scholarly journals MDM2-Driven Ubiquitination Rapidly Removes p53 from Its Cognate Promoters

Biomolecules ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 22
Author(s):  
Kester Mo Henningsen ◽  
Valentina Manzini ◽  
Anna Magerhans ◽  
Sabrina Gerber ◽  
Matthias Dobbelstein
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Culture Medium ◽  
Target Genes ◽  
Breast Adenocarcinoma ◽  
Transactivation Domain ◽  
Chromatin Binding ◽  
Tumor Suppressor P53 ◽  
Mdm2 Protein ◽  
Chromatin Interactions ◽  
Target Promoters

MDM2 is the principal antagonist of the tumor suppressor p53. p53 binds to its cognate DNA element within promoters and activates the transcription of adjacent genes. These target genes include MDM2. Upon induction by p53, the MDM2 protein binds and ubiquitinates p53, triggering its proteasomal degradation and providing negative feedback. This raises the question whether MDM2 can also remove p53 from its target promoters, and whether this also involves ubiquitination. In the present paper, we employ the MDM2-targeted small molecule Nutlin-3a (Nutlin) to disrupt the interaction of MDM2 and p53 in three different cancer cell lines: SJSA-1 (osteosarcoma), 93T449 (liposarcoma; both carrying amplified MDM2), and MCF7 (breast adenocarcinoma). Remarkably, removing Nutlin from the culture medium for less than five minutes not only triggered p53 ubiquitination, but also dissociated most p53 from its chromatin binding sites, as revealed by chromatin immunoprecipitation. This also resulted in reduced p53-responsive transcription, and it occurred much earlier than the degradation of p53 by the proteasome, arguing that MDM2 removes p53 from promoters prior to and thus independent of degradation. Accordingly, the short-term pharmacological inhibition of the proteasome did not alter the removal of p53 from promoters by Nutlin washout. However, when the proteasome inhibitor was applied for several hours, depleting non-conjugated ubiquitin prior to eliminating Nutlin, this compromised the removal of DNA-bound p53, as did an E1 ubiquitin ligase inhibitor. This suggests that the ubiquitination of p53 by MDM2 is necessary for its clearance from promoters. Depleting the MDM2 cofactor MDM4 in SJSA cells did not alter the velocity by that p53 was removed from promoters upon Nutlin washout. We conclude that MDM2 antagonizes p53 not only by covering its transactivation domain and by destabilization, but also by the rapid, ubiquitin-dependent termination of p53–chromatin interactions.

Accumulation of soluble and nucleolar-associated p53 proteins following cellular stress

2001 ◽  
Vol 114 (10) ◽  
pp. 1867-1873 ◽  
Author(s):  
S.A. Klibanov ◽  
H.M. O'Hagan ◽  
M. Ljungman
Keyword(s):  
Target Genes ◽  
Uv Light ◽  
Human Fibroblasts ◽  
Proteasome Inhibition ◽  
Tumor Suppressor P53 ◽  
Mdm2 Protein ◽  
Transcription Inhibitor ◽  
Cellular Stresses ◽  
In Cells

The tumor suppressor p53 is a nucleocytoplasmic shuttling protein that accumulates in the nucleus of cells exposed to various cellular stresses. One important role of nuclear p53 is to mobilize a stress response by transactivating target genes such as the p21(Waf1) gene. In this study, we investigated more closely the localization of p53 in cells following various stresses. Immunocytochemistry of fixed human fibroblasts treated with either UV light, the kinase and transcription inhibitor DRB or the proteasome inhibitor MG132 revealed abundant p53 localized to the nucleus. When cells treated with UV or DRB were permeabilized prior to fixation to allow soluble proteins to diffuse, the nuclear p53 signal was abolished. However, in cells treated with MG132, residual p53 localized to distinct large foci. Furthermore, nucleolin co-localized with p53 to these foci, suggesting that these foci were nucleolar structures. Interestingly, the MDM2 protein was found to co-localize with p53 to nucleolar structures following proteasome inhibition. Our results suggest that the p53 proteins accumulating in the nucleus following UV-irradiation or blockage of transcription are freely soluble and, thus, should be able to roam the nucleus to ensure high occupancy of p53 binding sites. However, inhibition of proteasome activity may be a unique stress in that it leads to the sequestering of p53 proteins to the nucleolus, thereby blunting the p53-mediated transactivation of target genes.

scholarly journals Bag1-L Is a Phosphorylation-Dependent Coactivator of c-Jun during Neuronal Apoptosis

2010 ◽  
Vol 30 (15) ◽  
pp. 3842-3852 ◽  
Author(s):  
Clive R. Da Costa ◽  
Javier Villadiego ◽  
Rocio Sancho ◽  
Xavier Fontana ◽  
Graham Packham ◽  
...  
Keyword(s):  
Cell Death ◽  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Neuronal Apoptosis ◽  
Target Genes ◽  
Critical Role ◽  
Transactivation Domain ◽  
Multifunctional Protein ◽  
System Cell ◽  
Factor C

ABSTRACT In the nervous system, cell death by apoptosis plays a critical role during normal development and pathological neurodegeneration. Jun N-terminal kinases (JNKs) are essential regulators of neuronal apoptosis. The AP-1 transcription factor c-Jun is phosphorylated at multiple sites within its transactivation domain by the JNKs, and c-Jun phosphorylation is required for JNK-induced neurotoxicity. While the importance of c-Jun as a mediator of apoptotic JNK signaling in neurons is firmly established, the molecular mechanism underlying the requirement for c-Jun N-terminal phosphorylation is enigmatic. Here we identify the multifunctional protein Bag1-L as a coactivator of phosphorylated c-Jun. Bag1-L preferentially interacts with N-terminally phosphorylated c-Jun, and Bag1-L greatly augments transcriptional activation by phosphorylated c-Jun. Chromatin immunoprecipitation experiments revealed binding of Bag1-L to the promoters of proapoptotic AP-1 target genes, and overexpression of Bag1-L augmented cell death in primary neurons. Therefore, Bag1-L functions as a coactivator regulating neurotoxicity mediated by phosphorylated c-Jun.

scholarly journals Androgen receptor enhancer usage and the chromatin regulatory landscape in human prostate cancers

2019 ◽  
Vol 26 (5) ◽  
pp. R267-R285 ◽  
Author(s):  
Suzan Stelloo ◽  
Andries M Bergman ◽  
Wilbert Zwart
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Target Genes ◽  
Binding Capacity ◽  
Association Studies ◽  
Clinical Samples ◽  
Gene Promoters ◽  
Chromatin Binding ◽  
Chromatin Interactions ◽  
Genome Wide

The androgen receptor (AR) is commonly known as a key transcription factor in prostate cancer development, progression and therapy resistance. Genome-wide chromatin association studies revealed that transcriptional regulation by AR mainly depends on binding to distal regulatory enhancer elements that control gene expression through chromatin looping to gene promoters. Changes in the chromatin epigenetic landscape and DNA sequence can locally alter AR-DNA-binding capacity and consequently impact transcriptional output and disease outcome. The vast majority of reports describing AR chromatin interactions have been limited to cell lines, identifying numerous other factors and interacting transcription factors that impact AR chromatin interactions. Do these factors also impact AR cistromics – the genome-wide chromatin-binding landscape of AR – in vivo? Recent technological advances now enable researchers to identify AR chromatin-binding sites and their target genes in human specimens. In this review, we provide an overview of the different factors that influence AR chromatin binding in prostate cancer specimens, which is complemented with knowledge from cell line studies. Finally, we discuss novel perspectives on studying AR cistromics in clinical samples.

scholarly journals Caspase cleavage of Ets-1 p51 generates fragments with transcriptional dominant-negative function

2010 ◽  
Vol 426 (2) ◽  
pp. 229-241 ◽  
Author(s):  
Souhaila Choul-Li ◽  
Catherine Leroy ◽  
Gabriel Leprivier ◽  
Clélia Laitem ◽  
David Tulasne ◽  
...  
Keyword(s):  
Target Genes ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Transactivation Domain ◽  
Caspase Cleavage ◽  
Terminal Fragment ◽  
Dominant Negative Form ◽  
Target Promoters ◽  
Spliced Variant

Ets-1 is a transcription factor that plays an important role in various physiological and pathological processes, such as development, angiogenesis, apoptosis and tumour invasion. In the present study, we have demonstrated that Ets-1 p51, but not the spliced variant Ets-1 p42, is processed in a caspase-dependent manner in Jurkat T-leukaemia cells undergoing apoptosis, resulting in three C-terminal fragments Cp20, Cp17 and Cp14 and a N-terminal fragment, Np36. In vitro cleavage of Ets-1 p51 by caspase 3 produces fragments consistent with those observed in cells undergoing apoptosis. These fragments are generated by cleavage at three sites located in the exon VII-encoded region of Ets-1 p51. This region is absent from the Ets-1 p42 isoform, which therefore cannot be cleaved by caspases. In Ets-1 p51, cleavage generates C-terminal fragments containing the DNA-binding domain, but lacking the transactivation domain. The Cp17 fragment, the major cleavage product generated during apoptosis, is devoid of transcriptional activity and inhibits Ets-1 p51-mediated transactivation of target genes by competing with Ets-1 p51 for binding to Ets-binding sites present in the target promoters. In the present study, we have demonstrated that caspase cleavage of Ets-1 within the exon VII-encoded region leads to specific down-regulation of the Ets-1 p51 isoform during apoptosis. Furthermore, our results establish that caspase cleavage generates a stable C-terminal fragment that acts as a natural dominant-negative form of the full-length Ets-1 p51 protein.

scholarly journals PARP7 and Mono-ADP-Ribosylation Negatively Regulate Estrogen Receptor α Signaling in Human Breast Cancer Cells

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 623
Author(s):  
Marit Rasmussen ◽  
Susanna Tan ◽  
Venkata S. Somisetty ◽  
David Hutin ◽  
Ninni Elise Olafsen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Protein Modification ◽  
Target Genes ◽  
Estrogen Receptor Α ◽  
Transactivation Domain ◽  
Adp Ribosylation ◽  
Protein Levels ◽  
17Β Estradiol ◽  
Mcf 7

ADP-ribosylation is a post-translational protein modification catalyzed by a family of proteins known as poly-ADP-ribose polymerases. PARP7 (TIPARP; ARTD14) is a mono-ADP-ribosyltransferase involved in several cellular processes, including responses to hypoxia, innate immunity and regulation of nuclear receptors. Since previous studies suggested that PARP7 was regulated by 17β-estradiol, we investigated whether PARP7 regulates estrogen receptor α signaling. We confirmed the 17β-estradiol-dependent increases of PARP7 mRNA and protein levels in MCF-7 cells, and observed recruitment of estrogen receptor α to the promoter of PARP7. Overexpression of PARP7 decreased ligand-dependent estrogen receptor α signaling, while treatment of PARP7 knockout MCF-7 cells with 17β-estradiol resulted in increased expression of and recruitment to estrogen receptor α target genes, in addition to increased proliferation. Co-immunoprecipitation assays revealed that PARP7 mono-ADP-ribosylated estrogen receptor α, and mass spectrometry mapped the modified peptides to the receptor’s ligand-independent transactivation domain. Co-immunoprecipitation with truncated estrogen receptor α variants identified that the hinge region of the receptor is required for PARP7-dependent mono-ADP-ribosylation. These results imply that PARP7-mediated mono-ADP-ribosylation may play an important role in estrogen receptor positive breast cancer.

scholarly journals Direct Examination of Histone Acetylation on Myc Target Genes Using Chromatin Immunoprecipitation

2000 ◽  
Vol 275 (43) ◽  
pp. 33798-33805 ◽  
Author(s):  
Scott R. Eberhardy ◽  
Caroline A. D'Cunha ◽  
Peggy J. Farnham
Keyword(s):  
Histone Acetylation ◽  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Direct Examination

scholarly journals Structure of tumor suppressor p53 and its intrinsically disordered N-terminal transactivation domain

2008 ◽  
Vol 105 (15) ◽  
pp. 5762-5767 ◽  
Author(s):  
M. Wells ◽  
H. Tidow ◽  
T. J. Rutherford ◽  
P. Markwick ◽  
M. R. Jensen ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Transactivation Domain ◽  
Tumor Suppressor P53 ◽  
Intrinsically Disordered

Transcriptional transactivation functions localized to the glucocorticoid receptor N terminus are necessary for steroid induction of lymphocyte apoptosis

1992 ◽  
Vol 12 (2) ◽  
pp. 589-597
Author(s):  
E S Dieken ◽  
R L Miesfeld
Keyword(s):  
Transcriptional Regulation ◽  
Glucocorticoid Receptor ◽  
Target Genes ◽  
Dominant Negative ◽  
Receptor Expression ◽  
Transactivation Domain ◽  
Lymphocyte Apoptosis ◽  
Murine Cell Line ◽  
N Terminus ◽  
Simplex Virus

Genetic studies have suggested that transcriptional regulation of specific target genes (by either induction or repression) is the molecular basis of glucocorticoid-mediated lymphocyte apoptosis. To examine the role of transcriptional regulation more directly, we developed a complementation assay utilizing stable transfection of wild-type (wt) and mutant (nti) glucocorticoid receptor (GR) cDNA constructs into a GR-deficient S49 murine cell line (7r). Our data confirm that the level of functional GR is rate limiting for S49 apoptosis and moreover that the GR amino terminus (N terminus), which as been deleted from the nti GR, is absolutely required for complementation in this system. Surprisingly, we found that at physiological levels of receptor, expression of the nti GR in cells containing wt GR results in enhanced dexamethasone sensitivity rather than a dominant negative phenotype. One interpretation of these data is that DNA binding by wt-nti heterodimers may be functionally similar to that of wt-wt homodimers, indicating that GRE occupancy by at least one transactivation domain may be sufficient to induce the hormonal response. To determine whether acidic activating sequences such as those localized to the GR N terminus are important in the induction of lymphocyte apoptosis, we tested the activity of a chimeric receptor in which we replaced the entire GR N terminus with sequences from the herpes simplex virus VP16 protein. Our results demonstrate that 7r cells expressing VP-GR fusions are indeed steroid sensitive, strongly supporting the idea that S49 apoptosis is dependent on transcriptional regulation of specific genes which respond to acidic activating domains, implying that induction, rather than repression, may be the critical initiating event.

Suppression of the rbf null mutants by a de2f1 allele that lacks transactivation domain

Development ◽  
2000 ◽  
Vol 127 (2) ◽  
pp. 367-379 ◽  
Author(s):  
W. Du
Keyword(s):  
Cell Proliferation ◽  
Target Genes ◽  
Suppressor Gene ◽  
Genetic Interactions ◽  
Transactivation Domain ◽  
Transcription Activator ◽  
Drosophila Development ◽  
Mitotic Wave ◽  
E2f Transcription Factors ◽  
Null Mutants

In mammals, a large number of proteins including E2F transcription factors have been shown to interact with the tumor suppressor gene product pRB, but it is not clear to what extend the function of pRB is mediated by E2F. In addition, E2F was shown to mediate both transcription activation and repression; it remains to be tested which function of E2F is critical for normal development. Drosophila homologs of the RB and E2F family of proteins RBF and dE2F1 have been identified. The genetic interactions between rbf and de2f1 were analyzed during Drosophila development, and the results presented here showed that RBF is required at multiple stages of development. Unexpectedly, rbf null mutants can develop until late pupae stage when the activity of dE2F1 is reduced, and can develop into viable adults with normal adult appendages in the presence of a de2f1 mutation that retains the DNA binding domain but lacks the transactivation domain. These results indicate that most, if not all, of the function of RBF during development is mediated through E2F. In turn, the genetic interactions shown here also suggest that dE2F1 functions primarily as a transcription activator rather than a co-repressor of RBF during Drosophila development. Analysis of the expression of an E2F target gene PCNA in eye discs showed that the expression of PCNA is activated by dE2F1 in the second mitotic wave and repressed in the morphogenetic furrow and posterior to the second mitotic wave by RBF. Interestingly, reducing the level of RBF restored the normal pattern of cell proliferation in de2f1 mutant eye discs but not the expression of E2F target genes, suggesting that the coordinated transcription of E2F target genes does not significantly affect the pattern of cell proliferation.

scholarly journals Differential Regulation of Estrogen-Inducible Proteolysis and Transcription by the Estrogen Receptor α N Terminus

2005 ◽  
Vol 25 (13) ◽  
pp. 5417-5428 ◽  
Author(s):  
Christopher C. Valley ◽  
Raphaël Métivier ◽  
Natalia M. Solodin ◽  
Amy M. Fowler ◽  
Mara T. Mashek ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Transcriptional Activity ◽  
Target Genes ◽  
Estrogen Receptor Α ◽  
Transactivation Domain ◽  
Related Factors ◽  
Additional Element ◽  
Transactivation Domains ◽  
Ubiquitin Proteasome ◽  
Transcriptional Complex

ABSTRACT The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor α (ERα) is also subjected to rapid ubiquitin-proteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ERα proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERα and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERα. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERα are mechanistically separable functions of ERα. We find that proteolysis of ERα correlates with the ability of ERα mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligand-induced receptor degradation which permits the uncoupling of estrogen-regulated ERα proteolysis and transcription.

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