HEXIM1 Expression Research Articles

Abstract A61: Hexim-1 protein expression levels modulate TGFβ activity in prostate cancer

Abstract Hexim-1 is a highly conserved protein from human to mouse, and a well-established inhibitor of RNA polymerase II-dependent transcription elongation and cell proliferation. Others and we have proposed and provided evidence that changes in the expression and activity in Hexim-1 significantly influence the progression of cancer. Here we show that changes in expression levels of Hexim-1 modulate TGFβ1 signaling by inducing posttranslational modifications in the SMADs proteins (small mother against decapentaplegic) at the polylinker region of the Smad1 and Smad3 proteins. Cdk9 serine activity not only targets specific serine residues in the carboxyl terminus of RNA polymerase II during transcriptional elongation but also serine residues in the polylinker region of Smad3. Hexim-1 directly inhibits Cdk9 activity, and therefore, transcription elongation and TGFβ1/Smad3 activity. Our results indicate that prostate cancer accompanies significant changes in Hexim-1 expression as well as sub cellular redistribution. Similar observations were obtained in the TRAMP mouse model of prostate cancer. In order to understand the role of Hexim-1 during prostate cancer progression, we generated a TRAMP mouse model heterozygous for Hexim-1. Our studies in the TRAMP mouse model revealed that lowered levels in Hexim-1 protein expression triggered a more aggressive form of prostate cancer. TGFβ1/Smad3 plays a significant role during proliferation, cell migration, as well as in epithelial mesenchymal transition, which is critical during metastasis. To test the role of Hexim-1 as a modulator of TGF/Smad3 in vitro, we use the epithelial mouse prostate cancer cell line TRAMP-C2 and derived the TRAMP-C2 heterozygous for Hexim-1 (HT). We observed increased serine 208 phosphorylation of Smad3 as well as enhanced transcriptional activity of TGF β/Smad3 target genes involved in EMT such as SM22alpha and alpha smooth muscle actin in the HT-TRAMP-C2 cell line. These findings suggest that Hexim-1 is a critical regulator of prostate cancer progression. Citation Format: Manya Dhar-Mascareno, Eduardo Mascareno, Fang Liu. Hexim-1 protein expression levels modulate TGFβ activity in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A61.

Vascular Endothelial Growth Factor Gene Regulation by HEXIM1 in Heart

See related article, pages 415–422 Hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) was cloned as an upregulated gene in vascular smooth muscle cells after treatment with the differentiating agent HMBA.1 A recent report has shown that HEXIM1 is an inhibitor of positive transcription elongation factor (P-TEF)b, which plays an important role in regulation of RNA polymerase II elongation.2–4 HEXIM1 was previously cloned as a potential cardiac transcriptional regulatory factor suppressing the cardiac myosin light chain-2v promoter and termed cardiac lineage protein (CLP)-1.5 HEXIM1 was also cloned as the novel inhibitor of breast cell growth estrogen-downregulated gene (EDG)1.6 HEXIM1 protein level is downregulated by estrogens. HEXIM1 also suppresses estrogen receptor-α transcriptional activity.6,7 Gene and protein expression indicate the broad expression of HEXIM1 during postnatal development, with highest levels in heart, skeletal muscle, and brain. Protein localization of endogenous HEXIM1 in vascular smooth muscle cells and primary cardiomyocytes suggests that HEXIM1 is a nuclear protein. Furthermore, HEXIM1 has been shown to be involved in many biological processes, including cancers, AIDS, cardiac hypertrophy, and inflammation, through transcriptional repression.4 In vitro experiments demonstrate that HEXIM1 functions as a transcriptional repressor by inhibiting P-TEFb, a protein complex composed of cyclin-dependent kinase 9 and a cyclin partner that regulates RNA polymerase II elongation.2–4 HEXIM1 interacts with 7SK small nuclear RNA (snRNA), a component of P-TEFb complex containing cyclin-dependent kinase 9 and cyclin T1. This interaction occurs through the KHRR 7SK snRNA binding motif of …

Modulation of a P-TEFb Functional Equilibrium for the Global Control of Cell Growth and Differentiation

P-TEFb phosphorylates RNA polymerase II and negative elongation factors to stimulate general transcriptional elongation. It is kept in a functional equilibrium through alternately interacting with its positive (the Brd4 protein) and negative (the HEXIM1 protein and 7SK snRNA) regulators. To investigate the physiological significance of this phenomenon, we analyzed the responses of HeLa cells and murine erythroleukemia cells (MELC) to hexamethylene bisacetamide (HMBA), which inhibits growth and induces differentiation of many cell types. For both cell types, an efficient, albeit temporary disruption of the 7SK-HEXIM1-P-TEFb snRNP and enhanced formation of the Brd4-P-TEFb complex occurred soon after the treatment started. When the P-TEFb-dependent HEXIM1 expression markedly increased as the treatment continued, the abundant HEXIM1 pushed the P-TEFb equilibrium back toward the 7SK/HEXIM1-bound state. For HeLa cells, as HMBA produced only a minor, temporary effect on their growth, the equilibrium gradually returned to its pretreatment level. In contrast, long-term treatment of MELC induced terminal division and differentiation. Concurrently, the P-TEFb equilibrium was shifted overwhelmingly toward the 7SK snRNP side. Together, these data link the P-TEFb equilibrium to the intracellular transcriptional demand and proliferative/differentiated states of cells.

Increased HEXIM1 expression during erythroleukemia and neuroblastoma cell differentiation

The HEXIM1 protein, in association with 7SK snRNA, binds and inhibits the kinase activity of P-TEFb (CDK9/cyclin T). P-TEFb activity is crucial for efficient transcription elongation of viral and cellular genes. HEXIM1 was originally isolated as a protein up-regulated by hexamethylene bisacetamide (HMBA), a prototypical inducer of differentiation. To determine the causative role of HEXIM1 during cell differentiation we analyzed the biochemical and functional consequences of HEXIM1 protein levels in several in vitro differentiation systems. We found that HEXIM1 mRNA and protein levels are up-regulated during differentiation of murine erythroleukemia cells upon treatment with HMBA or DMSO. Stimulation of HEXIM1 is not restricted to hematopoietic cells, as induction of phenotypic differentiation of neuroblastoma cells by retinoic acid results in up-regulation of HEXIM1. Moreover, ectopic expression of HEXIM1 causes growth inhibition and promotes neuronal differentiation. These findings highlight a crucial role of HEXIM1 protein during cell differentiation.

Transcription-dependent Association of Multiple Positive Transcription Elongation Factor Units to a HEXIM Multimer

The positive transcription elongation factor (P-TEFb) comprises a kinase, CDK9, and a Cyclin T1 or T2. Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA. HEXIM1 and HEXIM2 were found to form stable homo- and hetero-oligomers. Using yeast two-hybrid and transfection assays, we have now shown that the C-terminal domains of HEXIM proteins directly interact with each other. Hydrodynamic parameters measured by glycerol gradient ultracentrifugation and gel-permeation chromatography demonstrate that both purified recombinant and cellular HEXIM1 proteins form highly anisotropic particles. Chemical cross-links suggest that HEXIM1 proteins form dimers. The multimeric nature of HEXIM1 is maintained in P-TEFb.HEXIM1.7SK RNA complexes. Multiple P-TEFb modules are found in the inactive P-TEFb.HEXIM1.7SK complexes. It is proposed that 7SK RNA binding to a HEXIM1 multimer promotes the simultaneous recruitment and hence inactivation of multiple P-TEFb units.

Inhibition of Tat activity by the HEXIM1 protein

BackgroundThe positive transcription elongation factor b (P-TEFb) composed by CDK9/CyclinT1 subunits is a dedicated co-factor of HIV transcriptional transactivator Tat protein. Transcription driven by the long terminal repeat (LTR) of HIV involves formation of a quaternary complex between P-TEFb, Tat and the TAR element. This recruitment is necessary to enhance the processivity of RNA Pol II from the HIV-1 5' LTR promoter. The activity of P-TEFb is regulated in vivo and in vitro by the HEXIM1/7SK snRNA ribonucleic-protein complex.ResultsHere we report that Tat transactivation is effectively inhibited by co-expression of HEXIM1 or its paralog HEXIM2. HEXIM1 expression specifically represses transcription mediated by the direct activation of P-TEFb through artificial recruitment of GAL4-CycT1. Using appropriate HEXIM1 mutants we determined that effective Tat-inhibition entails the 7SK snRNA basic recognition motif as well as the C-terminus region required for interaction with cyclin T1. Enhanced expression of HEXIM1 protein modestly affects P-TEFb activity, suggesting that HEXIM1-mediated repression of Tat activity is not due to a global inhibition of cellular transcription.ConclusionThese results point to a pivotal role of P-TEFb for Tat's optimal transcription activity and suggest that cellular proteins that regulate P-TEFb activity might exert profound effects on Tat function in vivo.

HEXIM1 forms a transcriptionally abortive complex with glucocorticoid receptor without involving 7SK RNA and positive transcription elongation factor b

The HEXIM1 protein has been shown to form a protein-RNA complex composed of 7SK small nuclear RNA and positive transcription elongation factor b (P-TEFb), which is composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1, and to inhibit the kinase activity of CDK9, thereby suppressing RNA polymerase II-dependent transcriptional elongation. Here, we biochemically demonstrate that HEXIM1 forms a distinct complex with glucocorticoid receptor (GR) without RNA, CDK9, or cyclin T1. HEXIM1, through its arginine-rich nuclear localization signal, directly associates with the ligand-binding domain of GR. Introduction of HEXIM1 short interfering RNA and adenovirus-mediated exogenous expression of HEXIM1 positively and negatively modulated glucocorticoid-responsive gene activation, respectively. In the nucleus, HEXIM1 was shown to localize in a distinct compartment from that of the p160 coactivator transcriptional intermediary factor 2. Overexpression of HEXIM1 decreased ligand-dependent association between GR and transcriptional intermediary factor 2. Antisense-mediated disruption of 7SK blunted the negative effect of HEXIM1 on arylhydrocarbon receptor-dependent transcription but not on GR-mediated one, indicating that a class of transcription factors are direct targets of HEXIM1. These results indicate that HEXIM1 has dual roles in transcriptional regulation: inhibition of transcriptional elongation dependent on 7SK RNA and positive transcription elongation factor b and interference with the sequence-specific transcription factor GR via a direct protein-protein interaction. Moreover, the fact that the central nuclear localization signal of HEXIM1 is essential for both of these actions may argue the crosstalk of these functions.

Inhibition of P-TEFb (CDK9/Cyclin T) Kinase and RNA Polymerase II Transcription by the Coordinated Actions of HEXIM1 and 7SK snRNA

The positive transcriptional elongation factor b (P-TEFb), consisting of CDK9 and cyclin T, stimulates transcription by phosphorylating RNA polymerase II. It becomes inactivated when associated with the abundant 7SK snRNA. Here, we show that the 7SK binding alone was not sufficient to inhibit P-TEFb. P-TEFb was inhibited by the HEXIM1 protein in a process that specifically required 7SK for mediating the HEXIM1:P-TEFb interaction. This allowed HEXIM1 to inhibit transcription both in vivo and in vitro. P-TEFb dissociated from HEXIM1 and 7SK in cells undergoing stress response, increasing the level of active P-TEFb for stress-induced transcription. P-TEFb was the predominant HEXIM1-associated protein factor, and thus likely to be the principal target of inhibition coordinated by HEXIM1 and 7SK. Since HEXIM1 expression is induced in cells treated with hexamethylene bisacetamide, a potent inducer of cell differentiation, targeting the general transcription factor P-TEFb by HEXIM1/7SK may contribute to the global control of cell growth and differentiation.