Molecular Cell

Extensive DNA Damage-Induced Sumoylation Contributes to Replication and Repair and Acts in Addition to the Mec1 Checkpoint.

Publication Date: 2012 Jan 24 PMID: 22285753
Authors: Cremona, C. A. - Sarangi, P. - Yang, Y. - Hang, L. E. - Rahman, S. - Zhao, X.
Journal: Mol Cell

The cellular response to DNA damage employs multiple dynamic protein modifications to exert rapid and adaptable effects. Substantial work has detailed the roles of canonical checkpoint-mediated phosphorylation in this program. Recent studies have also implicated sumoylation in the DNA damage response; however, a systematic view of the contribution of sumoylation to replication and repair and its interplay with checkpoints is lacking. Here, using a biochemical screen in yeast, we establish that DNA damage-induced sumoylation occurs on a large scale. We identify MRX (Mre11-Rad50-Xrs2) as a positive regulator of this induction for a subset of repair targets. In addition, we find that defective sumoylation results in failure to complete replication of a damaged genome and impaired DNA end processing, highlighting the importance of the SUMO-mediated response in genome integrity. We also show that DNA damage-induced sumoylation does not require Mec1 checkpoint signaling, and the presence of both enables optimal DNA damage resistance.

post to: CiteULike

The WTX Tumor Suppressor Enhances p53 Acetylation by CBP/p300.

Publication Date: 2012 Jan 24 PMID: 22285752
Authors: Kim, W. J. - Rivera, M. N. - Coffman, E. J. - Haber, D. A.
Journal: Mol Cell

WTX encodes a tumor suppressor, frequently inactivated in Wilms tumor, with both plasma membrane and nuclear localization. WTX has been implicated in beta-catenin turnover, but its effect on nuclear proteins is unknown. We report an interaction between WTX and p53, derived from the unexpected observation of WTX, p53, and E1B 55K colocalization within the characteristic cytoplasmic body of adenovirus-transformed kidney cells. In other cells without adenovirus expression, the C-terminal domain of WTX binds to the DNA-binding domain of p53, enhances its binding to CBP, and increases CBP/p300-mediated acetylation of p53 at Lys 373/382. WTX knockdown accelerates CBP/p300 protein turnover and attenuates this modification of p53. In p53-reconstitution experiments, cell-cycle arrest, apoptosis, and p53 target-gene expression are suppressed by depletion of WTX. Together, these results suggest that WTX modulates p53 function, in part through regulation of its activator CBP/p300.

post to: CiteULike

Scd6 Targets eIF4G to Repress Translation: RGG Motif Proteins as a Class of eIF4G-Binding Proteins.

Publication Date: 2012 Jan 27 PMID: 22284680
Authors: Rajyaguru, P. - She, M. - Parker, R.
Journal: Mol Cell

The formation of mRNPs controls the interaction of the translation and degradation machinery with individual mRNAs. The yeast Scd6 protein and its orthologs regulate translation and mRNA degradation in yeast, C. elegans, D. melanogaster, and humans by an unknown mechanism. We demonstrate that Scd6 represses translation by binding the eIF4G subunit of eIF4F in a manner dependent on its RGG domain, thereby forming an mRNP repressed for translation initiation. Strikingly, several other RGG domain-containing proteins in yeast copurify with eIF4E/G and we demonstrate that two such proteins, Npl3 and Sbp1, also directly bind eIF4G and repress translation in a manner dependent on their RGG motifs. These observations identify the mechanism of Scd6 function through its RGG motif and indicate that eIF4G plays an important role as a scaffolding protein for the recruitment of translation repressors.

post to: CiteULike

Regulation of KLF4 Turnover Reveals an Unexpected Tissue-Specific Role of pVHL in Tumorigenesis.

Publication Date: 2012 Jan 27 PMID: 22284679
Authors: Gamper, A. M. - Qiao, X. - Kim, J. - Zhang, L. - Desimone, M. C. - Rathmell, W. K. - Wan, Y.
Journal: Mol Cell

The transcription factor Kruppel-like factor 4 (KLF4) is an important regulator of cell-fate decision, including cell-cycle regulation, apoptosis, and stem cell renewal, and plays an ambivalent role in tumorigenesis as a tissue-specific tumor suppressor or oncogene. Here, we report that the Von Hippel-Lindau gene product, pVHL, physically interacts with KLF4 and regulates its rapid turnover observed in both differentiated and stem cells. We provide mechanistic insights into KLF4 degradation and show that pVHL depletion in colorectal cancer cells leads to cell-cycle arrest concomitant with increased transcription of the KLF4-dependent p21 gene. Finally, immunohistochemical staining revealed elevated pVHL and reduced KLF4 levels in colon cancer tissues. We therefore propose that unexpectedly pVHL, via the degradation of KLF4, is a facilitating factor in colorectal tumorigenesis.

post to: CiteULike

Importin 7 and Exportin 1 Link c-Myc and p53 to Regulation of Ribosomal Biogenesis.

Publication Date: 2012 Jan 27 PMID: 22284678
Authors: Golomb, L. - Bublik, D. R. - Wilder, S. - Nevo, R. - Kiss, V. - Grabusic, K. - Volarevic, S. - Oren, M.
Journal: Mol Cell

Members of the beta-karyopherin family mediate nuclear import of ribosomal proteins and export of ribosomal subunits, both required for ribosome biogenesis. We report that transcription of the beta-karyopherin genes importin 7 (IPO7) and exportin 1 (XPO1), and several additional nuclear import receptors, is regulated positively by c-Myc and negatively by p53. Partial IPO7 depletion triggers p53 activation and p53-dependent growth arrest. Activation of p53 by IPO7 knockdown has distinct features of ribosomal biogenesis stress, with increased binding of Mdm2 to ribosomal proteins L5 and L11 (RPL5 and RPL11). Furthermore, p53 activation is dependent on RPL5 and RPL11. Of note, IPO7 and XPO1 are frequently overexpressed in cancer. Altogether, we propose that c-Myc and p53 counter each other in the regulation of elements within the nuclear transport machinery, thereby exerting opposing effects on the rate of ribosome biogenesis. Perturbation of this balance may play a significant role in promoting cancer.

post to: CiteULike

SUMO-Specific Protease 1 Is Critical for Early Lymphoid Development through Regulation of STAT5 Activation.

Publication Date: 2012 Jan 27 PMID: 22284677
Authors: Van Nguyen, T. - Angkasekwinai, P. - Dou, H. - Lin, F. M. - Lu, L. S. - Cheng, J. - Chin, Y. E. - Dong, C. - Yeh, E. T.
Journal: Mol Cell

Small ubiquitin-like modifier (SUMO) modification has emerged as an important regulatory mechanism during embryonic development. However, it is not known whether SUMOylation plays a role in the development of the immune system. Here, we show that SUMO-specific protease 1 (SENP1) is essential for the development of early T and B cells. STAT5, a key regulator of lymphoid development, is modified by SUMO-2 and is specifically regulated by SENP1. In the absence of SENP1, SUMO-2 modified STAT5 accumulates in early lymphoid precursors, resulting in a block in its acetylation and subsequent signaling. These results demonstrate a crucial role of SENP1 in the regulation of STAT5 activation during early lymphoid development.

post to: CiteULike

A Universal RNA Polymerase II CTD Cycle Is Orchestrated by Complex Interplays between Kinase, Phosphatase, and Isomerase Enzymes along Genes.

Publication Date: 2012 Jan 27 PMID: 22284676
Authors: Bataille, A. R. - Jeronimo, C. - Jacques, P. E. - Laramee, L. - Fortin, M. E. - Forest, A. - Bergeron, M. - Hanes, S. D. - Robert, F.
Journal: Mol Cell

Transcription by RNA polymerase II (RNAPII) is coupled to mRNA processing and chromatin modifications via the C-terminal domain (CTD) of its largest subunit, consisting of multiple repeats of the heptapeptide YSPTSPS. Pioneering studies showed that CTD serines are differentially phosphorylated along genes in a prescribed pattern during the transcription cycle. Genome-wide analyses challenged this idea, suggesting that this cycle is not uniform among different genes. Moreover, the respective role of enzymes responsible for CTD modifications remains controversial. Here, we systematically profiled the location of the RNAPII phosphoisoforms in wild-type cells and mutants for most CTD modifying enzymes. Together with results of in vitro assays, these data reveal a complex interplay between the modifying enzymes, and provide evidence that the CTD cycle is uniform across genes. We also identify Ssu72 as the Ser7 phosphatase and show that proline isomerization is a key regulator of CTD dephosphorylation at the end of genes.

post to: CiteULike

Immobilization of proteins in the nucleolus by ribosomal intergenic spacer noncoding RNA.

Publication Date: 2012 Jan 27 PMID: 22284675
Authors: Audas, T. E. - Jacob, M. D. - Lee, S.
Journal: Mol Cell

Cellular pathways are established and maintained by stochastic interactions of highly mobile molecules. The nucleolus plays a central role in the regulation of these molecular networks by capturing and immobilizing proteins. Here, we report a function for noncoding RNA (ncRNA) in the regulation of protein dynamics of key cellular factors, including VHL, Hsp70 and MDM2/PML. Stimuli-specific loci of the nucleolar intergenic spacer produce ncRNA capable of capturing and immobilizing proteins that encode a discrete peptidic code referred to as the nucleolar detention sequence (NoDS). Disruption of the NoDS/intergenic RNA interaction enables proteins to evade nucleolar sequestration and retain their dynamic profiles. Mislocalization of intergenic ncRNA triggers protein immobilization outside of the nucleolus, demonstrating that these ncRNA species can operate independently from the nucleolar architecture. We propose a model whereby protein immobilization by ncRNA is a posttranslational regulatory mechanism.

post to: CiteULike

A new role for the polycomb group protein ezh1 in promoting transcription.

Publication Date: 2012 Jan 27 PMID: 22284674
Authors: Riising, E. M. - Helin, K.
Journal: Mol Cell

In this issue, Mousavi et al. (2011) report a novel gene activation function of the H3K27 methyltransferase, Ezh1, in addition to its known role in transcriptional repression.

post to: CiteULike

Cellular signal processing: out of one, many.

Publication Date: 2012 Jan 27 PMID: 22284673
Authors: Kumar, R. M. - Collins, J. J.
Journal: Mol Cell

Cells can make fate decisions in response to information from the environment. In this issue of Molecular Cell, Chen et al. (2012) describe how the design of a signal-processing pathway allows a homogenous population of cells to display diverse responses to uniform growth factor cues.

post to: CiteULike

Policing Cells under Stress: Noncoding RNAs Capture Proteins in Nucleolar Detention Centers.

Publication Date: 2012 Jan 27 PMID: 22284672
Authors: Prasanth, K. V.
Journal: Mol Cell

In this issue of Molecular Cell,Audas et al. (2012) demonstrate that a class of stress-induced noncoding RNAs immobilizes proteins in the nucleolus in response to a specific stimulus.

post to: CiteULike

Direct Regulation of tRNA and 5S rRNA Gene Transcription by Polo-like Kinase 1.

Publication Date: 2012 Jan 24 PMID: 22281053
Authors: Fairley, J. A. - Mitchell, L. E. - Berg, T. - Kenneth, N. S. - von Schubert, C. - Sillje, H. H. - Medema, R. H. - Nigg, E. A. - White, R. J.
Journal: Mol Cell

Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase III (pol III) through direct binding and phosphorylation of transcription factor Brf1. During interphase, Plk1 promotes tRNA and 5S rRNA expression by phosphorylating Brf1 directly on serine 450. However, this stimulatory modification is overridden at mitosis, when elevated Plk1 activity causes Brf1 phosphorylation on threonine 270 (T270), which prevents pol III recruitment. Thus, although Plk1 enhances net tRNA and 5S rRNA production, consistent with its proliferation-stimulating function, it also suppresses untimely transcription when cells divide. Genomic instability is apparent in cells with Brf1 T270 mutated to alanine to resist Plk1-directed inactivation, suggesting that chromosome segregation is vulnerable to inappropriate pol III activity.

post to: CiteULike

Orientational Preferences of Neighboring Helices Can Drive ER Insertion of a Marginally Hydrophobic Transmembrane Helix.

Publication Date: 2012 Jan 24 PMID: 22281052
Authors: Ojemalm, K. - Halling, K. K. - Nilsson, I. - von Heijne, G.
Journal: Mol Cell

alpha-helical integral membrane proteins critically depend on the correct insertion of their transmembrane alpha helices into the lipid bilayer for proper folding, yet a surprisingly large fraction of the transmembrane alpha helices in multispanning integral membrane proteins are not sufficiently hydrophobic to insert into the target membrane by themselves. How can such marginally hydrophobic segments nevertheless form transmembrane helices in the folded structure? Here, we show that a transmembrane helix with a strong orientational preference (N(cyt)-C(lum) or N(lum)-C(cyt)) can both increase and decrease the hydrophobicity threshold for membrane insertion of a neighboring, marginally hydrophobic helix. This effect helps explain the "missing hydrophobicity" in polytopic membrane proteins.

post to: CiteULike

Mediator Complex Regulates Alternative mRNA Processing via the MED23 Subunit.

Publication Date: 2012 Jan 18 PMID: 22264826
Authors: Huang, Y. - Li, W. - Yao, X. - Lin, Q. J. - Yin, J. W. - Liang, Y. - Heiner, M. - Tian, B. - Hui, J. - Wang, G.
Journal: Mol Cell

Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its MED23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit, MED23, but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally, MED23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events, as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that MED23 can regulate hnRNP L occupancy at their coregulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, providing a molecular basis for coupling mRNA processing to transcription.

post to: CiteULike

Single-Cell Analysis Reveals that Noncoding RNAs Contribute to Clonal Heterogeneity by Modulating Transcription Factor Recruitment.

Publication Date: 2012 Jan 18 PMID: 22264825
Authors: Bumgarner, S. L. - Neuert, G. - Voight, B. F. - Symbor-Nagrabska, A. - Grisafi, P. - van Oudenaarden, A. - Fink, G. R.
Journal: Mol Cell

Mechanisms through which long intergenic noncoding RNAs (ncRNAs) exert regulatory effects on eukaryotic biological processes remain largely elusive. Most studies of these phenomena rely on methods that measure average behaviors in cell populations, lacking resolution to observe the effects of ncRNA transcription on gene expression in a single cell. Here, we combine quantitative single-molecule RNA FISH experiments with yeast genetics and computational modeling to gain mechanistic insights into the regulation of the Saccharomyces cerevisiae protein-coding gene FLO11 by two intergenic ncRNAs, ICR1 and PWR1. Direct detection of FLO11 mRNA and these ncRNAs in thousands of individual cells revealed alternative expression states and provides evidence that ICR1 and PWR1 contribute to FLO11's variegated transcription, resulting in Flo11-dependent phenotypic heterogeneity in clonal cell populations by modulating recruitment of key transcription factors to the FLO11 promoter.

post to: CiteULike

Intragenic Enhancers Act as Alternative Promoters.

Publication Date: 2012 Jan 18 PMID: 22264824
Authors: Kowalczyk, M. S. - Hughes, J. R. - Garrick, D. - Lynch, M. D. - Sharpe, J. A. - Sloane-Stanley, J. A. - McGowan, S. J. - De Gobbi, M. - Hosseini, M. - Vernimmen, D. - Brown, J. M. - Gray, N. E. - Collavin, L. - Gibbons, R. J. - Flint, J. - Taylor, S. - Buckle, V. J. - Milne, T. A. - Wood, W. G. - Higgs, D. R.
Journal: Mol Cell

A substantial amount of organismal complexity is thought to be encoded by enhancers which specify the location, timing, and levels of gene expression. In mammals there are more enhancers than promoters which are distributed both between and within genes. Here we show that activated, intragenic enhancers frequently act as alternative tissue-specific promoters producing a class of abundant, spliced, multiexonic poly(A)(+) RNAs (meRNAs) which reflect the host gene's structure. meRNAs make a substantial and unanticipated contribution to the complexity of the transcriptome, appearing as alternative isoforms of the host gene. The low protein-coding potential of meRNAs suggests that many meRNAs may be byproducts of enhancer activation or underlie as-yet-unidentified RNA-encoded functions. Distinguishing between meRNAs and mRNAs will transform our interpretation of dynamic changes in transcription both at the level of individual genes and of the genome as a whole.

post to: CiteULike

Inactivation of a Peroxiredoxin by Hydrogen Peroxide Is Critical for Thioredoxin-Mediated Repair of Oxidized Proteins and Cell Survival.

Publication Date: 2012 Jan 11 PMID: 22245228
Authors: Day, A. M. - Brown, J. D. - Taylor, S. R. - Rand, J. D. - Morgan, B. A. - Veal, E. A.
Journal: Mol Cell

Eukaryotic 2-Cys peroxiredoxins (Prx) are abundant antioxidant enzymes whose thioredoxin peroxidase activity plays an important role in protecting against oxidative stress, aging, and cancer. Paradoxically, this thioredoxin peroxidase activity is highly sensitive to inactivation by peroxide-induced Prx hyperoxidation. However, any possible advantage in preventing Prx from removing peroxides under oxidative stress conditions has remained obscure. Here we demonstrate that, in cells treated with hydrogen peroxide, the Prx Tpx1 is a major substrate for thioredoxin in the fission yeast Schizosaccharomyces pombe and, as such, competitively inhibits thioredoxin-mediated reduction of other oxidized proteins. Consequently, we reveal that the hyperoxidation of Tpx1 is critical to allow thioredoxin to act on other substrates ensuring repair of oxidized proteins and cell survival following exposure to toxic levels of hydrogen peroxide. We conclude that the inactivation of the thioredoxin peroxidase activity of Prx is important to maintain thioredoxin activity and cell viability under oxidative stress conditions.

post to: CiteULike

H2B Ubiquitylation Controls the Formation of Export-Competent mRNP.

Publication Date: 2012 Jan 13 PMID: 22244335
Authors: Vitaliano-Prunier, A. - Babour, A. - Herissant, L. - Apponi, L. - Margaritis, T. - Holstege, F. C. - Corbett, A. H. - Gwizdek, C. - Dargemont, C.
Journal: Mol Cell

Histone H2B ubiquitylation is a transcription-dependent modification that not only regulates nucleosome dynamics but also controls the trimethylation of histone H3 on lysine 4 by promoting ubiquitylation of Swd2, a component of both the histone methyltransferase COMPASS complex and the cleavage and polyadenylation factor(CPF). We show that preventing either H2B ubiquitylation or H2B-dependent modification of Swd2 results in nuclear accumulation of poly(A) RNA due to a defect in the integrity and stability of APT, a subcomplex of the CPF. Ubiquitin-regulated APT complex dynamics is required for the correct recruitment of the mRNA export receptor Mex67 to nuclear mRNPs. While H2B ubiquitylation controls the recruitment of the different Mex67 adaptors to mRNPs, the effect of Swd2 ubiquitylation is restricted to Yra1 and Nab2, which, in turn, controls poly(A) tail length. Modification of H2B thus participates in the crosstalk between cotranscriptional events and assembly of mRNPs linking nuclear processing and mRNA export.

post to: CiteULike

RNase H and Postreplication Repair Protect Cells from Ribonucleotides Incorporated in DNA.

Publication Date: 2012 Jan 13 PMID: 22244334
Authors: Lazzaro, F. - Novarina, D. - Amara, F. - Watt, D. L. - Stone, J. E. - Costanzo, V. - Burgers, P. M. - Kunkel, T. A. - Plevani, P. - Muzi-Falconi, M.
Journal: Mol Cell

The chemical identity and integrity of the genome is challenged by the incorporation of ribonucleoside triphosphates (rNTPs) in place of deoxyribonucleoside triphosphates (dNTPs) during replication. Misincorporation is limited by the selectivity of DNA replicases. We show that accumulation of ribonucleoside monophosphates (rNMPs) in the genome causes replication stress and has toxic consequences, particularly in the absence of RNase H1 and RNase H2, which remove rNMPs. We demonstrate that postreplication repair (PRR) pathways-MMS2-dependent template switch and Pol zeta-dependent bypass-are crucial for tolerating the presence of rNMPs in the chromosomes; indeed, we show that Pol zeta efficiently replicates over 1-4 rNMPs. Moreover, cells lacking RNase H accumulate mono- and polyubiquitylated PCNA and have a constitutively activated PRR. Our findings describe a crucial function for RNase H1, RNase H2, template switch, and translesion DNA synthesis in overcoming rNTPs misincorporated during DNA replication, and may be relevant for the pathogenesis of Aicardi-Goutieres syndrome.

post to: CiteULike

Ars2 promotes proper replication-dependent histone mRNA 3' end formation.

Publication Date: 2012 Jan 13 PMID: 22244333
Authors: Gruber, J. J. - Olejniczak, S. H. - Yong, J. - La Rocca, G. - Dreyfuss, G. - Thompson, C. B.
Journal: Mol Cell

Ars2 is a component of the nuclear cap-binding complex that contributes to microRNA biogenesis and is required for cellular proliferation. Here, we expand on the repertoire of Ars2-dependent microRNAs and determine that Ars2 regulates a number of mRNAs, the largest defined subset of which code for histones. Histone mRNAs are unique among mammalian mRNAs because they are not normally polyadenylated but, rather, are cleaved following a 3' stem loop. A significant reduction in correctly processed histone mRNAs was observed following Ars2 depletion, concurrent with an increase in polyadenylated histone transcripts. Furthermore, Ars2 physically associated with histone mRNAs and the noncoding RNA 7SK. Knockdown of 7SK led to an enhanced ratio of cleaved to polyadenylated histone transcripts, an effect dependent on Ars2. Together, the data demonstrate that Ars2 contributes to histone mRNA 3' end formation and expression and these functional properties of Ars2 are negatively regulated by interaction with 7SK RNA.

post to: CiteULike

Transcriptional Regulation by Pol II(G) Involving Mediator and Competitive Interactions of Gdown1 and TFIIF with Pol II.

Publication Date: 2012 Jan 13 PMID: 22244332
Authors: Jishage, M. - Malik, S. - Wagner, U. - Uberheide, B. - Ishihama, Y. - Hu, X. - Chait, B. T. - Gnatt, A. - Ren, B. - Roeder, R. G.
Journal: Mol Cell

Pol II(G) is a distinct form of RNA polymerase II that contains the tightly associated Gdown1 polypeptide (encoded by POLR2M). Unlike Pol II, Pol II(G) is highly dependent upon Mediator for robust activator-dependent transcription in a biochemically defined in vitro system. Here, in vitro studies show that Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II, thereby inhibiting an essential function of TFIIF in preinitiation complex assembly, but also that Mediator can actually facilitate Pol II(G) binding to the promoter prior to subsequent Mediator functions. Complementary ChIP and RNAi analyses reveal that Pol II(G) is recruited to promoter regions of subsets of actively transcribed genes, where it appears to restrict transcription. These and other results suggest that Pol II(G) may act to modulate some genes while simultaneously, as a poised (noninitiated) polymerase, setting the stage for Mediator-dependent enhancement of their activity.

post to: CiteULike

Functional Association of Gdown1 with RNA Polymerase II Poised on Human Genes.

Publication Date: 2012 Jan 13 PMID: 22244331
Authors: Cheng, B. - Li, T. - Rahl, P. B. - Adamson, T. E. - Loudas, N. B. - Guo, J. - Varzavand, K. - Cooper, J. J. - Hu, X. - Gnatt, A. - Young, R. A. - Price, D. H.
Journal: Mol Cell

Most human genes are loaded with promoter-proximally paused RNA polymerase II (Pol II) molecules that are poised for release into productive elongation by P-TEFb. We present evidence that Gdown1, the product of the POLR2M gene that renders Pol II responsive to Mediator, is involved in Pol II elongation control. During in vitro transcription, Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. Supporting these mechanistic insights, ChIP-Seq demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Our results establish that Gdown1 stabilizes poised polymerases while maintaining their responsiveness to P-TEFb and suggest that Mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function.

post to: CiteULike

Star-PAP Control of BIK Expression and Apoptosis Is Regulated by Nuclear PIPKIalpha and PKCdelta Signaling.

Publication Date: 2012 Jan 13 PMID: 22244330
Authors: Li, W. - Laishram, R. S. - Ji, Z. - Barlow, C. A. - Tian, B. - Anderson, R. A.
Journal: Mol Cell

BIK protein is an initiator of mitochondrial apoptosis, and BIK expression is induced by proapoptotic signals, including DNA damage. Here, we demonstrate that 3' end processing and expression of BIK mRNA are controlled by the nuclear PI4,5P(2)-regulated poly(A) polymerase Star-PAP downstream of DNA damage. Nuclear PKCdelta is a key mediator of apoptosis, and DNA damage stimulates PKCdelta association with the Star-PAP complex where PKCdelta is required for Star-PAP-dependent BIK expression. PKCdelta binds the PI4,5P(2)-generating enzyme PIPKIalpha, which is essential for PKCdelta interaction with the Star-PAP complex, and PKCdelta activity is directly stimulated by PI4,5P(2). Features in the BIK 3' UTR uniquely define Star-PAP specificity and may block canonical PAP activity toward BIK mRNA. This reveals a nuclear phosphoinositide signaling nexus where PIPKIalpha, PI4,5P(2), and PKCdelta regulate Star-PAP control of BIK expression and induction of apoptosis. This pathway is distinct from the Star-PAP-mediated oxidative stress pathway indicating signal-specific regulation of mRNA 3' end processing.

post to: CiteULike

Hydrogen Sulfide-Linked Sulfhydration of NF-kappaB Mediates Its Antiapoptotic Actions.

Publication Date: 2012 Jan 13 PMID: 22244329
Authors: Sen, N. - Paul, B. D. - Gadalla, M. M. - Mustafa, A. K. - Sen, T. - Xu, R. - Kim, S. - Snyder, S. H.
Journal: Mol Cell

Nuclear factor kappaB (NF-kappaB) is an antiapoptotic transcription factor. We show that the antiapoptotic actions of NF-kappaB are mediated by hydrogen sulfide (H(2)S) synthesized by cystathionine gamma-lyase (CSE). TNF-alpha treatment triples H(2)S generation by stimulating binding of SP1 to the CSE promoter. H(2)S generated by CSE stimulates DNA binding and gene activation of NF-kappaB, processes that are abolished in CSE-deleted mice. As CSE deletion leads to decreased glutathione levels, resultant oxidative stress may contribute to alterations in CSE mutant mice. H(2)S acts by sulfhydrating the p65 subunit of NF-kappaB at cysteine-38, which promotes its binding to the coactivator ribosomal protein S3 (RPS3). Sulfhydration of p65 predominates early after TNF-alpha treatment, then declines and is succeeded by a reciprocal enhancement of p65 nitrosylation. In CSE mutant mice, antiapoptotic influences of NF-kappaB are markedly diminished. Thus, sulfhydration of NF-kappaB appears to be a physiologic determinant of its antiapoptotic transcriptional activity.

post to: CiteULike

SIRT1 Regulation-It Ain't All NAD.

Publication Date: 2012 Jan 13 PMID: 22244328
Authors: Chao, L. C. - Tontonoz, P.
Journal: Mol Cell

SIRT1 is a phylogenetically conserved energy sensor. The molecular mechanisms that regulate its catalytic activity, however, are incompletely understood. Gerhart-Hines et al. (2011) have identified beta-adrenergic/cAMP signaling as a regulatory input that rapidly controls SIRT1 activity and fatty acid oxidation.

post to: CiteULike

Ramping Up Mitosis: An AMPKalpha2-Regulated Signaling Network Promotes Mitotic Progression.

Publication Date: 2012 Jan 13 PMID: 22244327
Authors: Robitaille, A. M. - Hall, M. N.
Journal: Mol Cell

In the December 23rd issue of Molecular Cell, Banko et al. (2011) describe a chemical genetic screen that identified 28 novel AMPKalpha2 direct substrates. A subset of these substrates comprise a signaling network by which AMPK, seemingly independent of cellular energy status, promotes mitotic progression.

post to: CiteULike

A Novel Antirecombinase Gains PARIty.

Publication Date: 2012 Jan 13 PMID: 22244326
Authors: Mankouri, H. W. - Chu, W. K. - Hickson, I. D.
Journal: Mol Cell

In this issue, Moldovan et al. (2012) report the identification of PARI, a putative human ortholog of the yeast Srs2 protein, which potentially regulates homologous recombination repair via its ability to disrupt the function of RAD51.

post to: CiteULike

Get Back TFIIF, Don't Let Me Gdown1.

Publication Date: 2012 Jan 13 PMID: 22244325
Authors: Espinosa, J. M.
Journal: Mol Cell

In this issue of Molecular Cell, papers by the Price and Roeder labs reveal how the Gdown1 protein antagonizes the general transcription factor TFIIF during RNAPII initiation and elongation and how the Mediator complex intervenes in this molecular tug-of-war to activate RNAPII.

post to: CiteULike

Cysteine 38 Holds the Key to NF-kappaB Activation.

Publication Date: 2012 Jan 13 PMID: 22244324
Authors: Perkins, N. D.
Journal: Mol Cell

The importance of parallel signaling pathways controlling NF-kappaB subunit posttranslational modifications is demonstrated by Sen et al. (2012), who reveal that RelA (p65) sulfhydration, at its highly conserved cysteine 38 residue, regulates association with the coactivator RPS3, DNA binding, and antiapoptotic gene expression.

post to: CiteULike

Assembly of Drosophila Centromeric Nucleosomes Requires CID Dimerization.

Publication Date: 2012 Jan 27 PMID: 22209075
Authors: Zhang, W. - Colmenares, S. U. - Karpen, G. H.
Journal: Mol Cell

Centromeres are essential chromosomal regions required for kinetochore assembly and chromosome segregation. The composition and organization of centromeric nucleosomes containing the essential histone H3 variant CENP-A (CID in Drosophila) is a fundamental, unresolved issue. Using immunoprecipitation of CID mononucleosomes and cysteine crosslinking, we demonstrate that centromeric nucleosomes contain CID dimers in vivo. Furthermore, CID dimerization and centromeric targeting require a residue implicated in formation of the four-helix bundle, which mediates intranucleosomal H3 dimerization and nucleosome integrity. Taken together, our findings suggest that CID nucleosomes are octameric in vivo and that CID dimerization is essential for correct centromere assembly.

post to: CiteULike

A Two-Dimensional ERK-AKT Signaling Code for an NGF-Triggered Cell-Fate Decision.

Publication Date: 2012 Jan 27 PMID: 22206868
Authors: Chen, J. Y. - Lin, J. R. - Cimprich, K. A. - Meyer, T.
Journal: Mol Cell

Growth factors activate Ras, PI3K, and other signaling pathways. It is not well understood how these signals are translated by individual cells into a decision to proliferate or differentiate. Here, using single-cell image analysis of nerve growth factor (NGF)-stimulated PC12 cells, we identified a two-dimensional phospho-ERK (pERK)-phospho-AKT (pAKT) response map with a curved boundary that separates differentiating from proliferating cells. The boundary position remained invariant when different stimuli were used or upstream signaling components perturbed. We further identified Rasa2 as a negative feedback regulator that links PI3K to Ras, placing the stochastically distributed pERK-pAKT signals close to the decision boundary. This allows for uniform NGF stimuli to create a subpopulation of cells that differentiates with each cycle of proliferation. Thus, by linking a complex signaling system to a simpler intermediate response map, cells gain unique integration and control capabilities to balance cell number expansion with differentiation.

post to: CiteULike

Polycomb Protein Ezh1 Promotes RNA Polymerase II Elongation.

Publication Date: 2012 Jan 27 PMID: 22196887
Authors: Mousavi, K. - Zare, H. - Wang, A. H. - Sartorelli, V.
Journal: Mol Cell

Polycomb group (PcG) proteins initiate the formation of repressed chromatin domains and regulate developmental gene expression. A mammalian PcG protein, enhancer of zeste homolog 2 (Ezh2), triggers transcriptional repression by catalyzing the addition of methyl groups onto lysine 27 of histone H3 (H3K27me2/3). This action facilitates the binding of other PcG proteins to chromatin for purposes of transcriptional silencing. Interestingly, there exists a paralog of Ezh2, termed Ezh1, whose primary function remains unclear. Here, we provide evidence for genome-wide association of Ezh1 complex with active epigenetic mark (H3K4me3), RNA polymerase II (Pol II), and mRNA production. Ezh1 depletion reduced global Pol II occupancy within gene bodies and resulted in delayed transcriptional activation during differentiation of skeletal muscle cells. Conversely, overexpression of wild-type Ezh1 led to premature gene activation and rescued Pol II occupancy defects in Ezh1-depleted cells. Collectively, these findings reveal a role for a PcG complex in promoting mRNA transcription.

post to: CiteULike

IKKalpha Activation of NOTCH Links Tumorigenesis via FOXA2 Suppression.

Publication Date: 2012 Jan 27 PMID: 22196886
Authors: Liu, M. - Lee, D. F. - Chen, C. T. - Yen, C. J. - Li, L. Y. - Lee, H. J. - Chang, C. J. - Chang, W. C. - Hsu, J. M. - Kuo, H. P. - Xia, W. - Wei, Y. - Chiu, P. C. - Chou, C. K. - Du, Y. - Dhar, D. - Karin, M. - Chen, C. H. - Hung, M. C.
Journal: Mol Cell

Proinflammatory cytokine TNFalpha plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFalpha-mediated tumor development remains unclear. Here, we show that IKKalpha, an important downstream kinase of TNFalpha, interacts with and phosphorylates FOXA2 at S107/S111, thereby suppressing FOXA2 transactivation activity and leading to decreased NUMB expression, and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKalpha, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKalpha and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFalpha/IKKalpha-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report a TNFalpha/IKKalpha/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.

post to: CiteULike

Activator-Induced Spread of Poly(ADP-Ribose) Polymerase Promotes Nucleosome Loss at Hsp70.

Publication Date: 2012 Jan 13 PMID: 22178397
Authors: Petesch, S. J. - Lis, J. T.
Journal: Mol Cell

Eukaryotic cells possess many transcriptionally regulated mechanisms to alleviate the nucleosome barrier. Dramatic changes to the chromatin structure of Drosophila melanogaster Hsp70 gene loci are dependent on the transcriptional activator, heat shock factor (HSF), and poly(ADP-ribose) polymerase (PARP). Here, we find that PARP is associated with the 5' end of Hsp70, and its enzymatic activity is rapidly induced by heat shock. This activation causes PARP to redistribute throughout Hsp70 loci and Poly(ADP-ribose) to concurrently accumulate in the wake of PARP's spread. HSF is necessary for both the activation of PARP's enzymatic activity and its redistribution. Upon heat shock, HSF triggers these PARP activities mechanistically by directing Tip60 acetylation of histone H2A lysine 5 at the 5' end of Hsp70, where inactive PARP resides before heat shock. This acetylation is critical for the activation and spread of PARP as well as for the rapid nucleosome loss over the Hsp70 loci.

post to: CiteULike

DNA Damage Signaling Triggers Degradation of Histone Methyltransferases through APC/C(Cdh1) in Senescent Cells.

Publication Date: 2012 Jan 13 PMID: 22178396
Authors: Takahashi, A. - Imai, Y. - Yamakoshi, K. - Kuninaka, S. - Ohtani, N. - Yoshimoto, S. - Hori, S. - Tachibana, M. - Anderton, E. - Takeuchi, T. - Shinkai, Y. - Peters, G. - Saya, H. - Hara, E.
Journal: Mol Cell

Both the DNA damage response (DDR) and epigenetic mechanisms play key roles in the implementation of senescent phenotypes, but very little is known about how these two mechanisms are integrated to establish senescence-associated gene expression. Here we show that, in senescent cells, the DDR induces proteasomal degradation of G9a and GLP, major histone H3K9 mono- and dimethyltransferases, through Cdc14B- and p21(Waf1/Cip1)-dependent activation of APC/C(Cdh1) ubiquitin ligase, thereby causing a global decrease in H3K9 dimethylation, an epigenetic mark for euchromatic gene silencing. Interestingly, induction of IL-6 and IL-8, major players of the senescence-associated secretory phenotype (SASP), correlated with a decline of H3K9 dimethylation around the respective gene promoters and knockdown of Cdh1 abolished IL-6/IL-8 expression in senescent cells, suggesting that the APC/C(Cdh1)-G9a/GLP axis plays crucial roles in aspects of senescent phenotype. These findings establish a role for APC/C(Cdh1) and reveal how the DDR integrates with epigenetic processes to induce senescence-associated gene expression.

post to: CiteULike

Epigenetic Mechanisms of Groucho/Grg/TLE Mediated Transcriptional Repression.

Publication Date: 2012 Jan 27 PMID: 22169276
Authors: Patel, S. R. - Bhumbra, S. S. - Paknikar, R. S. - Dressler, G. R.
Journal: Mol Cell

The repression of transcription, through the concerted actions of tissue specific DNA binding proteins, Polycomb repressor complexes, and DNA methylation, is essential for maintaining stem cell pluripotency and for cell fate specification in development. In this report, we show that recruitment of the co-repressor protein Grg4 to a Pax DNA-binding site displaces the adaptor protein PTIP and a histone H3K4me complex. Grg4 recruits the arginine methyltransferase PRMT5 to chromatin resulting in symmetric H4R3 dimethylation. PRMT5 is essential for recruiting Polycomb proteins, in a Pax2/Grg4 dependent manner, which results in H3K27 methylation. These data define the early epigenetic events in response to Pax/Grg mediated gene repression and demonstrate that a single DNA binding protein can recruit either an activator or a repressor complex depending on the availability of Grg4. These data suggest a model for understanding the initiation of Groucho/Grg/TLE mediated gene silencing.

post to: CiteULike

Inhibition of Homologous Recombination by the PCNA-Interacting Protein PARI.

Publication Date: 2012 Jan 13 PMID: 22153967
Authors: Moldovan, G. L. - Dejsuphong, D. - Petalcorin, M. I. - Hofmann, K. - Takeda, S. - Boulton, S. J. - D'Andrea, A. D.
Journal: Mol Cell

Inappropriate homologous recombination (HR) causes genomic instability and cancer. In yeast, the UvrD family helicase Srs2 is recruited to sites of DNA replication by SUMO-modified PCNA, where it acts to restrict HR by disassembling toxic RAD51 nucleofilaments. How human cells control recombination at replication forks is unknown. Here, we report that the protein PARI, containing a UvrD-like helicase domain, is a PCNA-interacting partner required for preservation of genome stability in human and DT40 chicken cells. Using cell-based and biochemical assays, we show that PARI restricts unscheduled recombination by interfering with the formation of RAD51-DNA HR structures. Finally, we show that PARI knockdown suppresses the genomic instability of Fanconi Anemia/BRCA pathway-deficient cells. Thus, we propose that PARI is a long sought-after factor that suppresses inappropriate recombination events at mammalian replication forks.

post to: CiteULike

Ser7 Phosphorylation of the CTD Recruits the RPAP2 Ser5 Phosphatase to snRNA Genes.

Publication Date: 2012 Jan 13 PMID: 22137580
Authors: Egloff, S. - Zaborowska, J. - Laitem, C. - Kiss, T. - Murphy, S.
Journal: Mol Cell

The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II) comprises multiple heptapeptide repeats of the consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Reversible phosphorylation of Ser2, Ser5, and Ser7 during the transcription cycle mediates the sequential recruitment of transcription/RNA processing factors. Phosphorylation of Ser7 is required for recruitment of the gene type-specific Integrator complex to the Pol II-transcribed small nuclear (sn)RNA genes. Here, we show that RNA Pol II-associated protein 2 (RPAP2) specifically recognizes the phospho-Ser7 mark on the Pol II CTD and also interacts with Integrator subunits. siRNA-mediated knockdown of RPAP2 and mutation of Ser7 to alanine cause similar defects in snRNA gene expression. In addition, we show that RPAP2 is a CTD Ser5 phosphatase. Taken together, our results indicate that during transcription of snRNA genes, Ser7 phosphorylation facilitates recruitment of RPAP2, which in turn both recruits Integrator and dephosphorylates Ser5.

post to: CiteULike