This study provides novel evidence for a physiological role of HEXIM1/P-TEFb interaction in attenuating E2/ERα driven transcription in the mammary gland and breast cancer cells. First, we demonstrated that increased HEXIM1 expression in the mammary gland of a transgenic mouse model decreased ductal branching, an E2-driven developmental process, due to changes in proliferation and apoptosis. We correlated these changes with a decrease in CCND1 and S2P RNAP II expression in vivo and in vitro. We also show that overexpression of HEXIM1 diminished E2-induced recruitment of ERα and cyclin T1 to the promoter and coding regions, respectively, of ER-responsive genes. Further, we show that E2 enhances the activity of the P-TEFb kinase, CDK9, which is inhibited by increased HEXIM1 expression. Surprisingly, increased HEXIM1 expression inhibited only E2-induced increases in P-TEFb activity and not basal P-TEFb kinase activity. In our studies, this inhibition of P-TEFb activity translates to a decrease in the recruitment of S2P RNAP II, and not other forms of RNAP II, to the coding regions of ER-responsive genes, pS2 and CCND1. These findings support a role for P-TEFb and transcription elongation in cell proliferation but more importantly, the data suggests a novel mechanism of action for HEXIM1 that can be recapitulated in vivo and a possible therapeutic role for HEXIM1 in hormone-dependent breast cancer.
Given that the regulation of CCND1
is complex (28
), we do not assume that our findings represent a comprehensive explanation regarding E2
-regulation of CCND1
. Also, other sites within CCND1
contribute to the transcriptional output (21
). We investigated the recruitment patterns of ERα, P-TEFb and RNAP II to two sites within the gene: an E2
-responsive region of the promoter and the coding region. Perhaps, a more extensive analysis of different sites within the genes, pS2
, and even other ER-responsive genes will reveal other insight into mechanism of HEXIM1 regulation of these genes. However, we believe that the information gathered from this study was sufficient to demonstrate the regulatory effects of HEXIM1 on ERα and P-TEFb recruitment to pS2
, suggesting a role for HEXIM1 and P-TEFb in ERα transcriptional regulation of some, but not all ERα target genes.
Based on our previous (19
) and current studies, we speculate that E2
enhances an ERα-P-TEFb interaction, and this increases the population of active P-TEFb at the gene locus of ER-responsive genes, which in turn phosphorylates the CTD of RNAP II. This phosphorylation event positions the gene in an active elongation state with increased S2P RNAP II occupancy at the coding regions, and enhanced recruitment of other forms of RNAP II marking the gene in an active transcription state (31
). However, increased HEXIM1 expression inhibits ERα and P-TEFb enrichment at the promoter and coding regions, respectively, of ER-responsive genes thus, decreasing the population of P-TEFb available to phosphorylate RNAP II to the serine 2 phosphorylated form associated with transcriptional elongation. In addition, we observed that increased HEXIM1 inhibits E2
-induced P-TEFb activity and postulated that this was due to an increase in the “free” form of HEXIM1, which serves to diminish any subsequent increases in PTEFb activity. Taken together, this scenario could represent the mechanism by which HEXIM1 modulates ERα-mediated transcription in the context of some ER-responsive genes (See for proposed model).
Proposed model for HEXIM1 action on ERα and P-TEFb at ER-responsive genes, pS2 and CCND1, in mammary cells.
The understanding of general mechanisms that control elongation stem from studies showing that HIV-1 harnesses P-TEFb as a cofactor to promote efficient mRNA transcript synthesis (12
). These and other studies have raised questions about whether P-TEFb acts as a general transcription elongation factor or serves in a gene-selective or context-dependent manner. The P-TEFb complex components, cyclin T1 and CDK9, have not been shown to have sequence-specific DNA binding activity, but transcription factors interact with and recruit P-TEFb to their respective promoter targets (12
). In addition, DNA microarray analyses of hearts from cyclin T1 transgenic mice indicate selective increases in subsets of genes, rather than a global increase in mRNA expression when compared to non-transgenic mice (42
). Our studies suggest that in breast epithelial cells, P-TEFb can be modulated by E2
/ERα and HEXIM1 in the context of some ERα target genes, although identical E2
-induced recruitment patterns for P-TEFb to pS2
genes suggest a general transcription elongation mechanism is also involved. In addition, the interaction of P-TEFb and E2
/ERα supports a positive aspect of ERα transcription elongation regulation, but E2
/ERα also interacts with negative elongation factor (NELF) and this interaction inhibits ERα-mediated transcription (43
HEXIM1 has been shown to have P-TEFb-independent action as seen with the glucocorticoid receptor (44
). We have also reported on P-TEFb-independent action of HEXIM1 in cardiovascular development (45
). Given this evidence, it is clear that HEXIM1 can inhibit transcription in a P-TEFb-dependent and -independent manner. Therefore, we cannot assume that the effect of HEXIM1 in the mammary gland is solely on ERα/P-TEFb, as other factors are involved in mammary gland development. However, we were able to demonstrate a specific inhibition pattern that HEXIM1 exerted on E2
-induced events at ER-responsive genes, pS2
, and increased HEXIM1 levels inhibited E2
-induced P-TEFb activity. Thus, based on our data, the HEXIM1 inhibition patterns observed in this study is largely P-TEFb-dependent in both our cell culture and animal models. Several studies also support an emerging role for HEXIM1 as a regulator of cell growth and differentiation (12
). Conversely, deletion of CLP-1, the mouse HEXIM1 gene, leads to pathological cardiac hypertrophy and perinatal death (47
In this study, a targeted increase in HEXIM1 expression in the mouse mammary gland driven by a mammary epithelial cell promoter (MMTV-LTR), led to a decrease in ductal branching (), an E2
/ERα driven mammary gland developmental process (25
). This observation was attributed to a decrease in proliferation and an increase in apoptosis (). The decrease in proliferation was linked to a concurrent decrease in CCND1 expression, further demonstrating that HEXIM1 regulates ER-responsive genes in vivo
. Future studies will not only aim to address HEXIM1 regulation of other E2
/ERα target genes, but also HEXIM1 regulation of other nuclear receptors relevant in mammary cell function and tumorigenesis.