TFIIF-associating Carboxyl-terminal Domain Phosphatase Dephosphorylates Phosphoserines 2 and 5 of RNA Polymerase II

Lin, Patrick S.; Dubois, Marie-Françoise; Dahmus, Michael E.

doi:10.1074/jbc.m208588200

Cited by 59 publications

(48 citation statements)

References 57 publications

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“…By using this system, we observed that Fcp1 can dephosphorylate serine 5 but not serine 2 of the RNAPII CTD. This result was surprising, given that purified human Fcp1 has been shown previously to dephosphorylate these substrates with similar efficiency (50). Moreover, yeast FCP1 affects the serine 2 phosphorylation levels, whereas it has little, if any, effect on serine 5 phosphorylation levels in genes in vivo (17).…”

Section: Discussioncontrasting

confidence: 38%

Interaction of Fcp1 Phosphatase with Elongating RNA Polymerase II Holoenzyme, Enzymatic Mechanism of Action, and Genetic Interaction with Elongator

Kong

Kobor

Krogan

et al. 2005

Journal of Biological Chemistry

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Fcp1 de-phosphorylates the RNA polymerase II (RNAPII) C-terminal domain (CTD) in vitro, and mutation of the yeast FCP1 gene results in global transcription defects and increased CTD phosphorylation levels in vivo. Here we show that the Fcp1 protein associates with elongating RNAPII holoenzyme in vitro. Our data suggest that the association of Fcp1 with elongating polymerase results in CTD de-phosphorylation when the native ternary RNAPII0-DNA-RNA complex is disrupted. Surprisingly, highly purified yeast Fcp1 dephosphorylates serine 5 but not serine 2 of the RNAPII CTD repeat. Only free RNAPII0(Ser-5) and not RNA-PII0-DNA-RNA ternary complexes act as a good substrate in the Fcp1 CTD de-phosphorylation reaction. In contrast, TFIIH CTD kinase has a pronounced preference for RNAPII incorporated into a ternary complex. Interestingly, the Fcp1 reaction mechanism appears to entail phosphoryl transfer from RNAPII0 directly to Fcp1. Elongator fails to affect the phosphatase activity of Fcp1 in vitro, but genetic evidence points to a functional overlap between Elongator and Fcp1 in vivo. Genetic interactions between Elongator and a number of other transcription factors are also reported. Together, these results shed new light on mechanisms that drive the transcription cycle and point to a role for Fcp1 in the recycling of RNAPII after dissociation from active genes. During the RNA polymerase II (RNAPII)1 transcription cycle, both the phosphorylation state of the polymerase and the proteins that RNAPII interacts with change dramatically. Throughout promoter recruitment and initiation where RNA-PII exists in a hypo-phosphorylated state, it interacts with general transcription factors, such as TFIID, TFIIB, TFIIF, TFIIE, TFIIH, and Mediator (1). In the process of promoter clearance and early transcript elongation where RNAPII becomes hyper-phosphorylated, interactions with initiation-specific factors are severed, and interactions with other proteins are established. Factors that have been shown to preferentially associate with hyper-phosphorylated elongating RNAPII include RNA processing factors (2) and factors thought to play a role in transcript elongation, such as Elongator, Set1, and Set2 (3-8).The C-terminal repeat domain (CTD) of RNAPII is the specific target for kinases that phosphorylates the polymerase at the transition from initiation to elongation. The CTD consists of the conserved heptapeptide, Tyr-Ser-Pro-Thr-Ser-Pro-Ser, repeated 26 times in yeast and up to 52 times in metazoans (9). After transcript initiation, the CTD is heavily phosphorylated on serine 2 and serine 5 by different protein kinases (pTEFb (yeast Bur1 or Ctdk1)) and TFIIH), respectively (10, 11). Later, during transcript elongation and at transcriptional termination, CTD phosphatases remove the phosphate groups, enabling recycling of RNAPII for a new round of transcription (11,12). A CTD phosphatase was first identified biochemically through its ability to specifically dephosphorylate the RNAPII CTD in vitro (13). The gene encoding a TFIIF-inter...

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Section: Discussioncontrasting

confidence: 38%

Interaction of Fcp1 Phosphatase with Elongating RNA Polymerase II Holoenzyme, Enzymatic Mechanism of Action, and Genetic Interaction with Elongator

Kong

Kobor

Krogan

et al. 2005

Journal of Biological Chemistry

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“…First, phosphorylated and 32 Plabeled Pol II substrates were generated by in vitro phosphorylation using the Ser͞Thr kinase mitogen-activated protein kinase 2. Mitogen-activated protein kinase 2 phosphorylates Ser-2 and Ser-5 sites of the CTD in vitro as has been demonstrated by phosphorylated CTD-specific antibodies (43), although with preference toward Ser-5 (44). We confirmed conversion of Pol IIA to Pol IIO by a mobility change in the largest subunit (Rpb1) of Pol II in SDS͞PAGE (Fig.…”

Section: Yeast Fcp1 Phosphatase Activity Is Independent Of the Globularsupporting

confidence: 66%

Fcp1 directly recognizes the C-terminal domain (CTD) and interacts with a site on RNA polymerase II distinct from the CTD

Suh

Zhang²,

Hausmann³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Fcp1 is an essential protein phosphatase that hydrolyzes phosphoserines within the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). Fcp1 plays a major role in the regulation of CTD phosphorylation and, hence, critically influences the function of Pol II throughout the transcription cycle. The basic understanding of Fcp1-CTD interaction has remained ambiguous because two different modes have been proposed: the ''dockingsite'' model versus the ''distributive'' mechanism. Here we demonstrate biochemically that Fcp1 recognizes and dephosphorylates the CTD directly, independent of the globular non-CTD part of the Pol II structure. We point out that the recognition of CTD by the phosphatase is based on random access and is not driven by Pol II conformation. Results from three different types of experiments reveal that the overall interaction between Fcp1 and Pol II is not stable but dynamic. In addition, we show that Fcp1 also interacts with a region on the polymerase distinct from the CTD. We emphasize that this non-CTD site is functionally distinct from the docking site invoked previously as essential for the CTD phosphatase activity of Fcp1. We speculate that Fcp1 interaction with the non-CTD site may mediate its stimulatory effect on transcription elongation reported previously.C-terminal domain dephosphorylation ͉ C-terminal domain hyperphosphorylation R NA polymerase II (Pol II) is distinguished from RNA polymerases I and III by the unique C-terminal domain (CTD) of its largest subunit, Rpb1 (1-3). The CTD, through its interaction with the Mediator complex, plays a central role in transcription activation (4-6), and it also plays critical roles in coupling mRNA synthesis to mRNA processing events, such as 5Ј capping, splicing, and 3Ј cleavage and polyadenylation (7,8). Composed of a tandemly repeated heptapeptide of the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , the CTD undergoes reversible phosphorylation (9-11). Whereas the hypophosphorylated Pol II isoform (Pol IIA) assembles with general transcription factors into the transcription preinitiation complex (12-14), the hyperphosphorylated isoform (Pol IIO) is the dominant species found in transcription elongation complexes in vivo (15)(16)(17). The phosphorylation state of the CTD is controlled by the activities of CTD-specific kinases and phosphatases. Several transcription factor-associated CTD kinases have been identified, including CDK7͞Kin28, CDK9͞Bur1͞Ctk1 and CDK8͞Srb10 (11,(18)(19)(20). Other kinases (e.g., Cdc2 kinase and mitogen-activated protein kinase 2) can phosphorylate the CTD in vitro, but their contributions to CTD phosphorylation in vivo are uncertain (18,(21)(22)(23).Pol IIO is believed to be dephosphorylated before it can recycle for another round of transcription initiation (13,14,21,24). Fcp1 was the first CTD phosphatase discovered to fulfill such a function (25, 26). Fcp1 was reported to remove phosphates specifically from the Pol II CTD, but not from the other phosphoproteins tested (25). The human and...

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“…In the present work, we show that full-length MINT enhances Runx2-dependent transcription, mediated via the autonomous Runx2 AD3. Consistent with a role in the activation of gene transcription, we demonstrate that MINT extensively co-localizes with RNAP IIo, the transcriptionally active and phosphorylated form of RNA polymerase (40).…”

Section: Discussionmentioning

confidence: 54%

MINT, the Msx2 Interacting Nuclear Matrix Target, Enhances Runx2-dependent Activation of the Osteocalcin Fibroblast Growth Factor Response Element

Sierra

Cheng

Loewy

et al. 2004

Journal of Biological Chemistry

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Msx2 promotes osteogenic lineage allocation from mesenchymal progenitors but inhibits terminal differentiation demarcated by osteocalcin (OC) gene expression. Msx2 inhibits OC expression by targeting the fibroblast growth factor responsive element (OCFRE), a 42-bp DNA domain in the OC gene bound by the Msx2 interacting nuclear target protein (MINT) and Runx2/ Cbfa1. To better understand Msx2 regulation of the OC-FRE, we have studied functional interactions between MINT and Runx2, a master regulator of osteoblast differentiation. In MC3T3E1 osteoblasts (with endogenous Runx2 and FGFR2), MINT augments transcription driven by the OCFRE that is further enhanced by FGF2 treatment. OCFRE regulation can be reconstituted in the naïve CV1 fibroblast cell background. In CV1 cells, MINT synergizes with Runx2 to enhance OCFRE activity in the presence of activated FGFR2. The RNA recognition motif domain of MINT (which binds the OCFRE) is required. Runx2 structural studies reveal that synergy with MINT uniquely requires Runx2 activation domain 3. In confocal immunofluorescence microscopy, MINT adopts a reticular nuclear matrix distribution that overlaps transcriptionally active osteoblast chromatin, extensively co-localizing with the phosphorylated RNA polymerase II meshwork. MINT only partially co-localizes with Runx2; however, co-localization is enhanced 2.5-fold by FGF2 stimulation. Msx2 abrogates Runx2-MINT OCFRE activation, and MINT-directed RNA interference reduces endogenous OC expression. In chromatin immunoprecipitation assays, Msx2 selectively inhibits Runx2 binding to OC chromatin. Thus, MINT enhances Runx2 activation of multiprotein complexes assembled by the OCFRE. Msx2 targets this complex as a mechanism of transcriptional inhibition. In osteoblasts, MINT may serve as a nuclear matrix platform that organizes and integrates osteogenic transcriptional responses.Bone formation arises via two overlapping yet distinct mechanisms (1). Endochondral ossification occurs via the calcification and vascularization of an initial cartilaginous template, best exemplified by long bone development and fracture repair. Non-endochondral ossification occurs during development in the flat bones of the skull, in teeth, and in the lateral portion of the clavicles. Direct bone deposition occurs in type I collagenbased extracellular matrix; no cartilage template precedes bone deposition. Both in vivo and in cell culture models, a characteristic gene expression program is elaborated as osteoblasts differentiate and mature from osteoprogenitors (2-4). Although transcription factors crucial to bone development (Runx2/Cbfa1, CBF-␤, Msx2, Msx1, Dlx5, Alx4, Osx, and Sox9) and metabolic/endocrine regulation (multiple nuclear receptors, Runx2/Cbfa1) have been identified, our understanding of the molecular details of stage-specific osteoblast gene expression is rudimentary (5). "Cross-talk" between these factors occurs in the osteoblast nucleus; an integrative model is lacking and sorely needed to better address unmet clinical needs in metabolic...

show abstract

TFIIF-associating Carboxyl-terminal Domain Phosphatase Dephosphorylates Phosphoserines 2 and 5 of RNA Polymerase II

Cited by 59 publications

References 57 publications

Interaction of Fcp1 Phosphatase with Elongating RNA Polymerase II Holoenzyme, Enzymatic Mechanism of Action, and Genetic Interaction with Elongator

Interaction of Fcp1 Phosphatase with Elongating RNA Polymerase II Holoenzyme, Enzymatic Mechanism of Action, and Genetic Interaction with Elongator

Fcp1 directly recognizes the C-terminal domain (CTD) and interacts with a site on RNA polymerase II distinct from the CTD

MINT, the Msx2 Interacting Nuclear Matrix Target, Enhances Runx2-dependent Activation of the Osteocalcin Fibroblast Growth Factor Response Element

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