scholarly journals Hex is a transcriptional repressor that contributes to anterior identity and suppresses Spemann organiser function

Development ◽  
2000 ◽  
Vol 127 (11) ◽  
pp. 2303-2315 ◽  
Author(s):  
J.M. Brickman ◽  
C.M. Jones ◽  
M. Clements ◽  
J.C. Smith ◽  
R.S. Beddington
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Es Cells ◽  
Transcriptional Repressor ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Lambda Repressor ◽  
Reading Frame ◽  
Transcriptional Activation Domain ◽  
Dorsal Mesoderm

One of the earliest markers of anterior asymmetry in vertebrate embryos is the transcription factor Hex. We find that Hex is a transcriptional repressor that can be converted to an activator by fusing full length Hex to two copies of the minimal transcriptional activation domain of VP16 together with the flexible hinge region of the (lambda) repressor (Hex-(lambda)VP2). Retention of the entire Hex open reading frame allows one to examine Hex function without disrupting potential protein-protein interactions. Expression of Hex-(lambda)VP2 in Xenopus inhibits expression of the anterior marker Cerberus and results in anterior truncations. Such embryos have multiple notochords and disorganised muscle tissue. These effects can occur in a cell non-autonomous manner, suggesting that one role of wild-type Hex is to specify anterior structures by suppressing signals that promote dorsal mesoderm formation. In support of this idea, over-expression of wild-type Hex causes cell non-autonomous dorso-anteriorization, as well as cell autonomous suppression of dorsal mesoderm. Suppression of dorsal mesoderm by Hex is accompanied by the down-regulation of Goosecoid and Chordin, while induction of dorsal mesoderm by Hex-(lambda)VP2 results in activation of these genes. Transient transfection experiments in ES cells suggest that Goosecoid is a direct target of Hex. Together, our results support a model in which Hex suppresses organiser activity and defines anterior identity.

scholarly journals Deletion of Open Reading Frame UL26 from the Human Cytomegalovirus Genome Results in Reduced Viral Growth, Which Involves Impaired Stability of Viral Particles

2006 ◽  
Vol 80 (11) ◽  
pp. 5423-5434 ◽  
Author(s):  
Kerstin Lorz ◽  
Heike Hofmann ◽  
Anja Berndt ◽  
Nina Tavalai ◽  
Regina Mueller ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Transcriptional Activation ◽  
Deletion Mutant ◽  
Artificial Chromosome ◽  
Open Reading Frame ◽  
Wild Type Virus ◽  
Wild Type ◽  
Reading Frame ◽  
Transcriptional Activation Domain ◽  
Viral Particles

ABSTRACT We previously showed that open reading frame (ORF) UL26 of human cytomegalovirus, a member of the US22 multigene family of betaherpesviruses, encodes a novel tegument protein, which is imported into cells in the course of viral infection. Moreover, we demonstrated that pUL26 contains a strong transcriptional activation domain and is capable of stimulating the major immediate-early (IE) enhancer-promoter. Since this suggested an important function of pUL26 during the initiation of the viral replicative cycle, we sought to ascertain the relevance of pUL26 by construction of a viral deletion mutant lacking the UL26 ORF using the bacterial artificial chromosome mutagenesis procedure. The resulting deletion virus was verified by PCR, enzyme restriction, and Southern blot analyses. After infection of human foreskin fibroblasts, the UL26 deletion mutant showed a small-plaque phenotype and replicated to significantly lower titers than wild-type or revertant virus. In particular, we noticed a striking decrease of infectious titers 7 days postinfection in a multistep growth experiment, whereas the release of viral DNA from infected cells was not impaired. A further investigation of this aspect revealed a significantly diminished stability of viral particles derived from the UL26 deletion mutant. Consistent with this, we observed that the tegument composition of the deletion mutant deviates from that of the wild-type virus. We therefore hypothesize that pUL26 plays a role not only in the onset of IE gene transcription but also in the assembly of the viral tegument layer in a stable and correct manner.

scholarly journals Antimorphic goosecoids

Development ◽  
1998 ◽  
Vol 125 (8) ◽  
pp. 1347-1359 ◽  
Author(s):  
B. Ferreiro ◽  
M. Artinger ◽  
K. Cho ◽  
C. Niehrs
Keyword(s):  
Transcriptional Activation ◽  
Low Dose ◽  
Marginal Zone ◽  
Homeobox Gene ◽  
Axis Formation ◽  
Wild Type ◽  
Transcriptional Activation Domain ◽  
Spemann Organizer ◽  
Axial Defects ◽  
Dorsal Mesoderm

goosecoid (gsc) is a homeobox gene expressed in the Spemann organizer that has been implicated in vertebrate axis formation. Here antimorphic gscs are described. One antimorphic gsc (MTgsc) was fortuitously created by adding 5 myc epitopes to the N terminus of gsc. The other antimorph (VP16gsc) contains the transcriptional activation domain of VP16. mRNA injection of either antimorph inhibits dorsal gastrulation movements and leads to embryos with severe axial defects. They upregulate ventral gene expression in the dorsal marginal zone and inhibit dorsal mesoderm differentiation. Like the VP16 domain, the N-terminal myc tags act by converting wild-type gsc from a transcriptional repressor into an activator. However, unlike MTgsc, VP16gsc is able at low dose to uncouple head from trunk formation, indicating that different antimorphs may elicit distinct phenotypes. The experiments reveal that gsc and/or gsc-related genes function in axis formation and gastrulation. Moreover, this work warns against using myc tags indiscriminately for labeling DNA-binding proteins.

scholarly journals Three new dominant C1 suppressor alleles in Zea mays

1998 ◽  
Vol 71 (2) ◽  
pp. 127-132 ◽  
Author(s):  
TATJANA SINGER ◽  
ALFONS GIERL ◽  
PETER A. PETERSON
Keyword(s):  
Amino Acids ◽  
Transcriptional Activation ◽  
Stop Codon ◽  
Suppressive Effect ◽  
Wild Type ◽  
Activation Domain ◽  
Reading Frame ◽  
Transcriptional Activation Domain ◽  
Imprecise Excision ◽  
Carboxy Terminal

Three new dominant suppressor mutations of the C1 transcription regulator gene in maize – C1-IΔ1, C1-IΔ2 and C1-IΔ3 – are described that suppress anthocyanin colouration in kernels similar to the function of the C1-I standard inhibitor. The C1-IΔ mutations were induced by imprecise excision of an En/Spm transposon in the third exon of the C1 gene. These transposon footprints cause a frameshift in the C1 open reading frame that leads to truncated proteins due to an early stop codon 30 amino acids upstream of the wild-type C1 protein. Therefore, the C1-IΔ gene products lack the carboxy-terminal transcriptional activation domain of C1. The C1-I standard allele also lacks this domain and in addition differs in 17 amino acids from the wild-type C1 allele. The new C1-IΔ alleles provide evidence that deletion of the carboxy-terminal activation domain alone is sufficient to generate a dominant suppressive effect on the function of wild-type C1.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

1996 ◽  
Vol 132 (3) ◽  
pp. 359-370 ◽  
Author(s):  
E F Smith ◽  
P A Lefebvre
Keyword(s):  
Protein Interactions ◽  
Insertional Mutagenesis ◽  
Flagellar Motility ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Central Pair ◽  
Central Apparatus ◽  
Immunofluorescence Labeling ◽  
Armadillo Repeats

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

scholarly journals A Specificity and Targeting Subunit of a Human SWI/SNF Family-Related Chromatin-Remodeling Complex

2000 ◽  
Vol 20 (23) ◽  
pp. 8879-8888 ◽  
Author(s):  
Zuqin Nie ◽  
Yutong Xue ◽  
Dafeng Yang ◽  
Sharleen Zhou ◽  
Bonnie J. Deroo ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Chromatin Remodeling ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Gene Activation ◽  
Dna Interaction ◽  
Protein Protein Interactions ◽  
Baf Complex ◽  
Chromatin Remodeling Complexes

ABSTRACT The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation by assisting transcription machinery to gain access to targets in chromatin. This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from humans and SWI/SNF and Rsc fromSaccharomyces cerevisiae. However, the relationship between the human and yeast complexes is unclear because all human subunits published to date are similar to those of both yeast SWI/SNF and Rsc. Also, the two human complexes have many identical subunits, making it difficult to distinguish their structures or functions. Here we describe the cloning and characterization of BAF250, a subunit present in human BAF but not PBAF. BAF250 contains structural motifs conserved in yeast SWI1 but not in any Rsc components, suggesting that BAF is related to SWI/SNF. BAF250 is also a homolog of the Drosophila melanogaster Osa protein, which has been shown to interact with a SWI/SNF-like complex in flies. BAF250 possesses at least two conserved domains that could be important for its function. First, it has an AT-rich DNA interaction-type DNA-binding domain, which can specifically bind a DNA sequence known to be recognized by a SWI/SNF family-related complex at the β-globin locus. Second, BAF250 stimulates glucocorticoid receptor-dependent transcriptional activation, and the stimulation is sharply reduced when the C-terminal region of BAF250 is deleted. This region of BAF250 is capable of interacting directly with the glucocorticoid receptor in vitro. Our data suggest that BAF250 confers specificity to the human BAF complex and may recruit the complex to its targets through either protein-DNA or protein-protein interactions.

scholarly journals Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo.

1997 ◽  
Vol 17 (1) ◽  
pp. 115-122 ◽  
Author(s):  
M B Sainz ◽  
S A Goff ◽  
V L Chandler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transcriptional Activation ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Activation Domain ◽  
Hydrophobic Residues ◽  
Transcriptional Activation Domain ◽  
Acidic Amino Acids

C1 is a transcriptional activator of genes encoding biosynthetic enzymes of the maize anthocyanin pigment pathway. C1 has an amino terminus homologous to Myb DNA-binding domains and an acidic carboxyl terminus that is a transcriptional activation domain in maize and yeast cells. To identify amino acids critical for transcriptional activation, an extensive random mutagenesis of the C1 carboxyl terminus was done. The C1 activation domain is remarkably tolerant of amino acid substitutions, as changes at 34 residues had little or no effect on transcriptional activity. These changes include introduction of helix-incompatible amino acids throughout the C1 activation domain and alteration of most single acidic amino acids, suggesting that a previously postulated amphipathic alpha-helix is not required for activation. Substitutions at two positions revealed amino acids important for transcriptional activation. Replacement of leucine 253 with a proline or glutamine resulted in approximately 10% of wild-type transcriptional activation. Leucine 253 is in a region of C1 in which several hydrophobic residues align with residues important for transcriptional activation by the herpes simplex virus VP16 protein. However, changes at all other hydrophobic residues in C1 indicate that none are critical for C1 transcriptional activation. The other important amino acid in C1 is aspartate 262, as a change to valine resulted in only 24% of wild-type transcriptional activation. Comparison of our C1 results with those from VP16 reveal substantial differences in which amino acids are required for transcriptional activation in vivo by these two acidic activation domains.

scholarly journals The Effect of Proline cis-trans Isomerization on the Folding of the C-Terminal SH2 Domain from p85

2019 ◽  
Vol 21 (1) ◽  
pp. 125
Author(s):  
Francesca Troilo ◽  
Francesca Malagrinò ◽  
Lorenzo Visconti ◽  
Angelo Toto ◽  
Stefano Gianni
Keyword(s):  
Protein Interactions ◽  
Specific Interaction ◽  
Sh2 Domain ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Sh2 Domains ◽  
Trans Isomerization ◽  
A Site ◽  
Kinetics Of

SH2 domains are protein domains that modulate protein–protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the βE and βF strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events.

scholarly journals PDTM-29. STRUCTURE FUNCTION AND CELL TYPE DETERMINANTS OF C11orf95-RELA DRIVEN TUMORS IN MICE

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi193-vi193
Author(s):  
Jesse Dunnack ◽  
Ericka Randazzo ◽  
Jahmique Caines ◽  
Jame He ◽  
Joseph LoTurco
Keyword(s):  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Point Mutations ◽  
Cell Types ◽  
Tumor Formation ◽  
Wild Type ◽  
Nuclear Localization Signals ◽  
Transcriptional Activation Domain ◽  
Neuronal Progenitor ◽  
Glial Progenitor

Abstract We used a new mouse model to better understand the cellular and molecular determinants of tumors driven by the C11orf95-RELA fusion. Our approach makes use of in utero electroporation and a binary transposase system to introduce human C11orf95-RELA sequence, wild type and mutant, into neural progenitors, and drive expression of the fusion in different glial and neuronal progenitor cell types. Our results indicate that truncations or point mutations in C11orf95 sequence which interfere with nuclear localization result in a complete loss of tumor-inducing activity. The mutations include truncations of the first 60 amino acids, internal truncations that delete possible mono and bipartite nuclear localization signals, and point mutations of two cysteines and histidines that make up a possible zinc finger domain in C11orf95. Interestingly, all of the mutations that block tumorigenesis also block signal independent nuclear localization of the wild type fusion, without blocking induction of NFKB response genes. We further found that over-expression of the NFKB1 subunit P50 which lacks a transcriptional activation domain significantly inhibits tumor formation by the fusion. In addition, we find that driving expression of the wild type fusion in glial progenitor types using promoters for either astrocytes or oligodendrocytes results in the formation of tumors with transcriptomes displaying significant similarities to human supratentorial ependymoma (ST-EPN), but with distinct patterns depending upon the glial progenitor promoter utilized. In contrast, promoters driving expression selectively in neuron restricted progenitors do not result in the formation of ST-EPN. Together our results reveal three new features of C11orf95-RELA driven tumorigenesis: i) multiple sequences within the C11orf95 domain are required for oncogenic driver activity of the fusion, ii) the P50 subunit of NFKB1 can inhibit fusion induced tumorigenesis, and iii) neuron-restricted precursors are less competent than glia-restricted precursors to form tumors induced by C11orf95-RELA.

The androgen receptor transactivation domain: the interplay between protein conformation and protein–protein interactions

2003 ◽  
Vol 31 (5) ◽  
pp. 1042-1046 ◽  
Author(s):  
J. Reid ◽  
R. Betney ◽  
K. Watt ◽  
I.J. McEwan
Keyword(s):  
Androgen Receptor ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Specific Protein ◽  
Transactivation Domain ◽  
Protein Protein Interactions ◽  
Induced Folding ◽  
Large N ◽  
Tryptophan Residues ◽  
Receptor Transactivation

The AR (androgen receptor) belongs to the nuclear receptor superfamily and directly regulates patterns of gene expression in response to the steroids testosterone and dihydrotestosterone. Sequences within the large N-terminal domain of the receptor have been shown to be important for transactivation and protein–protein interactions; however, little is known about the structure and folding of this region. Folding of the AR transactivation domain was observed in the presence of the helix-stabilizing solvent trifluorethanol and the natural osmolyte TMAO (trimethylamine N-oxide). TMAO resulted in the movement of two tryptophan residues to a less solvent-exposed environment and the formation of a protease-resistant conformation. Critically, binding to a target protein, the RAP74 subunit of the general transcription factor TFIIF, resulted in a similar resistance to protease digestion, consistent with induced folding of the receptor transactivation domain. Our current hypothesis is that the folding of the transactivation domain in response to specific protein–protein interactions creates a platform for subsequent interactions, resulting in the formation of a competent transcriptional activation complex.

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