Abstract
Hexamethylene bisacetamide inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which is composed of cyclin-dependent kinase 9 (CDK9)/cyclin T1. P-TEFb is an essential regulator for the transcriptional elongation by RNA polymerase II. A genome-wide study using human embryonic stem cells shows that most mRNA synthesis is regulated at the stage of transcription elongation, suggesting a possible role for P-TEFb/HEXIM1 in the gene regulation of stem cells. In this report, we detected a marked increase in HEXIM1 protein levels in the differentiated human pluripotent stem cells (hPSCs) induced by LY294002 treatment. Since no changes in CDK9 and cyclin T1 were observed in the LY294002-treated cells, increased levels of HEXIM1 might lead to inhibition of P-TEFb activity. However, treatment with a potent P-TEFb inhibiting compound, flavopiridol, failed to induce hPSC differentiation, ruling out the possible requirement for P-TEFb kinase activity in hPSC differentiation. Conversely, differentiation was observed when hPSCs were incubated with hexamethylene bisacetamide, a HEXIM1 inducing reagent. The involvement of HEXIM1 in the regulation of hPSCs was further supported when overexpression of HEXIM1 concomitantly induced hPSC differentiation. Collectively, our study demonstrates a novel role of HEXIM1 in regulating hPSC fate through a P-TEFb-independent pathway.
Highlights
Pluripotent stem cells (PSCs) such as human embryonic stem cells [1,2] and induced pluripotent stem cells [3,4] have enormous potential for regenerative medicine because of their ability to proliferate indefinitely and to differentiate into all three germ layers under appropriate conditions
It has been shown that many cellular genes required for human embryonic stem cells (hESCs) differentiation are regulated at the stage of transcription elongation, which is controlled by Positive transcription elongation factor b (P-TEFb)/Hexamethylene bisacetamide inducible protein 1 (HEXIM1) [20]
To investigate the possible involvement of P-TEFb/HEXIM1 in human pluripotent stem cells (hPSCs) pluripotency and differentiation, we examined the protein levels of HEXIM1, cyclin T1, and cyclin-dependent kinase 9 (CDK9) in HES-3 hPSC, HES-3-derived embryoid bodies (EB), and LY294002-treated HES-3 cells
Summary
Pluripotent stem cells (PSCs) such as human embryonic stem cells (hESCs) [1,2] and induced pluripotent stem (iPS) cells [3,4] have enormous potential for regenerative medicine because of their ability to proliferate indefinitely and to differentiate into all three germ layers under appropriate conditions. Treatment of P-TEFb inhibiting compounds, such as flavopiridol, blocks RNA Pol II at the pre-elongation phase and inhibits most of mRNA synthesis in cells [16,17,18,19] This observation clearly demonstrates that transcription of most cellular genes is regulated at the elongation stage, which is controlled by P-TEFb. Genome-wide analyses of Drosophila and hESCs reveal that many genes required for differentiation and development are regulated at the stage of transcription elongation, affirming the importance of P-TEFb in regulation of gene expression [20,21,22,23]
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