scholarly journals Activation of p53 Function by Human Transcriptional Coactivator PC4: Role of Protein-Protein Interaction, DNA Bending, and Posttranslational Modifications

2007 ◽  
Vol 27 (21) ◽  
pp. 7603-7614 ◽  
Author(s):  
Kiran Batta ◽  
Tapas K. Kundu
Keyword(s):  
Dna Binding ◽  
Posttranslational Modifications ◽  
Molecular Mechanisms ◽  
Double Helix ◽  
Dna Bending ◽  
Cell Cycle Checkpoints ◽  
Transcriptional Coactivator ◽  
Dna Double Helix

ABSTRACT Tumor suppressor p53 controls cell cycle checkpoints and apoptosis via the transactivation of several genes that are involved in these processes. The functions of p53 are regulated by a wide variety of proteins, which interact with it either directly or indirectly. The multifunctional human transcriptional coactivator PC4 interacts with p53 in vivo and in vitro and regulates its function. Here we report the molecular mechanisms of the PC4-mediated activation of p53 function. PC4 interacts with the DNA binding and C-terminal domains of p53 through its DNA binding domain, which is essential for the stimulation of p53 DNA binding. Remarkably, ligation-mediated circularization assays reveal that PC4 induces significant bending in the DNA double helix. Deletion mutants defective in DNA bending are found to be impaired in activating p53-mediated DNA binding and apoptosis. Furthermore, acetylation of PC4 enhances, while phosphorylation abolishes, its ability to bend DNA, activate p53 DNA binding, and, thereby, regulate p53 functions. In conclusion, PC4 activates p53 recruitment to p53-responsive promoters (Bax and p21) in vivo through its interaction with p53 and by providing bent substrate for p53 recruitment. These results elucidate the general molecular mechanisms of activation of p53 function, mediated by its coactivators.

scholarly journals A Paradoxical Mutant GATA Factor

2004 ◽  
Vol 3 (2) ◽  
pp. 393-405 ◽  
Author(s):  
M. Isabel Muro-Pastor ◽  
Joseph Strauss ◽  
Ana Ramón ◽  
Claudio Scazzocchio
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Double Helix ◽  
Constitutive Expression ◽  
Nitrogen Sources ◽  
Loss Of Function ◽  
Gata Factor ◽  
Dna Double Helix

ABSTRACT The niiA (nitrite reductase) and niaD (nitrate reductase) genes of Aspergillus nidulans are subject to both induction by nitrate and repression by ammonium or glutamine. The intergenic region between these genes functions as a bidirectional promoter. In this region, nucleosomes are positioned under nonexpression conditions. On nitrate induction under derepressing conditions, total loss of positioning occurs. This is independent of transcription and of the NirA-specific transcription factor but absolutely dependent on the wide-domain GATA-binding AreA factor. We show here that a 3-amino-acid deletion in the basic carboxy-terminal sequence of the DNA-binding domain results in a protein with paradoxical properties. Its weak DNA binding is consistent with its loss-of-function phenotype on most nitrogen sources. However, it results in constitutive expression and superinducibility of niiA and niaD. Nucleosome loss of positioning is also constitutive. The mutation partially suppresses null mutations in the transcription factor NirA. AreA binds NirA in vitro, and the mutation does not affect this interaction. The in vivo methylation pattern of the promoter is drastically altered, suggesting the recruitment of one or more unknown transcription factors and/or a local distortion on the DNA double helix.

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1056-1067 ◽  
Author(s):  
Mira T. Kassouf ◽  
Hedia Chagraoui ◽  
Paresh Vyas ◽  
Catherine Porcher
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Experimental Systems ◽  
Dna Binding Activity ◽  
Independent Functions

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

Role of the ligand in intracellular receptor function: receptor affinity determines activation in vitro of the latent dioxin receptor to a DNA-binding form

1991 ◽  
Vol 11 (1) ◽  
pp. 401-411
Author(s):  
S Cuthill ◽  
A Wilhelmsson ◽  
L Poellinger
Keyword(s):  
Dna Binding ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Rank Order ◽  
Receptor Ligands ◽  
Free System ◽  
Dioxin Receptor

To reconstitute the molecular mechanisms underlying the cellular response to soluble receptor ligands, we have exploited a cell-free system that exhibits signal- (dioxin-)induced activation of the latent cytosolic dioxin receptor to an active DNA-binding species. The DNA-binding properties of the in vitro-activated form were qualitatively indistinguishable from those of in vivo-activated nuclear receptor extracted from dioxin-treated cells. In vitro activation of the receptor by dioxin was dose dependent and was mimicked by other dioxin receptor ligands in a manner that followed the rank order of their relative affinities for the receptor in vitro and their relative potencies to induce target gene transcription in vivo. Thus, in addition to triggering the initial release of inhibition of DNA binding and presumably allowing nuclear translocation, the ligand appears to play a crucial role in the direct control of the level of functional activity of a given ligand-receptor complex.

scholarly journals Interaction between p53 N terminus and core domain regulates specific and nonspecific DNA binding

2019 ◽  
Vol 116 (18) ◽  
pp. 8859-8868 ◽  
Author(s):  
Fan He ◽  
Wade Borcherds ◽  
Tanjing Song ◽  
Xi Wei ◽  
Mousumi Das ◽  
...  
Keyword(s):  
Dna Binding ◽  
Posttranslational Modifications ◽  
Dynamic Control ◽  
Transactivation Domains ◽  
Binding Surface ◽  
N Terminus ◽  
Response To Stress ◽  
Stress Signals

The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. Dynamic control of p53 degradation and DNA binding in response to stress signals are critical for tumor suppression. The p53 N terminus (NT) contains two transactivation domains (TAD1 and TAD2), a proline-rich region (PRR), and multiple phosphorylation sites. Previous work revealed the p53 NT reduced DNA binding in vitro. Here, we show that TAD2 and the PRR inhibit DNA binding by directly interacting with the sequence-specific DNA binding domain (DBD). NMR spectroscopy revealed that TAD2 and the PRR interact with the DBD at or near the DNA binding surface, possibly acting as a nucleic acid mimetic to competitively block DNA binding. In vitro and in vivo DNA binding analyses showed that the NT reduced p53 DNA binding affinity but improved the ability of p53 to distinguish between specific and nonspecific sequences. MDMX inhibits p53 binding to specific target promoters but stimulates binding to nonspecific chromatin sites. The results suggest that the p53 NT regulates the affinity and specificity of DNA binding by the DBD. The p53 NT-interacting proteins and posttranslational modifications may regulate DNA binding, partly by modulating the NT–DBD interaction.

scholarly journals Current translational potential and underlying molecular mechanisms of necroptosis

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Tamás Molnár ◽  
Anett Mázló ◽  
Vera Tslaf ◽  
Attila Gábor Szöllősi ◽  
Gabriella Emri ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Posttranslational Modifications ◽  
Molecular Mechanisms ◽  
Inflammatory Diseases ◽  
Immune Surveillance ◽  
Kinase Domain ◽  
Tumor Formation

Abstract Cell death has a fundamental impact on the evolution of degenerative disorders, autoimmune processes, inflammatory diseases, tumor formation and immune surveillance. Over the past couple of decades extensive studies have uncovered novel cell death pathways, which are independent of apoptosis. Among these is necroptosis, a tightly regulated, inflammatory form of cell death. Necroptosis contribute to the pathogenesis of many diseases and in this review, we will focus exclusively on necroptosis in humans. Necroptosis is considered a backup mechanism of apoptosis, but the in vivo appearance of necroptosis indicates that both caspase-mediated and caspase-independent mechanisms control necroptosis. Necroptosis is regulated on multiple levels, from the transcription, to the stability and posttranslational modifications of the necrosome components, to the availability of molecular interaction partners and the localization of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Accordingly, we classified the role of more than seventy molecules in necroptotic signaling based on consistent in vitro or in vivo evidence to understand the molecular background of necroptosis and to find opportunities where regulating the intensity and the modality of cell death could be exploited in clinical interventions. Necroptosis specific inhibitors are under development, but >20 drugs, already used in the treatment of various diseases, have the potential to regulate necroptosis. By listing necroptosis-modulated human diseases and cataloging the currently available drug-repertoire to modify necroptosis intensity, we hope to kick-start approaches with immediate translational potential. We also indicate where necroptosis regulating capacity should be considered in the current applications of these drugs.

scholarly journals Subunit composition determines E2F DNA-binding site specificity.

1997 ◽  
Vol 17 (12) ◽  
pp. 6994-7007 ◽  
Author(s):  
Y Tao ◽  
R F Kassatly ◽  
W D Cress ◽  
J M Horowitz
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Binding Sites ◽  
Dna Bending ◽  
Susceptibility Gene ◽  
Specific Sequence ◽  
Cellular Genes ◽  
Cycle Dependent

The product of the retinoblastoma (Rb) susceptibility gene, Rb-1, regulates the activity of a wide variety of transcription factors, such as E2F, in a cell cycle-dependent fashion. E2F is a heterodimeric transcription factor composed of two subunits each encoded by one of two related gene families, denoted E2F and DP. Five E2F genes, E2F-1 through E2F-5, and two DP genes, DP-1 and DP-2, have been isolated from mammals, and heterodimeric complexes of these proteins are expressed in most, if not all, vertebrate cells. It is not yet clear whether E2F/DP complexes regulate overlapping and/or specific cellular genes. Moreover, little is known about whether Rb regulates all or a subset of E2F-dependent genes. Using recombinant E2F, DP, and Rb proteins prepared in baculovirus-infected cells and a repetitive immunoprecipitation-PCR procedure (CASTing), we have identified consensus DNA-binding sites for E2F-1/DP-1, E2F-1/DP-2, E2F-4/DP-1, and E2F-4/DP-2 complexes as well as an Rb/E2F-1/DP-1 trimeric complex. Our data indicate that (i) E2F, DP, and Rb proteins each influence the selection of E2F-binding sites; (ii) E2F sites differ with respect to their intrinsic DNA-bending properties; (iii) E2F/DP complexes induce distinct degrees of DNA bending; and (iv) complex-specific E2F sites selected in vitro function distinctly as regulators of cell cycle-dependent transcription in vivo. These data indicate that the specific sequence of an E2F site may determine its role in transcriptional regulation and suggest that Rb/E2F complexes may regulate subsets of E2F-dependent cellular genes.

scholarly journals Scanning Mutagenesis of Mcm1: Residues Required for DNA Binding, DNA Bending, and Transcriptional Activation by a MADS-Box Protein

2000 ◽  
Vol 20 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Thomas B. Acton ◽  
Janet Mead ◽  
Andrew M. Steiner ◽  
Andrew K. Vershon
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Essential Gene ◽  
Dna Bending ◽  
Mads Box ◽  
Dna Interactions ◽  
Protein Dna Interactions

ABSTRACT MCM1 is an essential gene in the yeastSaccharomyces cerevisiae and is a member of the MADS-box family of transcriptional regulatory factors. To understand the nature of the protein-DNA interactions of this class of proteins, we have made a series of alanine substitutions in the DNA-binding domain of Mcm1 and examined the effects of these mutations in vivo and in vitro. Our results indicate which residues of Mcm1 are important for viability, transcriptional activation, and DNA binding and bending. Substitution of residues in Mcm1 which are highly conserved among the MADS-box proteins are lethal to the cell and abolish DNA binding in vitro. These positions have almost identical interactions with DNA in both the serum response factor-DNA and α2-Mcm1-DNA crystal structures, suggesting that these residues make up a conserved core of protein-DNA interactions responsible for docking MADS-box proteins to DNA. Substitution of residues which are not as well conserved among members of the MADS-box family play important roles in contributing to the specificity of DNA binding. These results suggest a general model of how MADS-box proteins recognize and bind DNA. We also provide evidence that the N-terminal extension of Mcm1 may have considerable conformational freedom, possibly to allow binding to different DNA sites. Finally, we have identified two mutants at positions which are critical for Mcm1-mediated DNA bending that have a slow-growth phenotype. This finding is consistent with our earlier results, indicating that DNA bending may have a role in Mcm1 function in the cell.

scholarly journals Mos activates myogenic differentiation by promoting heterodimerization of MyoD and E12 proteins.

1997 ◽  
Vol 17 (2) ◽  
pp. 584-593 ◽  
Author(s):  
J L Lenormand ◽  
B Benayoun ◽  
M Guillier ◽  
M Vandromme ◽  
M P Leibovitch ◽  
...  
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Myogenic Differentiation ◽  
Hybrid Approach ◽  
Specific Gene ◽  
Physical Interaction ◽  
Muscle Creatine Kinase ◽  
Bhlh Factors

The activities of myogenic basic helix-loop-helix (bHLH) factors are regulated by a number of different positive and negative signals. Extensive information has been published about the molecular mechanisms that interfere with the process of myogenic differentiation, but little is known about the positive signals. We previously showed that overexpression of rat Mos in C2C12 myoblasts increased the expression of myogenic markers whereas repression of Mos products by antisense RNAs inhibited myogenic differentiation. In the present work, our results show that the rat mos proto-oncogene activates transcriptional activity of MyoD protein. In transient transfection assays, Mos promotes transcriptional transactivation by MyoD of the muscle creatine kinase enhancer and/or a reporter gene linked to MyoD-DNA binding sites. Physical interaction between Mos and MyoD, but not with E12, is demonstrated in vivo by using the two-hybrid approach with C3H10T1/2 cells and in vitro by using the glutathione S-transferase (GST) pull-down assays. Unphosphorylated MyoD from myogenic cell lysates and/or bacterially expressed MyoD physically interacts with Mos. This interaction occurs via the helix 2 region of MyoD and a highly conserved region in Mos proteins with 40% similarity to the helix 2 domain of the E-protein class of bHLH factors. Phosphorylation of MyoD by activated GST-Mos protein inhibits the DNA-binding activity of MyoD homodimers and promotes MyoD-E12 heterodimer formation. These data support a novel function for Mos as a mediator (coregulator) of muscle-specific gene(s) expression.

scholarly journals SMC1A promotes tumor growth in breast cancer by activation of the AKT/FOXM1/STMN1 Signaling Pathway

2021 ◽  
Author(s):  
kaichun li ◽  
Ping Dai ◽  
Lin Xue ◽  
Long Liu ◽  
Shiyu Cheng ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Molecular Mechanisms ◽  
Xenograft Model ◽  
Cell Cycle Checkpoints ◽  
Biological Functions ◽  
Tumor Tissues ◽  
Subcutaneous Xenograft ◽  
Novel Target

Abstract Background SMC1A (Structural maintenance of chromosomes 1) is overexpressed in various cancers and acts as an oncogene which has been implicated in critical biological functions (cell-cycle checkpoints regulation, cell division, and DNA repair). However, the mechanism and role of SMC1A in breast cancer are poorly understood. Methods TCGA database was utilized to explore the expression of SMC1A and the relationship between SMC1A and FOXM1 and STMN1. Subsequently, short hairpin RNA (shRNA) targeting SMC1A was used to examined the biological functions of it in MDA-MB-231 and MDA-MB-468 cells. Finally, subcutaneous xenograft model to verify the roles of SMC1A in vivo. Results In the present study, we demonstrated that SMC1A was significantly increased in breast cancer (BC) via TCGA database. Then loss and gain of function studies revealed that SMC1A contributed to BC cell survival, apoptosis, and invasion. Interestingly, we found that SMC1A triggered the AKT/FOXM1 cascade, which promoted BC cell proliferation. Furthermore, overexpression of FOXM1 abolished the inhibition of cell growth induced by SMC1A silencing in vitro. Clinically, the expression of SMC1A in BC tumor tissues is positively correlated with the expression of FOXM1. Conclusion Taken together, our findings not only enhanced our understanding of molecular mechanisms of SMC1A in BC, but also might provide a novel target for the development of therapeutic strategies.

scholarly journals The deaminase ADAL-pivoted catabolism checkpoint suppresses aberrant DNA N6-methyladenine incorporation

2021 ◽  
Author(s):  
Shaokun Chen ◽  
Weiyi Lai ◽  
Zhiyi Zhao ◽  
Ning Zhang ◽  
Yan Liu ◽  
...  
Keyword(s):  
Adenylate Kinase ◽  
Patient Survival ◽  
Double Helix ◽  
Intracellular Degradation ◽  
Dna Double Helix ◽  
Adenylate Kinase 1 ◽  
Phosphorylation Rate

Abstract Abundant RNA N6-methyladenine (m6A) is degraded in RNA decay and potentially induces aberrant DNA N6-methyladenine (6mA) misincorporation. Biophysically, like truly methylated product DNA 6mA, misincorporated 6mA also destabilizes the DNA double helix and thus ditto affects DNA replication and transcription. By heavy stable isotope tracing, we demonstrate that intracellular degradation of RNA m6A cannot induce any misincorporated DNA 6mA, unveiling the existence of a catabolism checkpoint that blocks DNA 6mA misincorporation. We further show that the deaminase ADAL preferentially catabolizes N6-methyl-2’-deoxyadenosine monophosphate (6mdAMP) in vitro and in vivo, and adenylate kinase 1 restricts the phosphorylation rate of 6mdAMP, together contributing to the identified checkpoint. Noteworthy, low ADAL expression reduces dramatically the patient survival in four cancers. Collectively, our data strongly support a pivotal role of ADAL in the suppression of 6mA misincorporation and implicate that both ADAL and misincorporated 6mA may mark cancer abnormalities.

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