Pol II-dependent Transcription Research Articles

Opposing Role of Condensin and Radiation-sensitive Gene RAD52 in Ribosomal DNA Stability Regulation

Blocking target of rapamycin signaling by starvation or rapamycin inhibits ribosomal DNA (rDNA) transcription and causes condensin-mediated rDNA condensation and nucleolar contraction. In the absence of condensin, however, repression of rDNA transcription leads to rDNA instability and elevated level of extrachromosomal rDNA circles and nucleolar fragmentation. Here, we show that mutations in the Rad52 homologous recombination machinery block rDNA instability. Rad52 is normally excluded from the nucleolus. In the absence of condensin, however, repression of rDNA transcription results in Rad52 localization to the nucleolus, association with rDNA and subsequent formation of extrachromosomal rDNA circles, and reduced cell survival. In contrast, deletion of RAD52 restores cell viability under the same conditions. These results reveal an opposing role of condensin and Rad52 in the control of rDNA stability under nutrient starvation conditions.

Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1

We have found earlier that Tor1 binds to 5S rDNA chromatin but the functional significance has not been established. Here, we show that association with 5S rDNA chromatin is necessary for TOR complex 1 (TORC1) to regulate the synthesis of 5S ribosomal RNA and transfer RNAs (tRNAs) by RNA polymerase (Pol) III, as well as the phosphorylation and binding to Pol III-transcribed genes of the Pol III repressor Maf1. Interestingly, TORC1 does not bind to tRNA genes, suggesting that TORC1 modulates tRNA synthesis indirectly through Maf1 phosphorylation at the rDNA loci. We also find that Maf1 cytoplasmic localization is dependent on the SSD1-v allele. In W303 cells that carry the SSD1-d allele, Maf1 is constitutively nuclear but its nucleolar localization is inhibited by TORC1, indicating that TORC1 regulates nucleoplasm-to-nucleolus transport of Maf1. Finally, we show that TORC1 interacts with Maf1 in vivo and phosphorylates Maf1 in vitro, and regulates Maf1 nucleoplasm-to-nucleolus translocation. Together, these observations provide new insights into the chromatin-dependent mechanism by which TORC1 controls transcription by Pol III.

A Phase 1 Trial of SNS-032, a Potent and Specific CDK 2, 7 and 9 Inhibitor, in Chronic Lymphocytic Leukemia and Multiple Myeloma

SNS-032, formerly BMS-387032, is a highly selective and potent inhibitor of cyclin-dependent kinases (CDK) 2, 7 and 9. CDK2 and CDK7 are involved in cell cycle regulation. CDK7, along with CDK9, regulate RNA polymerase (Pol) II-dependent transcription. Temporary inhibition of RNA Pol II-dependent transcription by SNS-032 has significant effects on short half-life transcripts and proteins, particularly survival factors, cell cycle regulatory proteins, and cytokines that are critical for the survival of malignant B-cells in chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). A phase 1 dose-escalation study in patients with MM and CLL is ongoing with separate dose escalations for each indication. The study is designed to evaluate safety, pharmacokinetics (PK) and preliminary evidence of activity of a loading dose (LD) followed by a 6 hour infusion of SNS-032 given weekly for 3 consecutive weeks of each 28-day cycle. Dose and schedule aim to maintain for 6 hours threshold plasma concentrations of 115 ng/mL (in vitro IC90) and higher. The study incorporates an exploratory analysis of potential pharmacodynamic (PD) biomarkers such as decreased phosphorylation of RNA Pol II C-terminal domain to demonstrate inhibition of CDK7 and CDK9, and decreased expression of survival factors to indicate transcriptional inhibition.Methods: Previously treated patients with advanced CLL or MM, measurable disease, and ECOG status 0–1 were eligible. Increasing doses of SNS-032 given as an LD followed by a 6 hr infusion were evaluated. The total starting dose was 15 mg/m2 comprised of a LD of 5 mg/m2 followed by 10 mg/m2 over 6 hr with dose escalation by modified Fibonacci. PD studies of target modulation were performed on peripheral blood mononuclear cells (PBMC) obtained pre- and post-dose. Direct target modulation or downstream effects of target inhibition were evaluated.Results: 35 patients have been treated to date, 18 MM patients and 17 CLL patients. Median age was 61 (range 45–82), with 13 females and 24 males. Median number of prior therapies was 5 (range: 1–11). MM patients have received total doses of 15 – 75 mg/m2. No drug-related dose limiting toxicities (DLTs) or objective responses have been reported thus far in MM. CLL patients have received total doses of 15 –100 mg/m2. No drug-related DLTs were observed through the 50 mg/m2 dose cohort. At 75 mg/m2, concentrations of SNS- 032 exceeded IC90 (mean maximum concentration during the 6 hr infusion was 261 ± 45 ng/mL). Evidence of biochemical tumor lysis syndrome (TLS) was observed in all CLL patients treated at this dose level. One patient experienced a DLT of vascular leak syndrome and failure to receive all 3 cycle 1 doses. One CLL patient has been treated thus far at 100 mg/m2. This patient experienced TLS with a DLT of elevated liver function enzymes for >48 hr and received only 2 of 3 doses in cycle 1. No objective responses have been observed. PD analyses showed evidence of decreased Mcl-1 or XIAP in several patients.Conclusions: The mechanism of action of SNS-032 supports testing this agent in B-cell malignancies such as MM and CLL. A pharmacologically-derived dose regimen that sustains IC90 SNS-032 concentrations or higher for 6 hr is being studied; target levels were achieved and exceeded in cohort 5 (75 mg/m2) for both MM and CLL. No DLTs or objective responses have been observed thus far in MM. AEs and DLTs related to mild to moderate TLS were observed in CLL patients at 75 mg/m2 and higher. No objective responses have been observed. Preliminary evidence of target-specific PD modulation has been demonstrated. Enrollment in this trial is continuing.

EEF1A Is a Novel Component of the Mammalian Nuclear Protein Export Machinery

The cytoplasmic translation factor eEF1A has been implicated in the nuclear export of tRNA species in lower eukaryotes. Here we demonstrate that eEF1A plays a central role in nuclear export of proteins in mammalian cells. TD-NEM (transcription-dependent nuclear export motif), a newly characterized nuclear export signal, mediates efficient nuclear export of several proteins including the von Hippel-Lindau (VHL) tumor suppressor and the poly(A)-binding protein (PABP1) in a manner that is dependent on ongoing RNA polymerase II (RNA PolII)-dependent transcription. eEF1A interacts specifically with TD-NEM of VHL and PABP1 and disrupting this interaction, by point mutations of key TD-NEM residues or treatment with actinomycin D, an inhibitor of RNA PolII-dependent transcription, prevents assembly and nuclear export. siRNA-induced knockdown or antibody-mediated depletion of eEF1A prevents in vivo and in vitro nuclear export of TD-NEM-containing proteins. Nuclear retention experiments and inhibition of the Exportin-5 pathway suggest that eEF1A stimulates nuclear export of proteins from the cytoplasmic side of the nuclear envelope, without entering the nucleus. Together, these data identify a role for eEF1A, a cytoplasmic mediator of tRNA export in yeast, in the nuclear export of proteins in mammalian cells. These results also provide a link between the translational apparatus and subcellular trafficking machinery demonstrating that these two central pathways in basic metabolism can act cooperatively.

RNA polymerase II-mediated transcription at active loci does not require histone H3S10 phosphorylation in Drosophila.

JIL-1 is the major kinase controlling the phosphorylation state of histone H3S10 at interphase in Drosophila. In this study, we used three different commercially available histone H3S10 phosphorylation antibodies, as well as an acid-free polytene chromosome squash protocol that preserves the antigenicity of the histone H3S10 phospho-epitope, to examine the role of histone H3S10 phosphorylation in transcription under both heat shock and non-heat shock conditions. We show that there is no redistribution or upregulation of JIL-1 or histone H3S10 phosphorylation at transcriptionally active puffs in such polytene squash preparations after heat shock treatment. Furthermore, we provide evidence that heat shock-induced puffs in JIL-1 null mutant backgrounds are strongly labeled by antibody to the elongating form of RNA polymerase II (Pol IIoser2), indicating that Pol IIoser2 is actively involved in heat shock-induced transcription in the absence of histone H3S10 phosphorylation. This is supported by the finding that there is no change in the levels of Pol IIoser2 in JIL-1 null mutant backgrounds compared with wild type. mRNA from the six genes that encode the major heat shock protein in Drosophila, Hsp70, is transcribed at robust levels in JIL-1 null mutants, as directly demonstrated by qRT-PCR. Taken together, these data are inconsistent with the model that Pol II-dependent transcription at active loci requires JIL-1-mediated histone H3S10 phosphorylation, and instead support a model in which transcriptional defects in the absence of histone H3S10 phosphorylation are a result of structural alterations of chromatin.

PCR-based detection of Pol III-transcribed transposons and its application to the rodent model of ultraviolet response

Cellular levels of RNAs containing transposable elements increase in response to various stresses. Polymerase III (Pol III)-dependent transcripts of transposons are different from transposon-containing RNAs generated by read-through Pol II-dependent transcription. Until now, Pol III transcripts were detected by primer extension followed by time-consuming gel electrophoresis. In this paper, we describe a more sensitive PCR-based method for the selective detection of Pol III-transcribed RNAs. The method is based on the difference in sequences at the 5' ends of the Pol II- and Pol III-dependent transcripts. We employed this method to quantify Pol III transcripts of transposon B1 in rodent cells and revealed that their levels are affected by UV irradiation. We therefore conclude that the abundance of the Pol III-transcribed fraction of cellular RNA may serve as marker of stress response and can be conveniently quantified by the method described.

The Werner Syndrome Helicase Is a Cofactor for HIV-1 Long Terminal Repeat Transactivation and Retroviral Replication

The Werner syndrome helicase (WRN) participates in DNA replication, double strand break repair, telomere maintenance, and p53 activation. Mutations of wrn cause Werner syndrome (WS), an autosomal recessive premature aging disorder associated with cancer predisposition, atherosclerosis, and other aging related symptoms. Here, we report that WRN is a novel cofactor for HIV-1 replication. Immortalized human WRN(-/-) WS fibroblasts, lacking a functional wrn gene, are impaired for basal and Tat-activated HIV-1 transcription. Overexpression of wild-type WRN transactivates the HIV-1 long terminal repeat (LTR) in the absence of Tat, and WRN cooperates with Tat to promote high-level LTR transactivation. Ectopic WRN induces HIV-1 p24(Gag) production and retroviral replication in HIV-1-infected H9(HIV-1IIIB) lymphocytes. A dominant-negative helicase-minus mutant, WRN(K577M), inhibits LTR transactivation and HIV-1 replication. Inhibition of endogenous WRN, through co-expression of WRN(K577M), diminishes recruitment of p300/CREB-binding protein-associated factor (PCAF) and positive transcription elongation factor b (P-TEFb) to Tat/transactivation response-RNA complexes, and immortalized WRN(-/-) WS fibroblasts exhibit comparable defects in recruitment of PCAF and P-TEFb to the HIV-1 LTR. Our results demonstrate that WRN is a novel cellular cofactor for HIV-1 replication and suggest that the WRN helicase participates in the recruitment of PCAF/P-TEFb-containing transcription complexes. WRN may be a plausible target for antiretroviral therapy.

Mitotic and Stress-induced Phosphorylation of HsPI3K-C2α Targets the Protein for Degradation

Activation of the phosphoinositide 3-kinases (PI 3-kinases) has been implicated in multiple cellular responses such as proliferation and survival, membrane and cytoskeletal reorganization, and intracellular vesicular trafficking. The activities and subcellular localization of PI 3-kinases were shown to be regulated by phosphorylation. Previously we demonstrated that class II HsPIK3-C2alpha becomes phosphorylated upon inhibition of RNA pol II-dependent transcription (Didichenko, S. A., and Thelen, M. (2001) J. Biol. Chem. 276, 48135-48142). In this study we investigated cell cycle-dependent and genotoxic stress-induced phosphorylation of HsPIK3-C2alpha. We find that the kinase becomes phosphorylated upon exposure of cells to UV irradiation and in proliferating cells at the G2/M transition of the cell cycle. Stress-dependent and mitotic phosphorylation of HsPIK3-C2alpha occurs on the same serine residue (Ser259) within a recognition motif for proline-directed kinases. Mitotic phosphorylation of HsPIK3-C2alpha can be attributed to Cdc2 activity, and stress-induced phosphorylation of HsPIK3-C2alpha is mediated by JNK/SAPK. The protein level of HsPIK3-C2alpha is regulated by proteolysis in a cell cycle-dependent manner and in response of cells to stress. Phosphorylation appears to be a prerequisite for proteasome-dependent degradation of HsPIK3-C2alpha and may therefore contribute indirectly to the regulation of the activity of the kinase.

Defects in yeast RNA polymerase II transcription elicit hypersensitivity to G1 arrest induced by Kluyveromyces lactis zymocin.

The G1 cell cycle arrest imposed by Kluyveromyces lactis zymocin on Saccharomyces cerevisiae requires a functional RNA polymerase II (pol II) TOT/Elongator complex. In a study of zymocin's mode of action, genetic scenarios known to impair transcription or affect the pol II machinery itself were found to elicit hypersensitivity to zymocin. Thus, mutations in components of SAGA, SWI/SNF, Mediator and Ccr4-Not, complexes involved in transcriptionally relevant functions such as nucleosome modification, chromatin remodelling and formation of the preinitiation complex, make yeast cells hypersensitive to the lethal effects of zymocin. The defects at the level of transcriptional elongation displayed by rtf1Delta, ctk1, fcp1 and rpb2 mutants also result in zymocin hypersensitivity. Intriguingly, inactivation of histone deacetylase (HDAC) activity, which is expected to reduce the demand for the histone acetyltransferase (HAT) function of TOT/Elongator, also reduces sensitivity to zymocin. Thus, zymocin interferes with pol II-dependent transcription, and this effect requires the HAT function of TOT, presumably while the Elongator complex is associated with pol II.

Differential transcriptional activity associated with chromatin configuration in fully grown mouse germinal vesicle oocytes.

It was previously shown that fully grown ovarian germinal vesicle (GV) oocytes of adult mice exhibit several nuclear configurations that differ essentially by the presence or absence of a ring of condensed chromatin around the nucleolus. These configurations have been termed, respectively, SN (surrounded nucleolus) and NSN (nonsurrounded nucleolus). Work from our and other laboratories has revealed ultrastructural and functional differences between these two configurations. The aims of the present study were 1) to analyze the equilibrium between the SN and the NSN population as a function of the age of the mice and the time after hCG-induced ovulation and 2) to study the polymerase I (pol I)- and polymerase II (pol II)-dependent transcription in both types of oocytes through the detection of bromouridine incorporated into nascent RNA. We show 1) that ovarian GV oocytes exhibiting the SN-type configuration can be found as soon as 17 days after birth in the C57/CBA mouse strain and 2) that the SN:NSN ratio of ovarian GV oocytes is very low just after hCG-induced ovulation and then increases progressively with the time after ovulation. Furthermore, we demonstrate that the SN configuration correlates strictly with the arrest of both pol I- and pol II-dependent transcription in mice at any age. Finally, we show that ribosomal genes are located at the outer periphery of the nucleolus in the NSN configuration and that pol I-dependent perinucleolar transcription sites correspond to specific ultrastructural features of the nucleolus. Altogether, these results provide clear-cut criteria delineating transcriptionally active GV oocytes from those that are inactive, and confirm that the SN-type configuration is mostly present in preovulatory oocytes.

Interaction of the Transcription Factor YY1 with Human Poly(ADP-Ribosyl) Transferase

Poly(ADP-ribosyl) transferase (ADPRT) is a nuclear enzyme that catalyzes the synthesis of ADP-ribose polymers from NAD+as well as the transfer of these polymers onto acceptor proteins. The function of ADPRT is thought to be related to a number of nuclear processes including DNA repair and transcription. The transcription factor Yin Yang 1 (YY1) is a potent regulator of RNA polymerase II (Pol II)-dependent transcription. In this study Alu-retroposon-associated binding sites for YY1 located in the distal region of the promoter of the human ADPRT gene have been identified suggesting a possible involvement of this protein in the regulation of ADPRT-gene expression. In the presence of the recombinant automodification domain of the ADPRT the formation of specific YY1 complexes, detected in gel-shift experiments, was strongly inhibited, indicating that this domain of the enzyme may interact directly with YY1. In accordance with this result YY1 was specifically precipitated from nuclear extracts by ADPRT immobilized on sepharose. These results suggest a direct ADPRT–YY1 interaction which may be of impotrance in the regulation of Pol II-dependent transcription. They also indicate that in some human promoters this regulation may be mediated by retroposons of the Alu family.