scholarly journals Creb-Binding Protein (Cbp/P300) and RNA Polymerase II Colocalize in Transcriptionally Active Domains in the Nucleus

2000 ◽  
Vol 150 (1) ◽  
pp. 265-274 ◽  
Author(s):  
Anna von Mikecz ◽  
Suisheng Zhang ◽  
Marc Montminy ◽  
Eng M. Tan ◽  
Peter Hemmerich
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Sites ◽  
Spatial Organization ◽  
Binding Protein ◽  
Nuclear Bodies ◽  
Laser Scanning Microscopy ◽  
Creb Binding Protein ◽  
Pol Ii ◽  
Pml Bodies

The spatial organization of transcription- associated proteins is an important control mechanism of eukaryotic gene expression. Here we analyzed the nuclear distribution of the transcriptional coactivators CREB-binding protein (CBP)/p300 in situ by confocal laser scanning microscopy, and in vivo complex formation by coimmunoprecipitation. A subpopulation of CBP and p300 is targeted to active sites of transcription and partially colocalizes with hyper- and hypophosphorylated RNA polymerase II (pol II) in discrete regions of variable size throughout the nucleus. However, the coactivators were found in tight association with hypophosphorylated, but not hyperphosphorylated pol II. Transcriptional inhibition induced a relocation of CBP/p300 and pol II into speckles. Moreover, double and triple immunofluorescence analyses revealed the presence of CBP, p300, and pol II in a subset of promyelocytic leukemia (PML) bodies. Our results provide evidence for a dynamic spacial link between coactivators of transcription and the basal transcription machinery in discrete nuclear domains dependent upon the transcriptional activity of the cell. The identification of pol II in CBP/PML-containing nuclear bodies supports the idea that transcription takes place at PML bodies.

scholarly journals Recruitment of an RNA Polymerase II Complex Is Mediated by the Constitutive Activation Domain in CREB, Independently of CREB Phosphorylation

2001 ◽  
Vol 21 (4) ◽  
pp. 1001-1010 ◽  
Author(s):  
Edward A. Felinski ◽  
Jeonga Kim ◽  
Jingfang Lu ◽  
Patrick G. Quinn
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Camp Response Element Binding ◽  
Creb Binding Protein ◽  
Transcription Activator ◽  
Activation Domain ◽  
Creb Phosphorylation ◽  
Constitutive Activation ◽  
Creb Activation

ABSTRACT The cAMP response element binding protein (CREB) is a bifunctional transcription activator, exerting its effects through a constitutive activation domain (CAD) and a distinct kinase inducible domain (KID), which requires phosphorylation of Ser-133 for activity. Both CAD and phospho-KID have been proposed to recruit polymerase complexes, but this has not been directly tested. Here, we show that the entire CREB activation domain or the CAD enhanced recruitment of a complex containing TFIID, TFIIB, and RNA polymerase II to a linked promoter. The nuclear extracts used mediated protein kinase A (PKA)-inducible transcription, but phosphorylation of CRG (both of the CREB activation domains fused to the Gal4 DNA binding domain) or KID-G4 did not mediate recruitment of a complex, and mutation of the PKA site in CRG abolished transcription induction by PKA but had no effect upon recruitment. The CREB-binding protein (CBP) was not detected in the recruited complex. Our results support a model for transcription activation in which the interaction between the CREB CAD and hTAFII130 of TFIID promotes the recruitment of a polymerase complex to the promoter.

scholarly journals RNA polymerase II condensate formation and association with Cajal and histone locus bodies in living human cells

2020 ◽  
Author(s):  
Takashi Imada ◽  
Takeshi Shimi ◽  
Ai Kaiho ◽  
Yasushi Saeki ◽  
Hiroshi Kimura
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Clusters ◽  
Nuclear Bodies ◽  
Histone Genes ◽  
Small Nuclear Rna ◽  
Pol Ii ◽  
Histone Locus Bodies ◽  
Dynamic Structures ◽  
Histone Locus

ABSTRACTIn eukaryotic nuclei, a number of phase-separated nuclear bodies (NBs) are present. RNA polymerase II (Pol II) is the main player in transcription and forms large condensates in addition to localizing at numerous transcription foci. Cajal bodies (CBs) and histone locus bodies (HLBs) are NBs that are involved in transcriptional and post-transcriptional regulation of small nuclear RNA and histone genes. By live-cell imaging using human HCT116 cells, we here show that Pol II condensates (PCs) nucleated near CBs and HLBs, and the number of PCs increased during S phase concomitantly with the activation period of histone genes. Ternary PC–CB– HLB associates were formed via three pathways: nucleation of PCs and HLBs near CBs, interaction between preformed PC–HLBs with CBs, and nucleation of PCs near preformed CB– HLBs. Coilin knockout increased the co-localization rate between PCs and HLBs, whereas the number, nucleation timing, and phosphorylation status of PCs remained unchanged. Depletion of PCs did not affect CBs and HLBs. Treatment with 1,6-hexanediol revealed that PCs were more liquid-like than CBs and HLBs. Thus, PCs are dynamic structures often nucleated following the activation of gene clusters associated with other NBs. (187 words)

scholarly journals Regulation of rRNA Synthesis by TATA-Binding Protein-Associated Factor Mot1

2007 ◽  
Vol 27 (8) ◽  
pp. 2886-2896 ◽  
Author(s):  
Arindam Dasgupta ◽  
Rebekka O. Sprouse ◽  
Sarah French ◽  
Pavel Aprikian ◽  
Robert Hontz ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rrna Synthesis ◽  
Direct Role ◽  
Pol Ii ◽  
Associated Factor ◽  
Pol I

ABSTRACT Mot1 is an essential, conserved, TATA-binding protein (TBP)-associated factor in Saccharomyces cerevisiae with well-established roles in the global control of RNA polymerase II (Pol II) transcription. Previous results have suggested that Mot1 functions exclusively in Pol II transcription, but here we report a novel role for Mot1 in regulating transcription by RNA polymerase I (Pol I). In vivo, Mot1 is associated with the ribosomal DNA, and loss of Mot1 results in decreased rRNA synthesis. Consistent with a direct role for Mot1 in Pol I transcription, Mot1 also associates with the Pol I promoter in vitro in a reaction that depends on components of the Pol I general transcription machinery. Remarkably, in addition to Mot1's role in initiation, rRNA processing is delayed in mot1 cells. Taken together, these results support a model in which Mot1 affects the rate and efficiency of rRNA synthesis by both direct and indirect mechanisms, with resulting effects on transcription activation and the coupling of rRNA synthesis to processing.

scholarly journals Mediator and RNA polymerase II clusters associate in transcription-dependent condensates

Science ◽  
2018 ◽  
Vol 361 (6400) ◽  
pp. 412-415 ◽  
Author(s):  
Won-Ki Cho ◽  
Jan-Hendrik Spille ◽  
Micca Hecht ◽  
Choongman Lee ◽  
Charles Li ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Spatial Organization ◽  
Embryonic Stem ◽  
Light Sheet ◽  
Gene Control ◽  
Pol Ii ◽  
Stable Clusters ◽  
Large Clusters

Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. We used live-cell superresolution and light-sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo.

scholarly journals Human Mediator Enhances Activator-Facilitated Recruitment of RNA Polymerase II and Promoter Recognition by TATA-Binding Protein (TBP) Independently of TBP-Associated Factors

2003 ◽  
Vol 23 (17) ◽  
pp. 6229-6242 ◽  
Author(s):  
Shwu-Yuan Wu ◽  
Tianyuan Zhou ◽  
Cheng-Ming Chiang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Associated Factors ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Dependent Manner ◽  
Basal Transcription ◽  
Pol Ii ◽  
Promoter Recognition ◽  
Stimulation Of

ABSTRACT Mediator is a general cofactor implicated in the functions of many transcriptional activators. Although Mediator with different protein compositions has been isolated, it remains unclear how Mediator facilitates activator-dependent transcription, independent of its general stimulation of basal transcription. To define the mechanisms of Mediator function, we isolated two forms of human Mediator complexes (Mediator-P.5 and Mediator-P.85) and demonstrated that Mediator-P.5 clearly functions by enhancing activator-mediated recruitment of RNA polymerase II (pol II), whereas Mediator-P.85 works mainly by stimulating overall basal transcription. The coactivator function of Mediator-P.5 was not impaired when TATA-binding protein (TBP) was used in place of TFIID, but it was abolished when another general cofactor, PC4, was omitted from the reaction or when Mediator-P.5 was added after pol II entry into the preinitiation complex. Moreover, Mediator- P.5 is able to enhance TBP binding to the TATA box in an activator-dependent manner. Our data provides biochemical evidence that Mediator functions by facilitating activator-mediated recruitment of pol II and also promoter recognition by TBP, both of which can occur in the absence of TBP-associated factors in TFIID.

scholarly journals An Array of Coactivators Is Required for Optimal Recruitment of TATA Binding Protein and RNA Polymerase II by Promoter-Bound Gcn4p

2004 ◽  
Vol 24 (10) ◽  
pp. 4104-4117 ◽  
Author(s):  
Hongfang Qiu ◽  
Cuihua Hu ◽  
Sungpil Yoon ◽  
Krishnamurthy Natarajan ◽  
Mark J. Swanson ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Pol Ii ◽  
Elongation Phase ◽  
Target Promoters

ABSTRACT Wild-type transcriptional activation by Gcn4p is dependent on multiple coactivators, including SAGA, SWI/SNF, Srb mediator, CCR4-NOT, and RSC, which are all recruited by Gcn4p to its target promoters in vivo. It was not known whether these coactivators are required for assembly of the preinitiation complex (PIC) or for subsequent steps in the initiation or elongation phase of transcription. We find that mutations in subunits of these coactivators reduce the recruitment of TATA binding protein (TBP) and RNA polymerase II (Pol II) by Gcn4p at ARG1, ARG4, and SNZ1, implicating all five coactivators in PIC assembly at Gcn4p target genes. Recruitment of Pol II at SNZ1 and ARG1 was eliminated by mutations in TBP or by deletion of the TATA box, indicating that TBP binding is a prerequisite for Pol II recruitment by Gcn4p. However, several mutations in SAGA subunits and deletion of SRB10 had a greater impact on promoter occupancy of Pol II versus TBP, suggesting that SAGA and Srb mediator can promote Pol II binding independently of their stimulatory effects on TBP recruitment. Our results reveal an unexpected complexity in the cofactor requirements for the enhancement of PIC assembly by a single activator protein.

scholarly journals Lamin A/C speckles mediate spatial organization of splicing factor compartments and RNA polymerase II transcription

2002 ◽  
Vol 159 (5) ◽  
pp. 783-793 ◽  
Author(s):  
R. Ileng Kumaran ◽  
Bhattiprolu Muralikrishna ◽  
Veena K. Parnaik
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hela Cells ◽  
Rna Splicing ◽  
Spatial Organization ◽  
Nuclear Periphery ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Lamin A ◽  
Pol Ii

The A-type lamins have been observed to colocalize with RNA splicing factors in speckles within the nucleus, in addition to their typical distribution at the nuclear periphery. To understand the functions of lamin speckles, the effects of transcriptional inhibitors known to modify RNA splicing factor compartments (SFCs) were examined. Treatment of HeLa cells with α-amanitin or 5,6-dichlorobenzimidazole riboside (DRB) inhibited RNA polymerase II (pol II) transcription and led to the enlargement of lamin speckles as well as SFCs. Removal of the reversible inhibitor DRB resulted in the reactivation of transcription and a rapid, synchronous redistribution of lamins and splicing factors to normal-sized speckles, indicating a close association between lamin speckles and SFCs. Conversely, the expression of NH2-terminally modified lamin A or C in HeLa cells brought about a loss of lamin speckles, depletion of SFCs, and down-regulation of pol II transcription without affecting the peripheral lamina. Our results suggest a unique role for lamin speckles in the spatial organization of RNA splicing factors and pol II transcription in the nucleus.

scholarly journals Structure of the p53/RNA polymerase II assembly

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shu-Hao Liou ◽  
Sameer K. Singh ◽  
Robert H. Singer ◽  
Robert A. Coleman ◽  
Wei-Li Liu
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
P53 Protein ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Pol Ii ◽  
Dna Binding Activity ◽  
Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

Sign in / Sign up

Export Citation Format

Share Document