Phosphorylation Of RNA Polymerase Research Articles

The CDK9 Tail Determines the Reaction Pathway of Positive Transcription Elongation Factor b

SummaryCDK9, the kinase of positive transcription elongation factor b (P-TEFb), stimulates transcription elongation by phosphorylating RNA polymerase II and transcription elongation factors. Using kinetic analysis of a human P-TEFb complex consisting of CDK9 and cyclin T, we show that the CDK9 C-terminal tail sequence is important for the catalytic mechanism and imposes an ordered binding of substrates and release of products. Crystallographic analysis of a CDK9/cyclin T complex in which the C-terminal tail partially blocks the ATP binding site reveals a possible reaction intermediate. Biochemical characterization of CDK9 mutants supports a model in which the CDK9 tail cycles through different conformational states. We propose that this mechanism is critical for the pattern of CTD Ser2 phosphorylation on actively transcribed genes.

DNA Ends Alter the Molecular Composition and Localization of Ku Multicomponent Complexes

The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.

PCBP1 is required for maintenance of the transcriptionally silent state in fully grown mouse oocytes

Global transcriptional silencing in fully grown oocytes is a critical event during mammalian oogenesis. However, how this event is regulated remains elusive. Here, we provide evidence that poly(rC)-binding protein 1 (PCBP1), a protein found by us previously to be present in metaphase II (MII) mouse oocytes, participates in maintenance of the transcriptionally silent state in fully grown mouse oocytes. Knocking down Pcbp1 by microinjection of its specific siRNAs into fully grown germinal vesicle (GV) oocytes resulted in remarkable changes in their transcriptional state, including the disequilibrium between the number of oocytes with an NSN (non-surrounded nucleolus) and those with a SN (surrounded nucleolus), and obvious transcriptional reactiviation in oocytes with a SN configuration as evidenced by BrUTP incorporation assay and immunofluorescent labeling of phosphorylated RNA polymerase II CTD and trimethylated H3 lysine 4, markers for active transcription. Furthermore, in a comprehensive microarray analysis of the preovulatory oocyte transcriptome, an incredible number of nearly 4,000 transcripts were upregulated in the Pcbp1 knockdown groups. These data indicate that lack of the function of PCBP1 disrupts the quiescent status of transcription in the fully grown oocytes, and hence supporting a role of this protein in the regulation of global transcriptional silcencing in fully grown mouse oocytes.

Cyclin-dependent kinases 7 and 9 specifically regulate neutrophil transcription and their inhibition drives apoptosis to promote resolution of inflammation

Terminally differentiated neutrophils are short-lived but the key effector cells of the innate immune response, and have a prominent role in the pathogenesis and propagation of many inflammatory diseases. Delayed apoptosis, which is responsible for their extended longevity, is critically dependent on a balance of intracellular survival versus pro-apoptotic proteins. Here, we elucidate the mechanism by which the cyclin-dependent kinase (CDK) inhibitor drugs such as R-roscovitine and DRB (5,6-dichloro-1-beta-𝒟-ribofuranosylbenzimidazole) mediate neutrophil apoptosis. We demonstrate (by a combination of microarray, confocal microscopy, apoptosis assays and western blotting) that the phosphorylation of RNA polymerase II by CDKs 7 and 9 is inhibited by R-roscovitine and that specific effects on neutrophil transcriptional capacity are responsible for neutrophil apoptosis. Finally, we show that specific CDK7 and 9 inhibition with DRB drives resolution of neutrophil-dominant inflammation. Thus, we highlight a novel mechanism that controls both primary human neutrophil transcription and apoptosis that could be targeted by selective CDK inhibitor drugs to resolve established inflammation.

Controlling Long-Range Genomic Interactions at a Native Locus by Targeted Tethering of a Looping Factor

Chromatin loops juxtapose distal enhancers with active promoters, but their molecular architecture and relationship with transcription remain unclear. In erythroid cells, the locus control region (LCR) and β-globin promoter form a chromatin loop that requires transcription factor GATA1 and the associated molecule Ldb1. We employed artificial zinc fingers (ZF) to tether Ldb1 to the β-globin promoter in GATA1 null erythroblasts, in which the β-globin locus is relaxed and inactive. Remarkably, targeting Ldb1 or only its self-association domain to the β-globin promoter substantially activated β-globin transcription in the absence of GATA1. Promoter-tethered Ldb1 interacted with endogenous Ldb1 complexes at the LCR to form a chromatin loop, causing recruitment and phosphorylation of RNA polymerase II. ZF-Ldb1 proteins were inactive at alleles lacking the LCR, demonstrating that their activities depend on long-range interactions. Our findings establish Ldb1 as a critical effector of GATA1-mediated loop formation and indicate that chromatin looping causally underlies gene regulation.

Cellular GCN5 Is a Novel Regulator of Human Adenovirus E1A-Conserved Region 3 Transactivation

The largest isoform of adenovirus early region 1A (E1A) contains a unique region termed conserved region 3 (CR3). This region activates viral gene expression by recruiting cellular transcription machinery to the early viral promoters. Recent studies have suggested that there is an optimal level of E1A-dependent transactivation required by human adenovirus (hAd) during infection and that this may be achieved via functional cross talk between the N termini of E1A and CR3. The N terminus of E1A binds GCN5, a cellular lysine acetyltransferase (KAT). We have identified a second independent interaction of E1A with GCN5 that is mediated by CR3, which requires residues 178 to 188 in hAd5 E1A. GCN5 was recruited to the viral genome during infection in an E1A-dependent manner, and this required both GCN5 interaction sites on E1A. Ectopic expression of GCN5 repressed transactivation by both E1A CR3 and full-length E1A. In contrast, RNA interference (RNAi) depletion of GCN5 or treatment with the KAT inhibitor cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH2) resulted in increased E1A CR3 transactivation. Moreover, activation of the adenovirus E4 promoter by E1A was increased during infection of homozygous GCN5 KAT-defective (hat/hat) mouse embryonic fibroblasts (MEFs) compared to wild-type control MEFs. Enhanced histone H3 K9/K14 acetylation at the viral E4 promoter required the newly identified binding site for GCN5 within CR3 and correlated with repression and reduced occupancy by phosphorylated RNA polymerase II. Treatment with CPTH2 during infection also reduced virus yield. These data identify GCN5 as a new negative regulator of transactivation by E1A and suggest that its KAT activity is required for optimal virus replication.

Destabilization of PDK1 by Hsp90 inactivation suppresses hepatitis C virus replication through inhibition of PRK2-mediated viral RNA polymerase phosphorylation

Heat shock protein 90 (Hsp90), which chaperones multiple client proteins, has been shown to be implicated in HCV replication. Pharmacological inhibitors of Hsp90 display an anti-HCV activity. However, little is known about the mechanisms of regulation of HCV replication by Hsp90. Here, we show that Hsp90 inhibition by 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) destabilizes phosphoinositide-dependent kinase-1 (PDK1), an upstream kinase of the protein kinase C-related kinase 2 (PRK2) responsible for phosphorylation of HCV RNA polymerase, through the proteosome pathway. Destabilization of PDK1 led to inhibition of phosphorylation of the viral RNA polymerase through a decrease in the abundance of active form PRK2 level. Consequently, Hsp90 inhibition resulted in suppression of HCV replication both in human hepatoma Huh7 cells harboring an HCV subgenomic replicon and in HCV-infected cells. 17-DMAG treatment did not interfere with HCV internal ribosome entry site-mediated translation and the cell cycle in Huh7 cells. Co-treatment of 17-DMAG with interferon-α or HA1077, an inhibitor of PRK2, enhanced the anti-HCV activity of 17-DMAG. Taken together, these findings suggest that Hsp90 plays a critical role in the regulation of HCV RNA polymerase phosphorylation via the PDK1–PRK2 signaling pathway.

O-GlcNAc Transferase Is Involved in Glucocorticoid Receptor-mediated Transrepression

Recruitment of O-GlcNAc transferase (OGT) to promoters plays an important role in gene repression. Glucocorticoid signaling represses the transcriptional activities of NF-κB and AP-1 through direct binding, yet the molecular mechanisms remain to be elucidated. Here we report that OGT is an important component of GR-mediated transrepression. OGT associates with ligand-bound GR in a multi-protein repression complex. Overexpression of OGT potentiates the GR transrepression pathway, whereas depletion of endogenous OGT by RNA interference abolishes the repression. The recruitment of OGT by GR leads to increased O-GlcNAcylation and decreased phosphorylation of RNA polymerase II on target genes. Functionally, overexpression of OGT enhances glucocorticoid-induced apoptosis in resistant cell lines while knockdown of OGT prevents sensitive cell lines from apoptosis. These studies identify a molecular mechanism of GR transrepression, and highlight the function of O-GlcNAc in hormone signaling.

Angiotensin II-Dependent Transcriptional Activation of Human Steroidogenic Acute Regulatory Protein Gene by a 25-kDa cAMP-Responsive Element Modulator Protein Isoform and Yin Yang 1

Transcriptional activation of the steroidogenic acute regulatory protein (STAR) gene is a critical component in the angiotensin II (Ang II)-dependent increase in aldosterone biosynthesis in the adrenal gland. The purpose of this study was to define the molecular mechanisms that mediate the Ang II-dependent increase in STARD1 gene (STAR) expression in H295R human adrenocortical cells. Mutational analysis of the STAR proximal promoter revealed that a nonconsensus cAMP-responsive element located at -78 bp relative to the transcription start site (-78CRE) is required for the Ang II-stimulated STAR reporter gene activity. DNA immunoaffinity chromatography identified a 25-kDa cAMP-responsive element modulator isoform and Yin Yang 1 (YY1) as -78CRE DNA-binding proteins, and Ang II treatment of H295R cells increased expression of that 25-kDa CREM isoform. Small interfering RNA silencing of CREM and YY1 attenuated the Ang II-dependent increases in STAR reporter gene activity and STAR mRNA levels. Conversely, overexpression of CREM and YY1 in COS-1 cells resulted in transactivation of STAR reporter gene activity. Chromatin immunoprecipitation analysis demonstrated recruitment of CREM and YY1 to the STAR promoter along with increased association of the coactivator cAMP response element-binding protein-binding protein (CBP) and increased phosphorylated RNA polymerase II after Ang II treatment. Together our data reveal that the Ang II-stimulated increase in STAR expression in H295R cells requires 25 kDa CREM and YY1. The recruitment of these transcription factors to the STAR proximal promoter results in association of CBP and activation of RNA polymerase II leading to increased STAR transcription.

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

Phosphorylation of RNA polymerase II carboxy-terminal domain (CTD) in hepta-repeats YSPTSPS regulates eukaryotic transcription. Whereas Ser5 is phosphorylated in the initiation phase, Ser2 phosphorylation marks the elongation state. Here we show that the positive transcription elongation factor P-TEFb is a Ser5 CTD kinase that is unable to create Ser2/Ser5 double phosphorylations, while it exhibits fourfold higher activity on a CTD substrate pre-phosphorylated at Ser7 compared with the consensus hepta-repeat or the YSPTSPK variant. Mass spectrometry reveals an equal number of phosphorylations to the number of hepta-repeats provided, yet the mechanism of phosphorylation is distributive despite the repetitive nature of the substrate. Inhibition of P-TEFb activity is mediated by two regions in Hexim1 that act synergistically on Cdk9 and Cyclin T1. HIV-1 Tat/TAR abrogates Hexim1 inhibition to stimulate transcription of viral genes but does not change the substrate specificity. Together, these results provide insight into the multifaceted pattern of CTD phosphorylation.

Anti-Myeloma Activity of LDC-000067, a Novel Selective Inhibitor of Cyclin Dependent Kinase 9: A Targeted Approach

Abstract 5110 Background:Cyclin-dependent kinase (CDK) inhibitors are reported to induce apoptosis in myeloma cells (Gojo et al., 2002; MacCallum et al., 2005). Here we investigated pro-apoptotic and anti-proliferative effects of LDC-000067, a novel selective CDK9-inhibitor, in three multiple myeloma (MM) cell lines (L-363; RPMI-8226; U-266). In addition, we studied consequences of selective CDK 9-inhibition regarding transcriptional activity (RNA-polymerase II phosphorylation) and related anti-apoptotic pathways (Mcl-1 and NFkB). Methods:L-363, RPMI-8226 and U-266 cells were exposed to either DMSO (vehicle control) or serial dilutions of LDC-000067 [0.5–20μM]. Apoptosis was quantified by flow cytometry analyses (FITC Annexin V / propidium iodide staining) after 24 hours in corresponding samples. To further investigate mechanisms of induced apoptosis by LDC-000067 we examined corresponding protein extracts by immunoblot analyses. Results:We found that LDC-000067 showed anti-myeloma activity in all myeloma cell lines tested. The proportion of induced apoptosis by exposure to LDC-000067 was equipotent (RPMI-8226) or higher (L-363 and U-266) compared to Seliciclib, a selective CDK 2-, 7- and 9-inhibitor. The IC50 for LDC-000067 was 4.0 μmol/L for L-363, 6.9 μmol/L for U-266 and 11.5 μmol/L for RPMI-8226. LDC-000067 inhibited phosphorylation of RNA polymerase II at Ser2 of the carboxyl-terminal domain in all MM lines tested. In addition, exposure to LDC-000067 resulted in a rapid decline of the protein levels of Mcl-1, known as an anti-apoptotic protein in MM. Furthermore, it is reported that in 40% of MM cell lines and 17% of MM patients the NFkB pathway is constitutionally activated by mutations (Demchenko et al., 2010) and inhibition of the NFkB activity causes induction of apoptosis (Hongyu et al., 2001). In our study we showed that LDC-000067 potently inhibited the NFkB pathway. Immunoblot analyses revealed a decreased phosphorylation and degradation of the NFkB inhibitor IkBa and an inhibition of phosphorylation of the NFkB subunit RelA (p65). Moreover, our data demonstrated that exposure to LDC-000067 caused a significant reduction of the protein levels of cyclin D1 and cyclin D2 strongly correlated to cell cycle arrest. Conclusion:In conclusion, our data corroborate that LDC-000067, a novel selective CDK 9-inhibitor, shows anti-myeloma activity by targeting three key pathways in the molecular pathogenesis of MM: firstly, it induces consecutive decline of Mcl-1 protein and inhibition of RNA polymerase II phosphorylation; secondly, it inhibits constitutively activated NFkB pathway; and thirdly, it reduces levels of D-type cyclins. Disclosures:No relevant conflicts of interest to declare.

Solution Structure of Tandem SH2 Domains from Spt6 Protein and Their Binding to the Phosphorylated RNA Polymerase II C-terminal Domain

Spt6 is a highly conserved transcription elongation factor and histone chaperone. It binds directly to the RNA polymerase II C-terminal domain (RNAPII CTD) through its C-terminal region that recognizes RNAPII CTD phosphorylation. In this study, we determined the solution structure of the C-terminal region of Saccharomyces cerevisiae Spt6, and we discovered that Spt6 has two SH2 domains in tandem. Structural and phylogenetic analysis revealed that the second SH2 domain was evolutionarily distant from canonical SH2 domains and represented a novel SH2 subfamily with a novel binding site for phosphoserine. In addition, NMR chemical shift perturbation experiments demonstrated that the tandem SH2 domains recognized Tyr(1), Ser(2), Ser(5), and Ser(7) phosphorylation of RNAPII CTD with millimolar binding affinities. The structural basis for the binding of the tandem SH2 domains to different forms of phosphorylated RNAPII CTD and its physiological relevance are discussed. Our results also suggest that Spt6 may use the tandem SH2 domain module to sense the phosphorylation level of RNAPII CTD.

A Maternal Factor Unique to Ascidians Silences the Germline via Binding to P-TEFb and RNAP II Regulation

Suppression of zygotic transcription in early embryonic germline cells is tightly linked to their separation from the somatic lineage. Many invertebrate embryos utilize localized maternal factors that are successively inherited by the germline cells for silencing the germline. Germline quiescence has also been associated with the underphosphorylation of Ser2 of the C-terminal domain (CTD-Ser2) of RNA polymerase II [1-3]. Here, using the ascidian Halocynthia roretzi, we identified a first deuterostome example of a maternally localized factor, posterior end mark (PEM), which globally represses germline transcription. PEM knockdown resulted in ectopic transcription and ectopic phosphorylation of CTD-Ser2 in the germline. Overexpression of PEM abolished all transcription and led to the underphosphorylation of CTD-Ser2 in the somatic cells. PEM protein was reiteratively detected in the nucleus of the germline cells and coimmunoprecipitated with CDK9, a component of posterior transcription elongation factor b (P-TEFb). These results suggest that nonhomologous proteins, PEM and Pgc of Drosophila [3-5] and PIE-1 of C. elegans [1, 6, 7], repress germline gene expression through analogous functions: by keeping CTD-Ser2 underphosphorylated through binding to the P-TEFb complex. The present study is an interesting example of evolutionary constraint on how a mechanism of germline silencing can evolve in diverse animals.

Structural Insights to How Mammalian Capping Enzyme Reads the CTD Code

Physical interaction between the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD) and cellular capping enzymes is required for efficient formation of the 5' mRNA cap, the first modification of nascent mRNA. Here, we report the crystal structure of the RNA guanylyltransferase component of mammalian capping enzyme (Mce) bound to a CTD phosphopeptide. The CTD adopts an extended β-like conformation that docks Tyr1 and Ser5-PO(4) onto the Mce nucleotidyltransferase domain. Structure-guided mutational analysis verified that the Mce-CTD interface is a tunable determinant of CTD binding and stimulation of guanylyltransferase activity, and of Mce function invivo. The location and composition of the CTD binding site on mammalian capping enzyme is distinct from that of a yeast capping enzyme that recognizes the same CTD primary structure. Thus, capping enzymes from different taxa have evolved different strategies to read the CTD code.

Analogues and Derivatives of Oncrasin-1, a Novel Inhibitor of the C-Terminal Domain of RNA Polymerase II and Their Antitumor Activities

To optimize the antitumor activity of oncrasin-1, a small molecule RNA polymerase II inhibitor, we evaluated 69 oncrasin-1 analogues for their cytotoxic activity against normal human epithelial cells and K-Ras mutant tumor cells. About 40 of those compounds were as potent as or more potent than oncrasin-1 in tumor cells and had a minimal cytotoxic effect on normal cells. Structure-activity relationship analysis revealed that most of the active compounds contained either a hydroxymethyl group or an aldehyde group as a substitute at the 3-position of the indole. Both electron-donating and electron-withdrawing groups in the benzene ring were well tolerated. The hydroxymethyl compounds ranged from equipotent with to 100 times as potent as the corresponding aldehyde compounds. We tested three active analogues' effect on RNA polymerase phosphorylation and found that they all inhibited phosphorylation of the C-terminal domain of RNA polymerase II, suggesting that the active compounds might act through the same mechanisms as oncrasin-1.

NOVEL MECHANISM AND HOST COFACTORS FOR REGULATION OF HIV‐1 TRANSCRIPTION

Recruited by HIV Tat and TAR to the viral LTR, human transcription elongation factor P-TEFb phosphorylates RNA polymerase II and negative elongation factors to stimulate HIV transcription and replication. Since 1997, this discovery has provided the basic framework for our understanding of Tat function in the HIV life cycle and a paradigm for studying eukaryotic elongation control in general. Although P-TEFb remains the widely accepted Tat partner till this day, accumulating evidence suggests that Tat-transactivation involves more than the interaction between Tat and P-TEFb. In an effort to identify additional cellular factors that associate with Tat and P-TEFb, we have identified transcription elongation factor ELL2 and transcription factors AFF4, ENL and AF9 as new Tat partners. These factors join Tat and P-TEFb to form a multi-subunit complex called BFEC. Data will be presented to demonstrate how BFEC interacts with a chromatin template, stimulates basal and Tat-activated HIV transcription, and is stabilized in response to signals that activate HIV gene expression. The identification and characterization of BFEC will enable a better understanding of the mechanisms controlling HIV and cellular transcriptional elongation and may reveal novel strategies to reactivate latent HIV for eradication.

HEXIM1 is a critical determinant of the response to tamoxifen

Tamoxifen resistance is a major problem in the treatment of Estrogen Receptor (ER) positive patients. We have previously reported that Hexamethylene bis-acetamide Inducible Protein 1 (HEXIM1) inhibits ERα activity by competing with ERα for binding to cyclin T1, a subunit of Positive Transcription Elongation b (P-TEFb). This results in the inhibition of the phosphorylation of RNA polymerase II (RNAPII) at serine 2 and the inhibition of transcription elongation of ERα target genes. Since HEXIM1 can inhibit ER activity, we examined whether it plays a critical role in the inhibitory effects of tamoxifen on ER. We observed that tamoxifen induced HEXIM1 recruitment to the promoter region of ER target genes and decreased the recruitment of cyclin T1 and serine 2 phosphorylated RNAPII to the coding regions of these genes. Conversely, in cells wherein HEXIM1 expression has been downregulated we observed attenuation of the inhibitory effects of tamoxifen on estrogen-induced cyclin T1 recruitment to coding regions of ER target genes. As a consequence, downregulation of HEXIM1 resulted in the attenuation of the repressive effects of tamoxifen on estrogen-induced gene expression and proliferation. Conferring clinical relevance to our studies is our analysis of human breast cancer tissue samples that indicated association of lower expression of HEXIM1 with tumor recurrence in patients who received tamoxifen. Our studies provide a better understanding of the mechanistic basis for the inhibitory effect of tamoxifen on ER activity and may suggest new therapeutic targets for the treatment of tamoxifen resistant breast cancer.

Requirement of TFIIH kinase subunit Mat1 for RNA Pol II C-terminal domain Ser5 phosphorylation, transcription and mRNA turnover

The relevance of serine 5 phosphorylation of RNA polymerase II carboxy-terminal domain during initiation has been difficult to determine in mammalian cells as no general in vivo Ser5 kinase has been identified. Here, we demonstrate that deletion of the TFIIH kinase subunit Mat1 in mouse fibroblasts leads to dramatically reduced Pol II Ser5 phosphorylation. This is associated with defective capping and reduced Ser2 phosphorylation, decreased Pol II progression into elongation and severely attenuated transcription detected through analysis of nascent mRNAs, establishing a general requirement for mammalian Mat1 in transcription. Surprisingly, the general defect in Pol II transcription in Mat1−/− fibroblasts is not reflected in the majority of steady-state mRNAs. This indicates widespread stabilization of mRNAs and points to the existence of a regulatory mechanism to stabilize mRNAs following transcriptional attenuation, thus revealing a potential caveat in similar studies limited to analysis of steady-state mRNAs.

Roscovitine, a selective CDK inhibitor, reduces the basal and estrogen‐induced phosphorylation of ER‐α in human ER‐positive breast cancer cells

Roscovitine (ROSC), a selective cyclin-dependent kinase (CDK) inhibitor, arrests human estrogen receptor-α (ER-α) positive MCF-7 breast cancer cells in the G(2) phase of the cell cycle and concomitantly induces apoptosis via a p53-dependent pathway. The effect of ROSC is markedly diminished in MCF-7 cells maintained in the presence of estrogen-mimicking compounds. Therefore, we decided to examine whether ROSC has any effect on the functional status of the ER-α transcription factor. Exposure of MCF-7 cells to ROSC abolished the activating phosphorylation of CDK2 and CDK7 in a concentration and time-dependent manner. This inhibition of site-specific modification of CDK7 at Ser164/170 prevented phosphorylation of RNA polymerase II and reduced basal phosphorylation of ER-α at Ser118 in non-stimulated MCF-7 cells (resulting in its down-regulation). In MCF-7 cells, estrogen induced strong phosphorylation of ER-α at Ser118 but not at Ser104/Ser106. ROSC prevented this estrogen-promoted activating modification of ER-α. Furthermore, we sought to determine whether the activity of ROSC could be enhanced by combining it with an anti-estrogen. Tamoxifen (TAM), a selective estrogen receptor modulator (SERM), affected breast cancer cell lines irrespective of their ER status. In combination with ROSC, however, it had a different impact, enhancing G(1) or G(2) arrest. Our results indicate that ROSC prevents the activating phosphorylation of ER-α and that its mode of action is strongly dependent on the cellular context. Furthermore, our data show that ROSC can be combined with anti-estrogen therapy. The inhibitory effect of TAM on ER-negative cancer cells indicates that SERMs crosstalk with other steroid hormone receptors.

The F-actin severing protein cofilin-1 is required for RNA polymerase II transcription elongation

In mammals actin contributes to transcription elongation by facilitating establishment of permissive chromatin. Here we report that the F-actin severing protein cofilin-1 is part of the same complex with actin and phosphorylated RNA polymerase (pol) II. In chromatin immunoprecipitation assays cofilin-1 was found selectively associated with transcribed regions of active genes, its occupancy being influenced by the polymerization state of actin. Cofilin-1 gene silencing led to a drop in FUrd incorporation into nascent transcripts. In cofilin-1 silenced cells chromatin immunoprecipitations showed that active genes were devoid of actin, phosphorylated pol II and displayed low histone H3 acetylation levels on K9. These findings suggest that cofilin-1 plays a major role in pol II transcription, facilitating association of elongating pol II and actin with active genes. We speculate that cofilin-1 performs its function in pol II transcription by regulating polymerization of gene-associated actin.