Compensatory Contributions of HEXIM1 and HEXIM2 in Maintaining the Balance of Active and Inactive Positive Transcription Elongation Factor b Complexes for Control of Transcription

Jasper H.N Yik,Ruichuan Chen,Andrea C Pezda,Qiang Zhou

doi:10.1074/jbc.m500912200

Abstract

Human positive transcriptional elongation factor b (P-TEFb), consisting of a cyclin-dependent kinase 9-cyclin T heterodimer, stimulates general and disease-specific transcriptional elongation by phosphorylating RNA polymerase II. The HEXIM1 protein, aided by the 7SK snRNA, sequesters P-TEFb into an inactive 7SK.HEXIM1.P-TEFb small nuclear ribonucleic acid particle for inhibition of transcription and, consequently, cell proliferation. Here we show that, like HEXIM1, a highly homologous protein named HEXIM2 also possesses the ability to inactivate P-TEFb to suppress transcription through a 7SK-mediated interaction with P-TEFb. Furthermore, HEXIM1 and HEXIM2 can form stable homo- and hetero-oligomers (most likely dimers), which may nucleate the formation of the 7SK small nuclear ribonucleic acid particle. Despite their similar functions, HEXIM1 and HEXIM2 exhibit distinct expression patterns in various human tissues and established cell lines. In HEXIM1-knocked down cells, HEXIM2 can functionally and quantitatively compensate for the loss of HEXIM1 to maintain a constant level of the 7SK/HEXIM-bound P-TEFb. Our results demonstrate that there is a tightly regulated cellular process to maintain the balance between active and inactive P-TEFb complexes, which controls global transcription as well as cell growth and differentiation.

Highlights

RNA polymerase II mediates transcription of all class II protein-coding genes in a tightly regulated process that can be divided into five stages: preinitiation, initiation, promoter clearance, elongation, and termination (1)
In this study we show that the HEXIM1 homologue, HEXIM2, is a novel and integral component of the 7SK1⁄7P-TEFb snRNP, which plays an important role in sequestering positive transcriptional elongation factor b (P-TEFb) into an inactive complex for suppression of transcriptional elongation
We have previously shown that the two regions most critical for HEXIM1 function are the arginine-rich 7SKbinding motif located within the central nuclear localization signal (NLS) and the P-TEFbbinding and inhibitory domain found in the carboxyl-terminal half (7, 11)

Summary

The abbreviations used are

P-TEFb, positive transcription elongation factor b; CDK, cyclin-dependent kinase; CycT1, cyclin T1; DRB, 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole; HMBA, hexamethylene bisacetamide; NE, nuclear extract; NLS, nuclear localization signal; snRNP, small nuclear ribonucleic acid particle; GST, glutathione Stransferase; HIV, human immunodeficiency virus; HA, hemagglutinin. Disruption of the 7SK ribonucleic acid particle by hypertrophic signals has been shown to activate P-TEFb in cardiac myocytes, leading to an increase in phosphorylation of RNA polymerase II that results in a global elevation of RNA and protein contents (12). This chain of events eventually causes the enlargement of heart cells that leads to cardiac hypertrophy. The observed anti-growth function of HEXIM1 in many cell types suggests that regulation of the activity of P-TEFb through changes in HEXIM1 expression or its P-TEFb-targeting capability could potentially play a key role in directing the cells toward either proliferation or differentiation. We examined the expression patterns of HEXIM1 and HEXIM2 in various human tissues and compared the inhibitory effects of HEXIM1 and HEXIM2 on P-TEFb activity in order to gain insights into the physiological function of HEXIM2

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION