Brd4 Regulation of Papillomavirus Protein E2 Stability

ABSTRACT The papillomavirus (PV) E2 protein is an important regulator of the viral life cycle. It has diverse roles in viral transcription, DNA replication, and genome maintenance. Our laboratory has previously identified the cellular bromodomain protein Brd4 as a key interacting partner of E2. Brd4 mediates the transcriptional activation function of E2 and plays an important role in viral genome maintenance in dividing cells. E2 interacts with the C-terminal domain (CTD) of Brd4, and the CTD functions in a dominant-negative manner through binding E2 and interfering with E2's interaction with the full-length Brd4 protein. Previous studies have shown that PV E2 proteins are short lived; however, the mechanisms regulating their stability and degradation have not yet been well established. In this study, we explored the role of Brd4 in the regulation of bovine PV 1 (BPV1) and human PV 16 (HPV16) E2 stability. Expression of the Brd4 CTD dramatically increases E2 levels. Both BPV1 E2 and HPV16 E2 are regulated by ubiquitylation, and Brd4 CTD expression blocks this ubiquitylation, thus stabilizing the E2 protein. Furthermore, we have identified the cullin-based E3 ligases and specifically cullin-3 as potential components of the ubiquitylation machinery that targets both BPV1 and HPV16 E2 for ubiquitylation. Expression of the Brd4 CTD blocks the interaction between E2 and the cullin-3 complex. In addition to Brd4's role in mediating E2 transcription and genome tethering activities, these data suggest a potential role for Brd4 in regulating E2 stability and protein levels within PV-infected cells.

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CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation

The Journal of Cell Biology ◽

10.1083/jcb.200711044 ◽

2008 ◽

Vol 181 (6) ◽

pp. 959-972 ◽

Cited By ~ 80

Author(s):

Xueni Li ◽

Mei Huang ◽

Huiling Zheng ◽

Yinyin Wang ◽

Fangli Ren ◽

...

Keyword(s):

Transcriptional Activation ◽

Osteoblast Differentiation ◽

Degradation Mechanism ◽

Interacting Protein ◽

E3 Ligases ◽

Protein Levels ◽

C Terminus ◽

Osteoblast Lineage

Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.

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The Naturally Occurring Variant of Estrogen Receptor (ER) ERΔE7 Suppresses Estrogen-Dependent Transcriptional Activation by Both Wild-Type ERα and ERβ

Endocrinology ◽

10.1210/en.2002-0027 ◽

2003 ◽

Vol 144 (7) ◽

pp. 2967-2976 ◽

Cited By ~ 41

Author(s):

Juana M. García Pedrero ◽

Pedro Zuazua ◽

Carlos Martínez-Campa ◽

Pedro S. Lazo ◽

Sofía Ramos

Keyword(s):

Estrogen Receptor ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Activation Function ◽

Inhibitory Effect ◽

Dominant Negative ◽

Wild Type ◽

Independent Manner ◽

Naturally Occurring ◽

Estrogen Response

Abstract We have isolated and functionally characterized the exon 7-skipped variant (ERΔE7) of estrogen receptor (ER)α, which has emerged as the predominant variant expressed in multiple normal and tumoral tissues. However, to date no function has been established for this variant in mammalian cells. ERΔE7 exhibits a negligible ability to bind ligands, insensitivity to allosteric modulation by estrogen and antiestrogens, and loss of estrogen-dependent interaction with p160 coactivators such as SRC-1 and AIB1. ERΔE7 is able to form heterodimers with both ERα and ERβ in a ligand-independent manner. Transient expression experiments in HeLa cells show that increasing amounts of ERΔE7 result in a progressive inhibition of the estrogen-dependent transcriptional activation by both wild-type ERα and ERβ on estrogen response element-driven promoters. The inhibitory effect of ERΔE7 is due to the inhibition of binding of wild-type receptors to their responsive elements. Surprisingly, the activation function (AF)-1-dependent transactivation triggered by epithelial growth factor and phorbol-12-myristate-13-acetate is also abolished in ERΔE7 despite AF1 integrity, suggesting a cross-talk between AF1 and AF2 regions of the receptor. These results indicate that the naturally occurring variant ERΔE7 is a dominant negative receptor that, when expressed at high levels relative to wild-type ERs, might have profound effects on several estrogen-dependent functions.

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Bromodomain Protein 4 Mediates the Papillomavirus E2 Transcriptional Activation Function

Journal of Virology ◽

10.1128/jvi.80.9.4276-4285.2006 ◽

2006 ◽

Vol 80 (9) ◽

pp. 4276-4285 ◽

Cited By ~ 89

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.

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E1 Protein of Bovine Papillomavirus Type 1 Interferes with E2 Protein-Mediated Tethering of the Viral DNA to Mitotic Chromosomes

Journal of Virology ◽

10.1128/jvi.76.7.3440-3451.2002 ◽

2002 ◽

Vol 76 (7) ◽

pp. 3440-3451 ◽

Cited By ~ 22

Author(s):

Christian Voitenleitner ◽

Michael Botchan

Keyword(s):

Negative Regulator ◽

Bovine Papillomavirus ◽

Wild Type ◽

Mitotic Chromosomes ◽

Viral Dna ◽

Nuclear Retention ◽

E2 Protein ◽

Protein Levels ◽

E1 Protein ◽

Dividing Cells

ABSTRACT Eukaryotic viruses can maintain latency in dividing cells as extrachromosomal plasmids. It is therefore of vital importance for viruses to ensure nuclear retention and proper segregation of their viral DNA. The bovine papillomavirus (BPV) E2 enhancer protein plays a key role in these processes by tethering the viral DNA to the host cell chromosomes. Viral genomes that harbor phosphorylation mutations in the E2 gene are transformation defective, and for these mutant genomes, neither the viral DNA nor the E2 protein is detected on mitotic chromosomes, while other key functions of E2 in transcription and replication were wild type. Moreover, secondary mutations in both the E2 and E1 proteins lead to suppression of the phosphorylation mutant phenotype and resulted in reattachment of the viral DNA and the E2 protein onto mitotic chromosomes, suggesting that E1 also plays a role in viral genome partitioning. The E1 protein was cytologically always excluded from mitotic chromatin, either as a suppressor allele or as the wild type. In the absence of other viral proteins, an E2 protein containing alanine substitutions for phosphorylation substrates in the hinge region (E2-A4) was detected as wild-type on mitotic chromosomes. However, when wild-type E1 protein levels were increased in cells expressing either the A4 mutant E2 proteins or wild-type E2, the E2-A4 protein was much more sensitive to chromosomal dislocation than was the wild-type protein. In contrast, suppressor alleles of E1 were not capable of such abrogation of E2 binding (A4 or wild-type) to chromosomes. These results suggest that wild-type E1 can be a negative regulator of the chromosomal attachment of E2.

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Direct control of caveolin-1 expression by FOXO transcription factors

Biochemical Journal ◽

10.1042/bj20041449 ◽

2005 ◽

Vol 385 (3) ◽

pp. 795-802 ◽

Cited By ~ 43

Author(s):

A. Pieter J. van den HEUVEL ◽

Almut SCHULZE ◽

Boudewijn M. T. BURGERING

Keyword(s):

Transcription Factors ◽

Transcriptional Activation ◽

Direct Interaction ◽

Dominant Negative ◽

Promoter Sequences ◽

Caveolin 1 ◽

Cellular Effects ◽

Protein Levels ◽

Epidermal Growth ◽

Foxo Transcription Factors

Protein kinase B can phoshorylate and thereby inactivate the FOXO (forkhead box O) family of transcription factors. When active, FOXO factors can bind to DNA in promoter sequences and subsequently regulate gene expression. We have used DNA microarray analysis to identify potential gene targets of FOXO. In the present study we demonstrate that caveolin-1 is directly controlled by FOXO. Firstly, caveolin-1 expression was increased upon induction or over-expression of FOXO factors at both mRNA and protein levels. Second, we show that endogenous regulation of FOXO activity regulates caveolin-1 levels and that this can be inhibited by dominant-negative FOXO. Third, FOXO activates transcription from the caveolin-1 promoter, and using chromatin immunoprecipitations we demonstrated that this activation occurs via direct interaction of FOXO with the promoter. Finally, we demonstrate FOXO-mediated attenuation of EGF (epidermal growth factor)-induced signalling, which in part is mediated by caveolin-1 expression, as suggested by previous studies [Park, Park, Cho, Kim, Ko, Seo and Park (2000) J. Biol. Chem. 275, 20847–20852]. These findings suggest a novel mechanism by which FOXO factors can exert their cellular effects via transcriptional activation of caveolin-1.

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Signal Transducer and Activator of Transcription 5 Activation Is Sufficient to Drive Transcriptional Induction of Cyclin D2 Gene and Proliferation of Rat Pancreatic β-Cells

Molecular Endocrinology ◽

10.1210/me.2002-0356 ◽

2003 ◽

Vol 17 (5) ◽

pp. 945-958 ◽

Cited By ~ 69

Author(s):

Birgitte N. Friedrichsen ◽

Henrijette E. Richter ◽

Johnny A. Hansen ◽

Christopher J. Rhodes ◽

Jens H. Nielsen ◽

...

Keyword(s):

Transcriptional Activation ◽

Fold Increase ◽

Dominant Negative ◽

Mrna Levels ◽

Dependent Manner ◽

Signal Transducer ◽

Β Cells ◽

Pancreatic Β Cells ◽

Cyclin D2 ◽

Protein Levels

Abstract Signal transducer and activator of transcription 5 (STAT5) activation plays a central role in GH- and prolactin-mediated signal transduction in the pancreatic β-cells. In previous experiments we demonstrated that STAT5 activation is necessary for human (h)GH-stimulated proliferation of INS-1 cells and hGH-induced increase of mRNA-levels of the cell cycle regulator cyclin D2. In this study we have further characterized the role of STAT5 in the regulation of cyclin D expression and β-cell proliferation by hGH. Cyclin D2 mRNA and protein levels (but not cyclin D1 and D3) were induced in a time-dependent manner by hGH in INS-1 cells. Inhibition of protein synthesis by coincubation with cycloheximide did not affect the hGH-induced increase of cyclin D2 mRNA levels at 4 h. Expression of a dominant negative STAT5 mutant, STAT5aΔ749, partially inhibited cyclin D2 protein levels. INS-1 cells transiently transfected with a cyclin D2 promoter-reporter construct revealed a 3- to 5-fold increase of transcriptional activity in response to hGH stimulation. Furthermore, coexpression of a constitutive active STAT5 mutant (either CA-STAT5a or CA-STAT5b) was sufficient to drive transactivation of the promoter. CA-STAT5b was stably expressed in INS-1 cells under the control of a doxycycline-inducible promoter. Gel retardation experiments using a probe representing a putative STAT5 binding site in the cyclin D2 promoter revealed binding of the doxycycline-induced CA-STAT5b. Furthermore, induction of CA-STAT5b stimulated transcriptional activation of the cyclin D2 promoter and induced hGH-independent proliferation in these cells. In primary β-cells, adenovirus-mediated expression of CA-STAT5b profoundly stimulated DNA-synthesis (5.3-fold over control) in the absence of hGH. Our studies indicate that STAT5 activation is sufficient to drive proliferation of the β-cells and that cyclin D2 may be a critical target gene for STAT5 in this process.

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Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

Journal of Virology ◽

10.1128/jvi.02426-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 523-534 ◽

Cited By ~ 35

Author(s):

Mayuri Sharma ◽

Brian J. Bender ◽

Jeremy P. Kamil ◽

Ming F. Lye ◽

Jean M. Pesola ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Continuous Distribution ◽

Virus Production ◽

Nuclear Lamina ◽

Dominant Negative ◽

Lamin A ◽

Nuclear Egress ◽

Dividing Cells ◽

Infected Cells

ABSTRACTHerpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with aUL97mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC.IMPORTANCEHuman cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. In validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and we obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses.

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Role of Metazoan Mediator Proteins in Interferon-Responsive Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.23.2.620-628.2003 ◽

2003 ◽

Vol 23 (2) ◽

pp. 620-628 ◽

Cited By ~ 56

Author(s):

Joe F. Lau ◽

Inna Nusinzon ◽

Darya Burakov ◽

Leonard P. Freedman ◽

Curt M. Horvath

Keyword(s):

Transcriptional Activation ◽

Transcriptional Activity ◽

Activation Function ◽

Dominant Negative ◽

Gene Promoters ◽

A Subunit ◽

Induced Signal ◽

Necessary And Sufficient ◽

Coactivator Complex

ABSTRACT The interferon (IFN)-induced signal transduction and transcription activation complex, ISGF3, is assembled from three proteins, STAT1, STAT2, and IRF9. Of these components, STAT2 provides a fundamental and essential transcriptional activation function for ISGF3. In the present study, we show that ISGF3-mediated transcription is dependent on STAT2 interactions with DRIP150, a subunit of the multimeric Mediator coactivator complex. Other Mediator subunits, DRIP77 and DRIP130, were found either to bind STAT2 without augmenting ISGF3 transcriptional activity or to enhance ISGF3 transcription without binding STAT2, but only DRIP150 both enhanced IFN-dependent transcription and coimmunoprecipitated with STAT2. Endogenous DRIP150 and STAT2 were able to interact in solution, and DNA affinity chromatography and chromatin immunoprecipitation assays demonstrated that DRIP150 binds to the mature, activated ISGF3-DNA complex and is recruited to target gene promoters in an IFN-dependent fashion. IFN-dependent recruitment of DRIP130 to an ISGF3 target promoter and SRB10-STAT2 coprecipitation suggest indirect association with a multisubunit Mediator complex. The site of STAT2 interaction was mapped to DRIP150 residues 188 to 566, which are necessary and sufficient for interaction with STAT2. Expression of this DRIP150 fragment, but not DRIP150 fragments outside the STAT2 interaction region, suppressed ISGF3-mediated transcriptional activity in a dominant-negative fashion, suggesting a direct functional role of this domain in mediating STAT2-DRIP150 interactions. These findings indicate that the IFN-activated ISGF3 transcription factor regulates transcription through contact with DRIP150 and implicate the Mediator coactivator complex in IFN-activated gene regulation.

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Abstract 065: Expression of a Dominant Negative Cullin 3 Mutant (Cul3[[Unable to Display Character: ∆]]9) Impairs Endogenous Cullin 3 Activity and Causes Impaired Vasorelaxation of Aorta

Hypertension ◽

10.1161/hyp.66.suppl_1.065 ◽

2015 ◽

Vol 66 (suppl_1) ◽

Author(s):

Larry N Agbor ◽

Stella-Rita C Ibeawuchi ◽

Frederick W Quelle ◽

Curt D Sigmund

Keyword(s):

Transgenic Mice ◽

Genome Editing ◽

Dominant Negative ◽

Nitric Oxide Donor ◽

Chromosome 2 ◽

Loss Of Function ◽

Protein Levels ◽

Cullin 3 ◽

Rhoa Protein ◽

Exon 9

Patients with dominant loss-of-function mutations in Cullin 3 (Cul3) resulting in deletion of exon 9 (Cul3[[Unable to Display Character: ∆]]9) exhibit severe early onset hypertension correlated with reduced degradation of Cul3 substrates. We have shown that knockout of Cul3 induced by clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 in HEK293T cells resulted in accumulation of the Cul3 substrate RhoA. To gain insight into the mechanism of attenuated Cul3[[Unable to Display Character: ∆]]9-mediated protein degradation, we utilized CRISPR-Cas9 genome editing to generate Cul3[[Unable to Display Character: ∆]]9-expressing cell lines. We hypothesized that expression of the Cul3[[Unable to Display Character: ∆]]9 protein will impair endogenous Cul3 wildtype (WT) function and accumulate Cul3 substrates. CRISPR guide RNAs targeting endogenous Cul3 were cloned into a plasmid expressing humanized Cas9. HEK293T cells were transfected and single cell clones selected. Sequencing revealed that genome editing induced a 548 bp deletion spanning exon 9 (Chromosome 2:224503433-224503979). Sequencing of the mRNA transcript also revealed that exon 9 was deleted, and Cul3[[Unable to Display Character: ∆]]9 mRNA and protein was expressed. Although RhoA protein levels were normal, Cul3 substrates WNK4 and PLK1 were significantly increased in Cul3[[Unable to Display Character: ∆]]9-expressing cells (WNK4: 1.5±0.12 Cul3[[Unable to Display Character: ∆]]9 vs 1.0±0.02 WT; PLK1: 1.4±0.06 Cul3[[Unable to Display Character: ∆]]9 vs 1.0±0.03 WT, P<0.001). Interestingly, Cul3[[Unable to Display Character: ∆]]9-expressing cells exhibited impaired Cul3 ubiquitin ligase activity compared to WT. We also generated a novel transgenic mouse model inducibly expressing Cul3[[Unable to Display Character: ∆]]9 protein specifically in smooth muscle (termed S-Cul3[[Unable to Display Character: ∆]]9) and assessed vascular responses in the aorta using a wire myograph. Aorta from S-Cul3[[Unable to Display Character: ∆]]9 transgenic mice exhibited impaired ACh relaxation compared to WT (at 30 μM: 55±2% sCul3[[Unable to Display Character: ∆]]9 vs 71±7% WT, p<0.0001). In contrast, vasodilation to the nitric oxide donor, nitroprusside (SNP) was normal, as was constriction to endothelin-1. There was a significant increase in RhoA protein expression in aorta of S-Cul3[[Unable to Display Character: ∆]]9 transgenic mice compared to WT (1.6±0.2 sCul3[[Unable to Display Character: ∆]]9 vs 1.0±0.1 WT, P<0.05). These findings suggest a mechanism whereby Cul3[[Unable to Display Character: ∆]]9 protein may interfere with endogenous Cul3 and impair the degradation of Cul3 substrates such as RhoA or WNK4, contributing, at least in part-, to hypertension.

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Pirh2, an E3 ligase, regulates the AIP4–p73 regulatory pathway by modulating AIP4 expression and ubiquitination

Carcinogenesis ◽

10.1093/carcin/bgab009 ◽

2021 ◽

Author(s):

Rami Abou Zeinab ◽

H Helena Wu ◽

Yasser Abuetabh ◽

Sarah Leng ◽

Consolato Sergi ◽

...

Keyword(s):

Regulatory Protein ◽

Ectopic Expression ◽

E3 Ligase ◽

Regulatory Pathway ◽

Hect Domain ◽

E3 Ligases ◽

Multiple Substrates ◽

Protein Levels ◽

Cycle Arrest ◽

Negative Regulatory Pathway

Abstract Pirh2 is an E3 ligase belonging to the RING-H2 family and shown to bind, ubiquitinate and downregulate p73 tumor suppressor function without altering p73 protein levels. AIP4, an E3 ligase belonging to the HECT domain family, has been reported to be a negative regulatory protein that promotes p73 ubiquitination and degradation. Herein, we found that Pirh2 is a key regulator of AIP4 that inhibits p73 function. Pirh2 physically interacts with AIP4 and significantly downregulates AIP4 expression. This downregulation is shown to involve the ubiquitination of AIP4 by Pirh2. Importantly, we demonstrated that the ectopic expression of Pirh2 inhibits the AIP4–p73 negative regulatory pathway, which was restored when depleting endogenous Pirh2 utilizing Pirh2-siRNAs. We further observed that Pirh2 decreases AIP4-mediated p73 ubiquitination. At the translational level and specifically regarding p73 cell cycle arrest function, Pirh2 still ensures the arrest of p73-mediated G1 despite AIP4 expression. Our study reveals a novel link between two E3 ligases previously thought to be unrelated in regulating the same effector substrate, p73. These findings open a gateway to explain how E3 ligases differentiate between regulating multiple substrates that may belong to the same family of proteins, as it is the case for the p53 and p73 proteins.

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