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.

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.

Serial analysis of gene expression (SAGE) during porcine embryo development

2004 ◽  
Vol 16 (2) ◽  
pp. 87 ◽  
Author(s):  
Le Ann Blomberg ◽  
Kurt A. Zuelke
Keyword(s):  
Gene Expression ◽  
Functional Genomics ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Transgenic Animals ◽  
Specific Gene ◽  
Research Strategies

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

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 Adenovirus E1B 55-Kilodalton Oncoprotein Inhibits p53 Acetylation by PCAF

2000 ◽  
Vol 20 (15) ◽  
pp. 5540-5553 ◽  
Author(s):  
Yue Liu ◽  
April L. Colosimo ◽  
Xiang-Jiao Yang ◽  
Daiqing Liao
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Target Genes ◽  
Binding Activity ◽  
Physical Interaction ◽  
Dna Binding Activity ◽  
C Terminus ◽  
Adenovirus E1b

ABSTRACT The adenovirus E1B 55-kDa protein binds to cellular tumor suppressor p53 and inactivates its transcriptional transactivation function. p53 transactivation activity is dependent upon its ability to bind to specific DNA sequences near the promoters of its target genes. It was shown recently that p53 is acetylated by transcriptional coactivators p300, CREB bidning protein (CBP), and PCAF and that acetylation of p53 by these proteins enhances p53 sequence-specific DNA binding. Here we show that the E1B 55-kDa protein specifically inhibits p53 acetylation by PCAF in vivo and in vitro, while acetylation of histones and PCAF autoacetylation is not affected. Furthermore, the DNA-binding activity of p53 is diminished in cells expressing the E1B 55-kDa protein. PCAF binds to the E1B 55-kDa protein and to a region near the C terminus of p53 encompassing Lys-320, the specific PCAF acetylation site. We further show that the E1B 55-kDa protein interferes with the physical interaction between PCAF and p53, suggesting that the E1B 55-kDa protein inhibits PCAF acetylase function on p53 by preventing enzyme-substrate interaction. These results underscore the importance of p53 acetylation for its function and suggest that inhibition of p53 acetylation by viral oncoproteins prevent its activation, thereby contributing to viral transformation.

scholarly journals Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding.

1991 ◽  
Vol 11 (7) ◽  
pp. 3633-3641 ◽  
Author(s):  
T Chakraborty ◽  
T J Brennan ◽  
L Li ◽  
D Edmondson ◽  
E N Olson
Keyword(s):  
Dna Binding ◽  
Gene Activation ◽  
Binding Activity ◽  
Specific Gene ◽  
Gene Products ◽  
Basic Domain ◽  
High Affinity ◽  
Dna Binding Activity

Myogenin is a muscle-specific transcription factor that can activate myogenesis; it belongs to a family of transcription factors that share homology within a basic region and an adjacent helix-loop-helix (HLH) motif. Although myogenin alone binds DNA inefficiently, in the presence of the widely expressed HLH proteins E12 and E47 (encoded by the E2A gene), it forms heterooligomers that bind with high affinity to a DNA sequence known as a kappa E-2 site. In contrast, E47 and to a lesser extent E12 are both able to bind the kappa E-2 site relatively efficiently as homooligomers. To define the relative contributions of the basic regions of myogenin and E12 to DNA binding and muscle-specific gene activation, we created chimeras of the two proteins by swapping their basic regions. We showed that myogenin's weak affinity for the kappa E-2 site is attributable to inefficient homooligomerization and that the myogenin basic domain alone can mediate high-affinity DNA binding when placed in E12. Within a heterooligomeric complex, two basic regions were required to form a high-affinity DNA-binding domain. Basic-domain mutants of myogenin or E2A gene products that cannot bind DNA retained the ability to oligomerize and could abolish DNA binding of the wild-type proteins in vitro. These myogenin and E2A mutants also acted as trans-dominant inhibitors of muscle-specific gene activation in vivo. These findings support the notion that muscle-specific gene activation requires oligomerization between myogenin and E2A gene products and that E2A gene products play an important role in myogenesis by enhancing the DNA-binding activity of myogenin, as well as other myogenic HLH proteins.

scholarly journals UME6, a Novel Filament-specific Regulator ofCandida albicansHyphal Extension and Virulence

2008 ◽  
Vol 19 (4) ◽  
pp. 1354-1365 ◽  
Author(s):  
Mohua Banerjee ◽  
Delma S. Thompson ◽  
Anna Lazzell ◽  
Patricia L. Carlisle ◽  
Christopher Pierce ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Pathogenic Fungi ◽  
Filamentous Growth ◽  
Specific Gene ◽  
Regulatory Pathways ◽  
Expression Of Genes ◽  
High Level ◽  
Germ Tubes

The specific ability of the major human fungal pathogen Candida albicans, as well as many other pathogenic fungi, to extend initial short filaments (germ tubes) into elongated hyphal filaments is important for a variety of virulence-related processes. However, the molecular mechanisms that control hyphal extension have remained poorly understood for many years. We report the identification of a novel C. albicans transcriptional regulator, UME6, which is induced in response to multiple host environmental cues and is specifically important for hyphal extension. Although capable of forming germ tubes, the ume6Δ/ume6Δ mutant exhibits a clear defect in hyphal extension both in vitro and during infection in vivo and is attenuated for virulence in a mouse model of systemic candidiasis. We also show that UME6 is an important downstream component of both the RFG1-TUP1 and NRG1-TUP1 filamentous growth regulatory pathways, and we provide evidence to suggest that Nrg1 and Ume6 function together by a negative feedback loop to control the level and duration of filament-specific gene expression in response to inducing conditions. Our results suggest that hyphal extension is controlled by a specific transcriptional regulatory mechanism and is correlated with the maintenance of high-level expression of genes in the C. albicans filamentous growth program.

scholarly journals 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.

Hemogenic and nonhemogenic endothelium can be distinguished by the activity of fetal liver kinase (Flk)–1promoter/enhancer during mouse embryogenesis

Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 886-893 ◽  
Author(s):  
Hideyo Hirai ◽  
Minetaro Ogawa ◽  
Norio Suzuki ◽  
Masayuki Yamamoto ◽  
Georg Breier ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Specific Gene ◽  
Vascular Endothelial ◽  
Myb Genes ◽  
Definitive Hematopoiesis

Abstract Accumulating evidence in various species has suggested that the origin of definitive hematopoiesis is associated with a special subset of endothelial cells (ECs) that maintain the potential to give rise to hematopoietic cells (HPCs). In this study, we demonstrated that a combination of 5′-flanking region and 3′ portion of the first intron of the Flk-1 gene (Flk-1 p/e) that has been implicated in endothelium-specific gene expression distinguishes prospectively the EC that has lost hemogenic activity. We assessed the activity of this Flk-1 p/e by embryonic stem (ES) cell differentiation culture and transgenic mice by using theGFP gene conjugated to this unit. The expression ofGFP differed from that of the endogenous Flk-1gene in that it is active in undifferentiated ES cells and inactive in Flk-1+ lateral mesoderm. Flk-1 p/e becomes active after generation of vascular endothelial (VE)–cadherin+ ECs. Emergence of GFP− ECs preceded that of GFP+ ECs, and, finally, most ECs expressed GFP both in vitro and in vivo. Cell sorting experiments demonstrated that only GFP− ECs could give rise to HPCs and preferentially expressed Runx1 and c-Myb genes that are required for the definitive hematopoiesis. Integration of both GFP+ and GFP− ECs was observed in the dorsal aorta, but cell clusters appeared associated only to GFP−ECs. These results indicate that activation of Flk-1 p/e is associated with a process that excludes HPC potential from the EC differentiation pathway and will be useful for investigating molecular mechanisms underlying the divergence of endothelial and hematopoietic lineages.

scholarly journals Myocyte Enhancer Factor 2 Acetylation by p300 Enhances Its DNA Binding Activity, Transcriptional Activity, and Myogenic Differentiation

2005 ◽  
Vol 25 (9) ◽  
pp. 3575-3582 ◽  
Author(s):  
Kewei Ma ◽  
Jonathan K. L. Chan ◽  
Guang Zhu ◽  
Zhenguo Wu
Keyword(s):  
Dna Binding ◽  
Transcriptional Activity ◽  
Myogenic Differentiation ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Myocyte Enhancer Factor 2 ◽  
Myocyte Enhancer Factor ◽  
Enhancer Factor

ABSTRACT Myocyte enhancer factor 2 (MEF2) family proteins are key transcription factors controlling gene expression in myocytes, lymphocytes, and neurons. MEF2 proteins are known to be regulated by phosphorylation. We now provide evidence showing that MEF2C is acetylated by p300 both in vitro and in vivo. In C2C12 myogenic cells, MEF2 is preferentially acetylated in differentiating myocytes but not in undifferentiated myoblasts. Several major acetylation sites are mapped to the transactivation domain of MEF2C, some of which are fully conserved in other MEF2 members from several different species. Mutation of these lysines affects MEF2 DNA binding and transcriptional activity, as well as its synergistic effect with myogenin in myogenic conversion assays. When introduced into C2C12 myoblasts, the nonacetylatable MEF2C inhibits myogenic differentiation. Thus, in addition to phosphorylation, MEF2 activity is also critically regulated by acetylation during myogenesis.

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.

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