Qintong Li

Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor

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.

HEXIM2, a HEXIM1-related Protein, Regulates Positive Transcription Elongation Factor b through Association with 7SK

The kinase activity of positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1 or T2, is required for the transition of RNA polymerase II into productive elongation. P-TEFb activity has been shown to be negatively regulated by association with the small nuclear RNA 7SK and the HEXIM1 protein. Here, we characterize HEXIM2, a previously predicted protein with sequence similarity to HEXIM1. HEXIM2 is expressed in HeLa and Jurkat cells, and glycerol gradient analysis and immunoprecipitations indicate that HEXIM2, like HEXIM1, has a regulated association with P-TEFb. As HEXIM1 is knocked down, HEXIM2 functionally compensates for its association with P-TEFb. Electrophoretic mobility shift assays and in vitro kinase assays demonstrate that HEXIM2 forms complexes containing 7SK and P-TEFb and, in conjunction with 7SK, inhibits P-TEFb kinase activity. Our results provide strong evidence that HEXIM2 is a regulator of P-TEFb function. Furthermore, our results support the idea that the utilization of HEXIM1 or HEXIM2 to bind and inhibit P-TEFb can be differentially regulated in vivo.

HEXIM1 is a promiscuous double-stranded RNA-binding protein and interacts with RNAs in addition to 7SK in cultured cells

P-TEFb regulates eukaryotic gene expression at the level of transcription elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA-binding protein HEXIM1 or HEXIM2. In an effort to determine the minimal region of 7SK needed to interact with HEXIM1 in vitro, we found that an oligo comprised of nucleotides 10–48 sufficed. A bid to further narrow down the minimal region of 7SK led to a surprising finding that HEXIM1 binds to double-stranded RNA in a sequence-independent manner. Both dsRNA and 7SK (10–48), but not dsDNA, competed efficiently with full-length 7SK for HEXIM1 binding in vitro. Upon binding dsRNA, a large conformational change was observed in HEXIM1 that allowed the recruitment and inhibition of P-TEFb. Both subcellular fractionation and immunofluorescence demonstrated that, while most HEXIM1 is found in the nucleus, a significant fraction is found in the cytoplasm. Immunoprecipitation experiments demonstrated that both nuclear and cytoplasmic HEXIM1 is associated with RNA. Interestingly, the one microRNA examined (mir-16) was found in HEXIM1 immunoprecipitates, while the small nuclear RNAs, U6 and U2, were not. Our study illuminates novel properties of HEXIM1 both in vitro and in vivo, and suggests that HEXIM1 may be involved in other nuclear and cytoplasmic processes besides controlling P-TEFb.

Insulin‐like growth factor‐1 regulates the SIRT1‐p53 pathway in cellular senescence

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.

Nucleophosmin Interacts with HEXIM1 and Regulates RNA Polymerase II Transcription

Hexamethylene bis-acetamide-inducible protein 1 (HEXIM1) was identified earlier as an inhibitor of positive transcription elongation factor b (P-TEFb), which is a key transcriptional regulator of RNA polymerase II (Pol II). Studies show that more than half of P-TEFb in cells is associated with HEXIM1, which results in the inactivation of P-TEFb. Here, we identify a nucleolar protein, nucleophosmin (NPM), as a HEXIM1-binding protein. NPM binds to HEXIM1 in vitro and in vivo, and functions as a negative regulator of HEXIM1. Over-expression of NPM leads to proteasome-mediated degradation of HEXIM1, resulting in activation of P-TEFb-dependent transcription. In contrast, an increase in HEXIM1 protein levels and a decrease in transcription are detected when NPM is knocked down. We show that a cytoplasmic mutant of NPM, NPMc+, associates with and sequesters HEXIM1 in the cytoplasm resulting in higher RNA Pol II transcription. Correspondingly, cytoplasmic localization of endogenous HEXIM1 is detected in an acute myeloid leukemia (AML) cell line containing the NPMc+ mutation, suggesting the physiological importance of HEXIM1-NPMc+ interaction. Over-expression of NPM has been detected in tumors of various histological origins and our results may provide a possible molecular mechanism for the proto-oncogenic function of NPM. Furthermore, considering that 35% of AML patients are diagnosed with NPMc+ mutation, our findings suggest that in some cases of AML, RNA Pol II transcription may be disregulated by the malfunction of NPM and the mislocation of HEXIM1.

PKC phosphorylates HEXIM1 and regulates P-TEFb activity

The positive transcription elongation factor b (P-TEFb) regulates RNA polymerase II elongation. In cells, P-TEFb partitions between small active and larger inactive states. In the latter, HEXIM1 binds to 7SK snRNA and recruits as well as inactivates P-TEFb in the 7SK snRNP. Several stimuli can affect this P-TEFb equilibrium. In this study, we demonstrate that protein kinase C (PKC) phosphorylates the serine at position158 (S158) in HEXIM1. This phosphorylated HEXIM1 protein neither binds to 7SK snRNA nor inhibits P-TEFb. Phorbol esters or the engagement of the T cell antigen receptor, which activate PKC and the expression of the constitutively active (CA) PKCθ protein, which is found in T cells, inhibit the formation of the 7SK snRNP. All these stimuli increase P-TEFb-dependent transcription. In contrast, the kinase-negative PKCθ and the mutant HEXIM1 (S158A) proteins block effects of these PKC-activating stimuli. These results indicate that the phosphorylation of HEXIM1 by PKC represents a major regulatory step of P-TEFb activity in cells.

MicroRNA Regulation of Stem Cell Fate

MicroRNAs modulate target gene expression and are essential for normal development, but how does this pathway impact cell fate decisions? In this issue of Cell Stem Cell, Ivey et al. (2008) find that muscle-specific microRNAs repress nonmuscle genes to direct embryonic stem cell differentiation to mesoderm and muscle.

Cyclin K-containing Kinase Complexes Maintain Self-renewal in Murine Embryonic Stem Cells

Background: The physiological function of cyclin K is poorly defined. Results: Cyclin K interacts with CDK12 and CDK13, and knockdown of cyclin K, CDK12, or CDK13 causes embryonic stem cell differentiation. Conclusion: Cyclin K, CDK12, and CDK13 are required for embryonic stem cell self-renewal. Significance: Novel kinase complexes are identified to maintain embryonic stem cell pluripotency. Protein phosphorylation plays an important role in the regulation of self-renewal and differentiation of embryonic stem cells. However, the responsible intracellular kinases are not well characterized. Here, we discovered that cyclin K protein was highly expressed in pluripotent embryonic stem cells but low in their differentiated derivatives or tissue-specific stem cells. Upon cell differentiation, the level of cyclin K protein was decreased. Furthermore, knockdown of cyclin K led to cell differentiation, which could be rescued by an expression construct resistant to RNA interference. Surprisingly, cyclin K did not interact with CDK9 protein in cells as thought previously. Instead, it associated with CrkRS (also known as CDK12) and CDC2L5 (also known as CDK13). Similar to cyclin K, both CDK12 and CDK13 proteins were highly expressed in murine embryonic stem cells and were decreased upon cell differentiation. Importantly, knockdown of either kinase resulted in differentiation. Thus, our studies have uncovered two novel protein kinase complexes that maintain self-renewal in embryonic stem cells.

Cyclin T2 Is Essential for Mouse Embryogenesis

ABSTRACT The positive transcription elongation factor b (P-TEFb) is essential for the elongation of transcription and cotranscriptional processing by RNA polymerase II. In mammals, it contains predominantly the C-type cyclin cyclin T1 (CycT1) or CycT2 and cyclin-dependent kinase 9 (Cdk9). To determine if these cyclins have redundant functions or affect distinct sets of genes, we genetically inactivated the CycT2 gene (Ccnt2) using the β-galactosidase-neomycin gene (β-geo) gene trap technology in the mouse. Visualizing β-galactosidase during mouse embryogenesis revealed that CycT2 is expressed abundantly during embryogenesis and throughout the organism in the adult. This finding was reflected in the expression of CycT2 in all adult tissues and organs. However, despite numerous matings of heterozygous mice, we observed no CycT2−/− embryos, pups, or adult mice. This early lethality could have resulted from decreased expression of critical genes, which were revealed by short interfering RNAs against CycT2 in embryonic stem cells. Thus, CycT1 and CycT2 are not redundant, and these different P-TEFb complexes regulate subsets of distinct genes that are important for embryonic development.

Histone methyltransferase SETD2 modulates alternative splicing to inhibit intestinal tumorigenesis

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.