scholarly journals The Mitotic Chromosome Binding Activity of the Papillomavirus E2 Protein Correlates with Interaction with the Cellular Chromosomal Protein, Brd4

2005 ◽  
Vol 79 (8) ◽  
pp. 4806-4818 ◽  
Author(s):  
Michael K. Baxter ◽  
Maria G. McPhillips ◽  
Keiko Ozato ◽  
Alison A. McBride
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Mitotic Chromosome ◽  
Structural Model ◽  
Binding Activity ◽  
Chromosomal Protein ◽  
Transactivation Domain ◽  
Mitotic Chromosomes ◽  
Functional Region ◽  
Chromosome Binding

ABSTRACT The papillomavirus transcriptional activator, E2, is involved in key functions of the viral life cycle. These include transcriptional regulation, viral DNA replication, and viral genome segregation. The transactivation domain of E2 is required for each of these functions. To identify the regions of the domain that mediate binding to mitotic chromosomes, a panel of mutations has been generated and their effect on various E2 functions has been analyzed. A structural model of the bovine papillomavirus type 1 (BPV1) E2 transactivation domain was generated based on its homology with the solved structure of the human papillomavirus type 16 (HPV16) domain. This model was used to identify distinct surfaces of the domain to be targeted by point mutation to further delineate the functional region of the transactivation domain responsible for mitotic chromosome association. The mutated E2 proteins were assessed for mitotic chromosome binding and, in addition, transcriptional activation and transcriptional repression activities. Mutation of amino acids R37 and I73, which are located on a surface of the domain that in HPV16 E2 is reported to mediate self-interaction, completely eliminated mitotic chromosome binding. Mitotic chromosome binding activity was found to correlate well with the ability to interact with the cellular chromosomal associated factor Brd4, which has recently been proposed to mediate the association between BPV1 E2 and mitotic chromosomes.

scholarly journals Brd4-Independent Transcriptional Repression Function of the Papillomavirus E2 Proteins

2007 ◽  
Vol 81 (18) ◽  
pp. 9612-9622 ◽  
Author(s):  
Michal-Ruth Schweiger ◽  
Matthias Ottinger ◽  
Jianxin You ◽  
Peter M. Howley
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Regulatory Protein ◽  
Single Amino Acid ◽  
Bovine Papillomavirus ◽  
Transactivation Domain ◽  
Mitotic Chromosomes ◽  
Interacting Protein ◽  
Shrna Knockdown

ABSTRACT The papillomavirus E2 protein is a critical viral regulatory protein with transcription, DNA replication, and genome maintenance functions. We have previously identified the cellular bromodomain protein Brd4 as a major E2-interacting protein and established that it participates in tethering bovine papillomavirus type 1 E2 and viral genomes to host cell mitotic chromosomes. We have also shown that Brd4 mediates E2-dependent transcriptional activation, which is strongly inhibited by the disruption of E2/Brd4 binding as well as by short hairpin RNA (shRNA) knockdown of Brd4 expression levels. Since several mutants harboring single amino acid substitutions within the E2 transactivation domain that are defective for both transcriptional transactivation and Brd4 binding are also defective for transcriptional repression, we examined the role of Brd4 in E2 repression of the human papillomavirus E6/E7 promoter. Surprisingly, in a variety of in vivo assays, including transcription reporter assays, HeLa cell proliferation and colony reduction assays, and Northern blot analyses, neither blocking of the binding of E2 to Brd4 nor shRNA knockdown of Brd4 affected the E2 repression function. Our study provides evidence for a Brd4-independent mechanism of E2-mediated repression and suggests that different cellular factors must be involved in E2-mediated transcriptional activation and repression functions.

scholarly journals An Af9 cis-element directly targets Dot1a to mediate transcriptional repression of the αENaC gene

2013 ◽  
Vol 304 (4) ◽  
pp. F367-F375 ◽  
Author(s):  
Wenzheng Zhang ◽  
Zhiyuan Yu ◽  
Hongyu Wu ◽  
Lihe Chen ◽  
Qun Kong ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Protein Complexes ◽  
Collecting Duct ◽  
Binding Activity ◽  
Mrna Levels ◽  
Cis Element ◽  
Connecting Tubule ◽  
The Impact

The epithelial Na+ channel subunit-α ( αENaC) of the distal nephron is essential for salt balance. We previously demonstrated that the histone methyltransferase Dot1a and its protein partner Af9 basally repress αENaC transcription in mouse inner medullary collecting duct type 3 (mIMCD3) cells and link aldosterone-elicited chromatin modifications to αENaC transcriptional activation. Af9 DNA-binding activity has never been demonstrated, and whether and where Af9 binds to the αENaC promoter to target Dot1a are unknown. The present study sought to identify functional Af9 cis-element(s) in the −57/+439 “R3” subregion of αENaC, the principal site for Dot1a-Af9 interaction, in mIMCD3 cells. We also exploited connecting tubule/collecting duct-specific Dot1l-deficient mice ( Dot1l AC) to determine the impact of Dot1l inactivation on renal αENaC expression in vivo. mIMCD3 cell lines expressing αENaC promoter-reporter constructs harboring deletion of +74/+107 demonstrated greatly reduced association of Af9 and Dot1a by ChIP/qPCR. Aldosterone treatment resulted in further decrements in Af9 and Dot1a association with the αENaC promoter. Gel shift and antibody competition assays using wild-type and mutant oligomers revealed Af9-containing +78/+92 αENaC DNA-protein complexes in nuclear extracts of mIMCD3 cells. Mutation of the +78/+92 element resulted in higher basal αENaC promoter activity and impaired Dot1a-mediated inhibition in trans-repression assays. In agreement, mice with connecting tubule/collecting duct-specific knockout of Dot1l exhibited greater αENaC mRNA levels in kidney compared with control. Thus, we conclude that +78/+92 of αENaC represents the primary Af9 binding site involved in recruiting Dot1a to repress basal and aldosterone-sensitive αENaC transcription and that Dot1l inactivation promotes αENaC mRNA expression by eliminating Dot1a-mediated repression.

scholarly journals Contribution of hCAP-D2, a Non-SMC Subunit of Condensin I, to Chromosome and Chromosomal Protein Dynamics during Mitosis

2005 ◽  
Vol 25 (2) ◽  
pp. 740-750 ◽  
Author(s):  
Erwan Watrin ◽  
Vincent Legagneux
Keyword(s):  
Rna Interference ◽  
Mitotic Spindle ◽  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosomal Protein ◽  
Structural Components ◽  
Mitotic Chromosomes ◽  
Sister Chromatids ◽  
Chromosome Alignment ◽  
Structural Maintenance Of Chromosomes

ABSTRACT Condensins are heteropentameric complexes that were first identified as structural components of mitotic chromosomes. They are composed of two SMC (structural maintenance of chromosomes) and three non-SMC subunits. Condensins play a role in the resolution and segregation of sister chromatids during mitosis, as well as in some aspects of mitotic chromosome assembly. Two distinct condensin complexes, condensin I and condensin II, which differ only in their non-SMC subunits, exist. Here, we used an RNA interference approach to deplete hCAP-D2, a non-SMC subunit of condensin I, in HeLa cells. We found that the association of hCAP-H, another non-SMC subunit of condensin I, with mitotic chromosomes depends on the presence of hCAP-D2. Moreover, chromatid axes, as defined by topoisomerase II and hCAP-E localization, are disorganized in the absence of hCAP-D2, and the resolution and segregation of sister chromatids are impaired. In addition, hCAP-D2 depletion affects chromosome alignment in metaphase and delays entry into anaphase. This suggests that condensin I is involved in the correct attachment between chromosome kinetochores and microtubules of the mitotic spindle. These results are discussed relative to the effects of depleting both condensin complexes.

scholarly journals Bromodomain Protein 4 Mediates the Papillomavirus E2 Transcriptional Activation Function

2006 ◽  
Vol 80 (9) ◽  
pp. 4276-4285 ◽  
Author(s):  
Michal-Ruth Schweiger ◽  
Jianxin You ◽  
Peter M. Howley
Keyword(s):  
Dna Replication ◽  
Transcriptional Activation ◽  
Viral Genome ◽  
Regulatory Protein ◽  
Activation Function ◽  
Transactivation Domain ◽  
Genome Maintenance ◽  
Mitotic Chromosomes ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACT The papillomavirus E2 regulatory protein has essential roles in viral transcription and the initiation of viral DNA replication as well as for viral genome maintenance. Brd4 has recently been identified as a major E2-interacting protein and, in the case of the bovine papillomavirus type 1, serves to tether E2 and the viral genomes to mitotic chromosomes in dividing cells, thus ensuring viral genome maintenance. We have explored the possibility that Brd4 is involved in other E2 functions. By analyzing the binding of Brd4 to a series of alanine-scanning substitution mutants of the human papillomavirus type 16 E2 N-terminal transactivation domain, we found that amino acids required for Brd4 binding were also required for transcriptional activation but not for viral DNA replication. Functional studies of cells expressing either the C-terminal domain of Brd4 that can bind E2 and compete its binding to Brd4 or short interfering RNA to knock down Brd4 protein levels revealed a role for Brd4 in the transcriptional activation function of E2 but not for its viral DNA replication function. Therefore, these studies establish a broader role for Brd4 in the papillomavirus life cycle than as the chromosome tether for E2 during mitosis.

scholarly journals Mitotic Chromosome-Binding Activity of Latency-Associated Nuclear Antigen 1 Is Required for DNA Replication from Terminal Repeat Sequence of Kaposi's Sarcoma-Associated Herpesvirus

2004 ◽  
Vol 78 (13) ◽  
pp. 7248-7256 ◽  
Author(s):  
Chunghun Lim ◽  
Changtaek Choi ◽  
Joonho Choe
Keyword(s):  
Dna Replication ◽  
Viral Genome ◽  
Kaposi's Sarcoma ◽  
Mitotic Chromosome ◽  
Kaposi’S Sarcoma ◽  
Binding Activity ◽  
Terminal Repeat ◽  
Nuclear Antigen ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Chromosome Binding

ABSTRACT Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the persistence of the viral genome during latent infection. It has been suggested that LANA1 tethers the viral genome to the host chromosome and also participates actively in DNA replication from the terminal repeat of KSHV. Here we show by mutational analysis that the mitotic chromosome-binding activity of LANA1 is tightly coupled to its replication activity. Thus, KSHV appears to have evolved a unique tactic for its stable maintenance.

scholarly journals Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division

2010 ◽  
Vol 84 (21) ◽  
pp. 11175-11188 ◽  
Author(s):  
Toomas Silla ◽  
Andres Männik ◽  
Mart Ustav
Keyword(s):  
Transcriptional Activation ◽  
Human Herpesvirus ◽  
Bovine Papillomavirus ◽  
Transactivation Domain ◽  
Mitotic Chromosomes ◽  
Herpesvirus Type ◽  
Barr Virus ◽  
Human Herpesvirus Type ◽  
Multiple Copies ◽  
Epstein Barr

ABSTRACT Effective segregation of the bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated human herpesvirus type 8 (KSHV) genomes into daughter cells is mediated by a single viral protein that tethers viral genomes to host mitotic chromosomes. The linker proteins that mediate BPV1, EBV, and KSHV segregation are E2, LANA1, and EBNA1, respectively. The N-terminal transactivation domain of BPV1 E2 is responsible for chromatin attachment and subsequent viral genome segregation. Because E2 transcriptional activation and chromatin attachment functions are not mutually exclusive, we aimed to determine the requirement of these activities during segregation by analyzing chimeric E2 proteins. This approach allowed us to separate the two activities. Our data showed that attachment of the segregation protein to chromatin is not sufficient for proper segregation. Rather, formation of a segregation-competent complex which carries multiple copies of the segregation protein is required. Complementation studies of E2 functional domains indicated that chromatin attachment and transactivation functions must act in concert to ensure proper plasmid segregation. These data indicate that there are specific interactions between linker molecules and transcription factors/complexes that greatly increase segregation-competent complex formation. We also showed, using hybrid E2 molecules, that restored segregation function does not involve interactions with Brd4.

scholarly journals Acetylation-Mediated Transcriptional Activation of the ETS Protein ER81 by p300, P/CAF, and HER2/Neu

2003 ◽  
Vol 23 (17) ◽  
pp. 6243-6254 ◽  
Author(s):  
Apollina Goel ◽  
Ralf Janknecht
Keyword(s):  
Protein Kinase ◽  
Transcriptional Activation ◽  
Binding Activity ◽  
Tumor Formation ◽  
Mitogen Activated Protein Kinase ◽  
Transactivation Domain ◽  
Dna Binding Activity ◽  
Mitogen Activated Protein

ABSTRACT The regulated expression of the ETS transcription factor ER81 is a prerequisite for normal development, and its dysregulation contributes to neoplasia. Here, we demonstrate that ER81 is acetylated by two coactivators/acetyltransferases, p300 and p300- and CBP-associated factor (P/CAF) in vitro and in vivo. Whereas p300 acetylates two lysine residues (K33 and K116) within the ER81 N-terminal transactivation domain, P/CAF targets only K116. Acetylation of ER81 not only enhances its ability to transactivate but also increases its DNA binding activity and in vivo half-life. Furthermore, oncogenic HER2/Neu, which induces phosphorylation and thereby activation of ER81, was less able to activate acetylation-deficient ER81 mutants, indicating that both acetyltransferase and protein kinase-specific regulatory mechanisms control ER81 activity. Importantly, HER2/Neu overexpression stimulates the ability of p300 to acetylate ER81, likely by inducing phosphorylation of p300 through the Ras→Raf→mitogen-activated protein kinase pathway. This represents a novel mechanism by which oncogenic HER2/Neu, Ras, or Raf may promote tumor formation by enhancing acetylation not only of ER81 but also of other downstream effector transcription factors as well as histones.

scholarly journals Alleviation of histone H1-mediated transcriptional repression and chromatin compaction by the acidic activation region in chromosomal protein HMG-14.

1997 ◽  
Vol 17 (10) ◽  
pp. 5843-5855 ◽  
Author(s):  
H F Ding ◽  
M Bustin ◽  
U Hansen
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Simian Virus 40 ◽  
Histone H1 ◽  
Simian Virus ◽  
Higher Order ◽  
Chromosomal Protein ◽  
C Terminus

Histone H1 promotes the generation of a condensed, transcriptionally inactive, higher-order chromatin structure. Consequently, histone H1 activity must be antagonized in order to convert chromatin to a transcriptionally competent, more extended structure. Using simian virus 40 minichromosomes as a model system, we now demonstrate that the nonhistone chromosomal protein HMG-14, which is known to preferentially associate with active chromatin, completely alleviates histone H1-mediated inhibition of transcription by RNA polymerase II. HMG-14 also partially disrupts histone H1-dependent compaction of chromatin. Both the transcriptional enhancement and chromatin-unfolding activities of HMG-14 are mediated through its acidic, C-terminal region. Strikingly, transcriptional and structural activities of HMG-14 are maintained upon replacement of the C-terminal fragment by acidic regions from either GAL4 or HMG-2. These data support the model that the acidic C terminus of HMG-14 is involved in unfolding higher-order chromatin structure to facilitate transcriptional activation of mammalian genes.

scholarly journals The Carboxy-Terminal Neh3 Domain of Nrf2 Is Required for Transcriptional Activation

2005 ◽  
Vol 25 (24) ◽  
pp. 10895-10906 ◽  
Author(s):  
Paul Nioi ◽  
Truyen Nguyen ◽  
Philip J. Sherratt ◽  
Cecil B. Pickett
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Redox Homeostasis ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Transactivation Domain ◽  
Endogenous Gene ◽  
Nrf2 Target Gene

ABSTRACT Nrf2 is a transcription factor critical for the maintenance of cellular redox homeostasis. We have previously found that Nrf2 is a labile protein, and its activation in cells under stress involves mechanisms leading to its stabilization. As a modular protein, Nrf2 possesses distinct transactivation and DNA binding domains essential for its transcriptional activity. In this study, we found that the C-terminal “Neh3” domain of Nrf2 is also important for its activity. Deletion of the last 16 amino acids of the protein completely abolishes its ability to activate both reporter and endogenous gene expression. Using site-directed mutagenesis, we have identified a stretch of amino acids within this region that are essential for its activity and that are found to be conserved across species and among other members of the CNC-bZIP family. Importantly, deletion of the final 16 amino acids of Nrf2 does not influence its dimerizing capability, DNA binding activity, or subcellular localization, although it does increase the half-life of the protein. In addition, this region was found to be important for interaction with CHD6 (a chromo-ATPase/helicase DNA binding protein) in a yeast two-hybrid screen. RNA interference-mediated knockdown of CHD6 reduced both the basal and tert-butylhydroquinone-inducible expression of NQO1, a prototypical Nrf2 target gene. These data suggest that the Neh3 domain may act as a transactivation domain and that it is possibly involved in interaction with components of the transcriptional apparatus to affect its transcriptional activity.

scholarly journals Interaction of Bovine Papillomavirus E2 Protein with Brd4 Stabilizes Its Association with Chromatin

2005 ◽  
Vol 79 (14) ◽  
pp. 8920-8932 ◽  
Author(s):  
Maria G. McPhillips ◽  
Keiko Ozato ◽  
Alison A. McBride
Keyword(s):  
Cell Types ◽  
Bovine Papillomavirus ◽  
Chromosomal Protein ◽  
Transactivation Domain ◽  
Mitotic Chromosomes ◽  
E2 Protein ◽  
Viral Genomes ◽  
Protein Protein Interaction ◽  
Binding Function ◽  
Interphase Cells

ABSTRACT The bovine papillomavirus E2 protein maintains and segregates the viral extrachromosomal genomes by tethering them to cellular mitotic chromosomes. E2 interacts with a cellular bromodomain protein, Brd4, to mediate the segregation of viral genomes into daughter cells. Brd4 binds acetylated histones and has been observed to diffusely coat mitotic chromosomes in several cell types. In this study, we show that in mitotic C127 cells, Brd4 diffusely coated the condensed chromosomes. However, in the presence of the E2 protein, E2 and Brd4 colocalized in punctate dots that were randomly distributed over the chromosomes. A similar pattern of E2 and Brd4 colocalization on mitotic chromosomes was observed in CV-1 cells, whereas only a faint chromosomal coating of Brd4 was detected in the absence of the E2 protein. Therefore, the viral E2 protein relocalizes and/or stabilizes the association of Brd4 with chromosomes in mitotic cells. The colocalization of E2 and Brd4 was also observed in interphase cells, indicating that this protein-protein interaction persists throughout the cell cycle. The interaction of E2 with Brd4 greatly stabilized the association of Brd4 with interphase chromatin. In both mitotic and interphase cells, this stabilization required a transcriptionally competent transactivation domain, but not the DNA binding function of the E2 protein. Thus, the E2 protein modulates the chromatin association of Brd4 during both interphase and mitosis. This study demonstrates that the segregation of papillomavirus genomes is not simply due to the passive hitchhiking of the E2/genome complex with a convenient cellular chromosomal protein.

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