The positive transcription elongation factor b (P‐TEFb) plays a pivotal role in productive elongation of nascent RNA molecules by RNA polymerase II. Core active P‐TEFb is composed of CDK9 and cyclin T. In addition, mammalian cell extracts contain an inactive P‐TEFb complex composed of four components, CDK9, cyclin T, the 7SK snRNA and the MAQ1/HEXIM1 protein. We now report an in vitro reconstitution of 7SK‐dependent HEXIM1 association to purified P‐TEFb and subsequent CDK9 inhibition. Yeast three‐hybrid tests and gel‐shift assays indicated that HEXIM1 binds 7SK snRNA directly and a 7SK snRNA‐recognition motif was identified in the central part of HEXIM1 (amino acids (aa) 152–155). Data from yeast two‐hybrid and pull‐down assay on GST fusion proteins converge to a direct binding of P‐TEFb to the HEXIM1 C‐terminal domain (aa 181–359). Consistently, point mutations in an evolutionarily conserved motif (aa 202–205) were found to suppress P‐TEFb binding and inhibition without affecting 7SK recognition. We propose that the RNA‐binding domain of HEXIM1 mediates its association with 7SK and that P‐TEFb then enters the complex through association with HEXIM1.

Positive transcription elongation factor b (P-TEFb) regulates eukaryotic gene expression at the level of elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA-binding protein, HEXIM1 or HEXIM2. To further understand how P-TEFb is regulated, we analyzed the stoichiometry of all the known components of the large, inactive P-TEFb complex. Mutational analyses of a putative coiled coil region in the carboxyl-terminal portion of HEXIM1 revealed that the protein is a dimer in solution and remains a dimer after binding to 7SK. Although a HEXIM1 dimer contains two potential RNA binding motifs and ultimately recruits two P-TEFb molecules, it associates with only one molecule of RNA. The first 172 nucleotides of the 330-nucleotide 7SK are sufficient to bind HEXIM1 or HEXIM2, and then recruit and inhibit P-TEFb. Deletion of the first 121 amino acids of HEXIM1 allowed it to inhibit P-TEFb partially in the absence of 7SK RNA. Mutation of a conserved tyrosine (Tyr(271) in HEXIM1) to alanine or glutamate or mutation of a conserved phenylalanine (Phe(208)) to alanine, aspartate, or lysine, resulted in loss of inhibition of P-TEFb, but did not affect formation of the 7SK.HEXIM.P-TEFb complex. Analysis of T-loop phosphorylation in Cdk9 indicated that phosphorylation of Thr(186), but not Ser(175), was essential for kinase activity and for recruitment of P-TEFb to the 7SK.HEXIM complex. A model illustrates what is currently known about how HEXIM proteins, 7SK, and P-TEFb assemble to maintain an activated kinase in a readily available, but inactive form.

Summary: Cellular senescence, which is known to halt proliferation of aged and stressed cells, plays a key role against cancer development and is also closely associated with organismal aging. While increased insulin‐like growth factor (IGF) signaling induces cell proliferation, survival and cancer progression, disrupted IGF signaling is known to enhance longevity concomitantly with delay in aging processes. The molecular mechanisms involved in the regulation of aging by IGF signaling and whether IGF regulates cellular senescence are still poorly understood. In this study, we demonstrate that IGF‐1 exerts a dual function in promoting cell proliferation as well as cellular senescence. While acute IGF‐1 exposure promotes cell proliferation and is opposed by p53, prolonged IGF‐1 treatment induces premature cellular senescence in a p53‐dependent manner. We show that prolonged IGF‐1 treatment inhibits SIRT1 deacetylase activity, resulting in increased p53 acetylation as well as p53 stabilization and activation, thus leading to premature cellular senescence. In addition, either expression of SIRT1 or inhibition of p53 prevented IGF‐1‐induced premature cellular senescence. Together, these findings suggest that p53 acts as a molecular switch in monitoring IGF‐1‐induced proliferation and premature senescence, and suggest a possible molecular connection involving IGF‐1‐SIRT1‐p53 signaling in cellular senescence and aging.

The histone H3K36 methyltransferase SETD2 is frequently mutated or deleted in a variety of human tumors. Nevertheless, the role of SETD2 loss in oncogenesis remains largely undefined. Here, we found that SETD2 counteracts Wnt signaling and its inactivation promotes intestinal tumorigenesis in mouse models of colorectal cancer (CRC). SETD2 was not required for intestinal homeostasis under steady state; however, upon irradiation, genetic inactivation of Setd2 in mouse intestinal epithelium facilitated the self-renewal of intestinal stem/progenitor cells as well as tissue regeneration. Furthermore, depletion of SETD2 enhanced the susceptibility to tumorigenesis in the context of dysregulated Wnt signaling. Mechanistic characterizations indicated that SETD2 downregulation affects the alternative splicing of a subset of genes implicated in tumorigenesis. Importantly, we uncovered that SETD2 ablation reduces intron retention of dishevelled segment polarity protein 2 (DVL2) pre-mRNA, which would otherwise be degraded by nonsense-mediated decay, thereby augmenting Wnt signaling. The signaling cascades mediated by SETD2 were further substantiated by a CRC patient cohort analysis. Together, our studies highlight SETD2 as an integral regulator of Wnt signaling through epigenetic regulation of RNA processing during tissue regeneration and tumorigenesis.