Alternative titles; symbols
HGNC Approved Gene Symbol: HEXIM1
Cytogenetic location: 17q21.31 Genomic coordinates (GRCh38) : 17:45,148,475-45,152,099 (from NCBI)
RNA polymerase II is a multisubunit polymerase that is regulated by phosphorylation of serines in the C-terminal domain (CTD) of the largest subunit (POLR2A; 180660). The CTD is phosphorylated by positive transcription factor b (P-TEFb), a complex containing CDK9 (603251) and either cyclin T1 (CCNT1; 143055) or T2 (CCNT2; 603862). HEXIM1 or HEXIM2 (615695), along with 7SK small nuclear RNA (RN7SK; 606515), interacts with P-TEFb in a reversible manner and inhibits CDK9 kinase activity, thereby inhibiting activation of RNA polymerase II (summary by Byers et al., 2005).
Huang et al. (2002) cloned mouse Clp1. The deduced 41-kD protein is 85.3% homologous to human HIS1. Northern and Western blot analyses revealed wide expression during postnatal development, with highest levels in heart, skeletal muscle, and brain. Immunofluorescence localization of endogenous Clp1 in rat vascular smooth muscle and rat primary cardiac cells indicated nuclear localization.
By mass spectrometric analysis of tryptic peptides that copurified with inactive P-TEFb/7SK complexes from HeLa cell lysates, Michels et al. (2003) identified HEXIM1, which they designated MAQ1. The deduced 359-amino acid protein has a central bipartite nuclear localization sequence. Database analysis revealed a paralog, HEXIM2, in mammals, but only a single MAQ1 ortholog was found in frogs, fish, and possibly insects. No orthologs were detected in worm or yeast. Immunofluorescence analysis detected MAQ1 in HeLa cell nuclei.
Byers et al. (2005) stated that human HEXIM1 has a calculated molecular mass of 41 kD. However, they found by SDS-PAGE that the HEXIM1 expressed in E. coli had an apparent molecular mass of 67 kD.
By Northern blot analysis, Yik et al. (2005) found HEXIM1 transcripts of approximately 4.0 and 2.4 kb in all 10 human tissues examined, with a particularly high level of the short form in placenta. The 2 forms differed in alternate usage of poly(A) signals.
By coimmunoprecipitation of HeLa cell lysates, Michels et al. (2003) found that MAQ1 associated with inactive P-TEFb/7SK complexes, but not with active P-TEFb complexes. 7SK RNA appeared to be required to maintain stable association between MAQ1 and P-TEFb. MAQ1 interacted directly with cyclins T1 and T2, but not with CDK9, in yeast 2-hybrid assays. Domain analysis revealed that the C-terminal region of MAQ1 interacted with the N terminus of cyclin T1 or T2.
Byers et al. (2005) stated that the PYNT motif of HEXIM1 is required for high-affinity binding of HEXIM1 to the P-TEFb complex. Western blot analysis of fractions of human cell lines separated by gradient sedimentation revealed a portion of HEXIM1 that cosedimented with CDK9 and cyclin T1 and another portion in the free form. A similar pattern was found for HEXIM2. The 2 proteins had similar affinity for 7SK RNA and P-TEFb, and both inhibited the in vitro kinase activity of P-TEFb. P-TEFb phosphorylated threonine within the PYNT motif of HEXIM1 or HEXIM2, and the HEXIM proteins required this motif for inhibition of P-TEFb. Inhibition of transcription elongation increased the amount of free HEXIM proteins, in addition to causing dissociation of the P-TEFb complex from RNA polymerase II. Knockdown of HEXIM1 in HeLa cells via small interfering RNA caused upregulation of HEXIM2.
Using epitope-tagged proteins expressed in HeLa cells, Yik et al. (2005) found that HEXIM1 and HEXIM2 formed stable homo- and heterodimers in a 7SK- and P-TEFb-independent manner. Either HEXIM dimer could interact with 7SK and P-TEFb independently of the other HEXIM protein, and both inhibited the kinase activity of P-TEFb and the transcriptional activity of RNA polymerase II. Both proteins were induced by differentiation in several human cell lines.
Li et al. (2005) found that HEXIM1 formed stable dimers via a coiled-coil region near its C terminus. HEXIM1 remained a dimer after binding to 7SK. Domain analysis revealed that the first 120 amino acids of HEXIM1 enabled it to inhibit P-TEFb in the absence of 7SK, suggesting that the N terminus of HEXIM1 is an autoinhibitory domain that masks the P-TEFb-binding domain. Mutation analysis revealed that tyr271 and phe208 of HEXIM1 had minimal involvement in binding of HEXIM1 to CDK9, but that they were critical for inhibition of CDK9 kinase activity. Phosphorylation of CDK9 on thr186 was required for efficient kinase activity, and mutation of this residue reduced the affinity of CDK9 for 7SK-HEXIM1 and 7SK-HEXIM2.
Kohoutek et al. (2006) found that HEXIM1 inhibited the ability of C2TA (MHC2TA; 600005) to activate transcription of MHC class II genes in HeLa cells. This inhibition depended on the CCNT1-binding domain of HEXIM1. In vitro binding experiments and chromatin immunoprecipitation analyses showed that HEXIM1 competed with C2TA for binding to CCNT1 and sequestered PTEFB from C2TA. HeLa cells depleted of HEXIM1 by small interfering RNA displayed increased C2TA activity and induction of C2TA-dependent genes.
Michels et al. (2003) determined that HEXIM1 is an intronless gene.
By genomic sequence analysis, Michels et al. (2003) found that the HEXIM1 and HEXIM2 genes are oriented in tandem on chromosome 17q21.32. The 2 genes are separated by 8.7 kb.
Byers, S. A., Price, J. P., Cooper, J. J., Li, Q., Price, D. H. HEXIM2, a HEXIM1-related protein, regulates positive transcription elongation factor b through association with 7SK. J. Biol. Chem. 280: 16360-16367, 2005. [PubMed: 15713662] [Full Text: https://doi.org/10.1074/jbc.M500424200]
Huang, F., Wagner, M., Siddiqui, M. A. Q. Structure, expression, and functional characterization of the mouse CLP-1 gene. Gene 292: 245-259, 2002. [PubMed: 12119119] [Full Text: https://doi.org/10.1016/s0378-1119(02)00596-6]
Kohoutek, J., Blazek, D., Peterlin, B. M. Hexim1 sequesters positive transcription elongation factor b from the class II transactivator on MHC class II promoters. Proc. Nat. Acad. Sci. 103: 17349-17354, 2006. [PubMed: 17088550] [Full Text: https://doi.org/10.1073/pnas.0603079103]
Li, Q., Price, J. P., Byers, S. A., Cheng, D., Peng, J., Price, D. H. Analysis of the large inactive P-TEFb complex indicates that it contains one 7SK molecule, a dimer of HEXIM1 or HEXIM2, and two P-TEFb molecules containing Cdk9 phosphorylated at threonine 186. J. Biol. Chem. 280: 28819-28826, 2005. [PubMed: 15965233] [Full Text: https://doi.org/10.1074/jbc.M502712200]
Michels, A. A., Nguyen, V. T., Fraldi, A., Labas, V., Edwards, M., Bonnet, F., Lania, L., Bensaude, O. MAQ1 and 7SK RNA interact with CDK9/cyclin T complexes in a transcription-dependent manner. Molec. Cell. Biol. 23: 4859-4869, 2003. Note: Erratum: Molec. Cell. Biol. 23: 9405 only, 2003. [PubMed: 12832472] [Full Text: https://doi.org/10.1128/MCB.23.14.4859-4869.2003]
Yik, J. H. N., Chen, R., Pezda, A. C., Zhou, Q. Compensatory contributions of HEXIM1 and HEXIM2 in maintaining the balance of active and inactive positive transcription elongation factor b complexes for control of transcription. J. Biol. Chem. 280: 16368-16376, 2005. [PubMed: 15713661] [Full Text: https://doi.org/10.1074/jbc.M500912200]