanthine-aminopterin-thymidine complex. Indeed, there is a reduction in levels of 19S ATPase at the MMTV promoter in the presence of hormone. Subsequently we find that depletion of Sug1 results in an increase in hormone-dependent transcription. In contrast to Sug1, the 20S complex is present at the 3′ end of the gene, consistent with hormone-dependent and -independent transcription. This observation supports recent studies in yeast showing that the presence of the 20S at the 3′ end of the gene facilitates readthrough of the transcription termination site (15
). Perhaps on the MMTV locus the 20S proteasome can decrease termination and facilitate hormone-dependent and -independent transcription, as seen in cells treated with proteasome inhibitor. RNAi experiments corroborate a role for the 20S proteasome as depletion of the PMSA3 subunit affects basal transcription. Our results are consistent with recent reports showing that the 20S is associated primarily with the 3′ ends of certain highly transcribed genes in yeast (2
). Additionally, the dynamic interplay between the 19S and 20S proteasome subunits at transcriptionally active loci was recently shown to dictate differential assembly of transcriptional complexes and activator-dependent transcription in embryonic stem cells and the human immunodeficiency virus type 1 (HIV-1) LTR (25
). In embryonic stem cells, the loss of the 19S subunit did not impede recruitment of the 20S subunit at transcriptionally active loci, suggesting the subunits can be targeted to different regulatory regions. This might then allow the recruitment of different transcriptional complexes and activities to modulate transcriptional output (50
). A specific prediction would be that the 20S complex could form a preinitiation complex that could lead to permissive transcription of certain loci in embryonic stem cells (50
). The finding that depletion of the 19S and 20S subunits has differential effects on basal MMTV transcription is echoed in a recent study on the HIV LTR locus. As shown for GR-mediated transactivation of the MMTV, ablation of the 19S ATPases affected TAT-mediated transcription of the HIV LTR without affecting basal transcription (25
). As shown for MMTV, knockdown of the 20S enhanced basal transcription of the HIV LTR independent of TAT, analogous to the effect seen for GR. The authors attribute these effects to a switch between the proteolytic and nonproteolytic effects of the proteasome subunits. Specifically, the 19S is involved in activator turnover, whereas the 20S may be involved in initiation and elongation processes and control of the mature transcript production. Thus, the redistribution of the proteasome subunits at the MMTV locus after proteasome inhibition may facilitate formation of different transcriptional or coregulator complexes. Such complexes would then modulate hormone-dependent and -independent transcriptional output by as-yet-uncharacterized mechanisms. In contrast to the MMTV locus, a recent study has shown that the 20S proteasome beta subunit LMP2 physically interacts with the p160 coactivators and enhances estrogen receptor-mediated transcription of the pS2 gene (54
). However, similar to our study and the studies cited above, the authors showed that the 20S subunit is specifically involved in the transcriptional elongation, supporting a role of the 20S at the 3′ end of the gene. Taken together, the evidence currently available supports a role of specific proteasome subunits in receptor-mediated transcriptional regulation.
Proteasome inhibition results in global changes in trimethyl histone H3K4, a mark recently shown to be associated with an active chromatin structure that is permissive to transcription (29
). Additionally, the trimethyl histone H3K4 is associated with genes that maintain a poised chromatin state, such as the β-globin locus (44
). Histone modifications can alter chromatin structure by acting as recognition marks for factors that recognize specific modifications and alter nucleosome structure (6
). For example, the chromodomain helicase binding protein 1 (CHD1), a member of the SNF2-like family of ATPases that mobilize nucleosomes, specifically recognizes the methyl histone H3K4 mark (13
). Notably, in human cells the nucleosome remodeling factor (NURF), another member of the ATP-dependent chromatin remodeling complex, seems to specifically recognize the trimethyl histone H3K4 mark (53
). While we have not looked directly at NURF in the context of these experiments, our previous studies suggest it is not able to remodel the promoter (11
). An important caveat is that in the previous studies the promoter would not have been expected to have any significant trimethyl histone H3K4, and thus its contribution is unknown.
The increase in both mRNA and protein expression for MLL, a histone H3K4me3-specific histone methyltransferase, in cells treated with proteasome inhibitor and RNAi of proteasome subunits is intriguing. This suggests that the proteasome functions to regulate MLL expression, although the mechanism is not clear. The increase in MLL expression and the presence of the trimethyl H3K4me3 on hormone-activated genes suggests a role of MLL in hormone response. MLL regulates mainly homeobox genes, but recent reports show that MLL regulates p27 (Kip 1) and p18 (ink4C) genes involved in suppression of cell growth and proliferation (34
). This function agrees well with our current finding that suggests that proteasome function can modulate hormone and biological response by changing factors that regulate transcription. In contrast to MLL, SMYD3, known to increase cell proliferation, is inhibited by proteasome inhibition (18
The observation that chromatin hypersensitivity and gene expression are increased independent of the hormone implies that the proteasome may function to regulate basal transcription (Fig. , lane 3). These data suggest that the proteasome, in the absence of hormonal stimulation, functions to maintain a closed chromatin environment at the MMTV promoter. The mechanisms by which this occurs are presently unknown, but we note that the MG132-dependent increase in transcription is accompanied by a modest recruitment of BRG1 at the promoter independent of hormone (Fig. ). Interestingly, there are concomitant increases in the chromatin structure sensitivity, gene expression, and H3K4me3 levels at the MMTV locus, suggesting that specific histone modifications at certain loci can initiate hormone-independent aberrant gene expression (Fig. ). Furthermore, this may specifically involve the 20S proteasome, since RNAi of this subunit increases gene expression independent of hormone (Fig. ). Our findings echo a recent report showing that histone modifications are important in modulating hormone-independent gene expression implicated in androgen insensitivity in tumor cells (19
Another important feature correlated with the enhanced gene expression after proteasome inhibition is the increase in global pools of phosphorylated RNA Pol II. RNA Pol II phosphorylation is essential for a number of transcriptional processes that lead to successful mature transcript (37
). That Pol II is hyperphosphorylated upon proteasome inhibition is consistent with recent findings suggesting that polymerase phosphorylation, particularly at Ser5, inhibits polymerase ubiquitylation and increases transcriptional efficiency (48
). Notably, despite the increase in global levels of hyperphosphorylated Pol II after proteasome inhibition, the transcriptional effect is exclusively hormone dependent, suggesting cooperativity of these forms of Pol II with additional factors that modulate transcriptional responses. Such cooperativity between the hyperphosphorylated forms of RNA Pol II and other transcriptional regulators would support differential regulation of receptor target genes after proteasome inhibition. Our findings are consistent with recent reports showing that the requirement of phosphorylated Pol II by p53 target genes is gene specific and dependent on the type of stimuli (16
). An interesting observation is the lower levels of nonphosphorylated CTD at the promoter and the coding region of the MMTV gene after proteasome inhibition. From Western blotting analysis, it is clear that global pools of RNA Pol II forms change after proteasome inhibition. Interestingly, some studies have attributed decreases in hormone response after proteasome inhibition to the lack of polymerase loading on the promoter of the target gene. For example, a recent study showed that proteasome inhibition suppresses progesterone receptor-mediated gene expression and attributed this to a decrease in RNA polymerase II recruitment, but the study did not analyze hyperphosphorylated forms of the polymerase (7
). On the same lines of evidence, Pol II was not detected in 60% of transcription-competent gene promoters using the same antibody as that in our study (21
). The authors attributed the lack of correlation between transcription and Pol II occupancy to the efficiency of immunoprecipitation of chromatin fragments with this antibody. Our observations may explain the diverse effects of proteasome inhibition on steroid hormone receptor-mediated gene transcription. Indeed, global analysis of gene expression by microarray indicates differential effects of proteasome inhibition on GR target genes (H. K. Kinyamu, J. Collins, S. Grissom, P. Hebbar, and T. K. Archer, unpublished data). Finally, our data support the hypothesis that the 20S proteasome complexes are present at sites of active transcription in conjunction with Pol II phosphorylated forms (2
In summary, our findings posit dynamic interplay between steroid hormone receptor-mediated gene transcription and proteasome activity that links proteasome activity with histone modifications and Pol II transcriptional machinery in mammalian cells (Fig. ). This postinitiation transcriptional role for the proteasome in regulating receptor-mediated gene expression represents a powerful mechanism for receptors to regulate a diverse array of genes involved in numerous physiological processes.
FIG. 9. Model for enhanced receptor-mediated transcriptional output by regulating chromatin modifications and RNA Pol II machinery upon proteasome inhibition. 1. In the absence of hormone or proteasome inhibition, the 19S subunit and nonphosphorylated Pol II (more ...)