scholarly journals Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription.

1996 ◽  
Vol 16 (11) ◽  
pp. 6372-6384 ◽  
Author(s):  
C Y Chen ◽  
R J Schwartz
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Binding Activity ◽  
Dominant Negative ◽  
Actin Gene ◽  
Response Factor ◽  
Dna Binding Activity ◽  
Actin Promoter ◽  
Alpha Actin ◽  
Serum Response

We recently showed that the cardiogenic homeodomain factor Nkx-2.5 served as a positive acting accessory factor for serum response factor (SRF) and that together they provided strong transcriptional activation of the cardiac alpha-actin promoter, depending upon intact serum response elements (SREs) (C. Y. Chen, J. Croissant, M. Majesky, S. Topouz, T. McQuinn, M. J. Frankovsky, and R. J. Schwartz, Dev. Genet. 19:119-130, 1996). As shown here, Nkx-2.5 and SRF collaborated to activate the endogenous murine cardiac alpha-actin gene in 10T1/2 fibroblasts by a mechanism in which SRF recruited Nkx-2.5 to the alpha-actin promoter. Activation of a truncated promoter consisting of the proximal alpha-actin SRE1 occurred even when Nkx-2.5 DNA-binding activity was blocked by a point mutation in the third helix of its homeodomain. Investigation of protein-protein interactions showed that Nkx-2.5 was bound to SRF in the absence of DNA in soluble protein complexes retrieved from cardiac myocyte nuclei but could also be detected in coassociated binding complexes on the proximal SRE1. Recruitment of Nkx-2.5 to an SRE depended upon SRF DNA-binding activity and was blocked by the dominant negative SRFpm1 mutant, which allowed for dimerization of SRF monomers but prevented DNA binding. Interactive regions shared by Nkx-2.5 and SRF were mapped to N-terminal/helix I and helix II/helix III regions of the Nkx-2.5 homeodomain and to the N-terminal extension of the MADS box. Our study suggests that physical association between Nkx-2.5 and SRF is one way that cardiac specified genes are activated in cardiac cell lineages.

Human and Drosophila Homeodomain Proteins That Enhance the DNA-Binding Activity of Serum Response Factor

Science ◽  
1992 ◽  
Vol 257 (5073) ◽  
pp. 1089-1095 ◽  
Author(s):  
D. A. Grueneberg ◽  
S. Natesan ◽  
C. Alexandre ◽  
M. Z. Gilman
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Binding Activity ◽  
Response Factor ◽  
Homeodomain Proteins ◽  
Dna Binding Activity ◽  
Serum Response

Mutation of serum response factor phosphorylation sites and the mechanism by which its DNA-binding activity is increased by casein kinase II

1991 ◽  
Vol 11 (7) ◽  
pp. 3652-3659
Author(s):  
J R Manak ◽  
R Prywes
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Casein Kinase Ii ◽  
Binding Activity ◽  
Casein Kinase ◽  
Binding Domain ◽  
Response Factor ◽  
Dna Binding Domain ◽  
Dna Binding Activity ◽  
Serum Response

Casein kinase II (CKII) phosphorylates the mammalian transcription factor serum response factor (SRF) on a serine residue(s) located within a region of the protein spanning amino acids 70 to 92, thereby enhancing its DNA-binding activity in vitro. We report here that serine 83 appears to be the residue phosphorylated by CKII but that three other serines in this region can also be involved in phosphorylation and the enhancement of DNA-binding activity. A mutant that contained glutamate residues in place of these serines had only low-level binding activity; however, when the serines were replaced with glutamates and further mutations were made that increased the negative charge of the region, the resulting mutant showed a constitutively high level of binding equal to that achieved by phosphorylation of wild-type SRF. We have investigated the mechanism by which phosphorylation of SRF increases its DNA-binding activity. We have ruled out the possibilities that phosphorylation affects SRF dimerization or relieves inhibition due to masking of the DNA-binding domain by an amino-terminal region of the protein. Rather, using partial proteolysis to probe SRF's structure, we find that the conformation of SRF's DNA-binding domain is altered by phosphorylation.

scholarly journals Mutation of serum response factor phosphorylation sites and the mechanism by which its DNA-binding activity is increased by casein kinase II.

1991 ◽  
Vol 11 (7) ◽  
pp. 3652-3659 ◽  
Author(s):  
J R Manak ◽  
R Prywes
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Casein Kinase Ii ◽  
Binding Activity ◽  
Casein Kinase ◽  
Binding Domain ◽  
Response Factor ◽  
Dna Binding Domain ◽  
Dna Binding Activity ◽  
Serum Response

Casein kinase II (CKII) phosphorylates the mammalian transcription factor serum response factor (SRF) on a serine residue(s) located within a region of the protein spanning amino acids 70 to 92, thereby enhancing its DNA-binding activity in vitro. We report here that serine 83 appears to be the residue phosphorylated by CKII but that three other serines in this region can also be involved in phosphorylation and the enhancement of DNA-binding activity. A mutant that contained glutamate residues in place of these serines had only low-level binding activity; however, when the serines were replaced with glutamates and further mutations were made that increased the negative charge of the region, the resulting mutant showed a constitutively high level of binding equal to that achieved by phosphorylation of wild-type SRF. We have investigated the mechanism by which phosphorylation of SRF increases its DNA-binding activity. We have ruled out the possibilities that phosphorylation affects SRF dimerization or relieves inhibition due to masking of the DNA-binding domain by an amino-terminal region of the protein. Rather, using partial proteolysis to probe SRF's structure, we find that the conformation of SRF's DNA-binding domain is altered by phosphorylation.

Functional antagonism between YY1 and the serum response factor

1992 ◽  
Vol 12 (9) ◽  
pp. 4209-4214
Author(s):  
A Gualberto ◽  
D LePage ◽  
G Pons ◽  
S L Mader ◽  
K Park ◽  
...  
Keyword(s):  
Serum Response Factor ◽  
Regulatory Element ◽  
Target Sequence ◽  
Response Factor ◽  
Basal Expression ◽  
Actin Promoter ◽  
Alpha Actin ◽  
Serum Response

The rapid, transient induction of the c-fos proto-oncogene by serum growth factors is mediated by the serum response element (SRE). The SRE shares homology with the muscle regulatory element (MRE) of the skeletal alpha-actin promoter. It is not known how these elements respond to proliferative and cell-type-specific signals, but the response appears to involve the binding of the serum response factor (SRF) and other proteins. Here, we report that YY1, a multifunctional transcription factor, binds to SRE and MRE sequences in vitro. The methylation interference footprint of YY1 overlaps with that of the SRF, and YY1 competes with the SRF for binding to these DNA elements. Overexpression of YY1 repressed serum-inducible and basal expression from the c-fos promoter and repressed basal expression from the skeletal alpha-actin promoter. YY1 also repressed expression from the individual SRE and MRE sequences upstream from a TATA element. Unlike that of YY1, SRF overexpression alone did not influence the transcriptional activity of the target sequence, but SRF overexpression could reverse YY1-mediated trans repression. These data suggest that YY1 and the SRF have antagonistic functions in vivo.

Activation of skeletal alpha-actin gene transcription: the cooperative formation of serum response factor-binding complexes over positive cis-acting promoter serum response elements displaces a negative-acting nuclear factor enriched in replicating myoblasts and nonmyogenic cells

1991 ◽  
Vol 11 (10) ◽  
pp. 5090-5100
Author(s):  
T C Lee ◽  
K L Chow ◽  
P Fang ◽  
R J Schwartz
Keyword(s):  
Gene Transcription ◽  
Inner Core ◽  
Serum Response Factor ◽  
Actin Gene ◽  
Response Factor ◽  
Cis Acting ◽  
Promoter Complex ◽  
Functional Analyses ◽  
Alpha Actin ◽  
Serum Response

Three upstream CBAR cis-acting promoter elements, containing the inner core CC(A/T)6GG of the serum response element (SRE), are required for myogenic cell type-restricted expression of the avian skeletal alpha-actin gene (K.L. Chow and R.J. Schwartz, Mol. Cell. Biol. 10:528-538, 1990). These actin SRE elements display differential binding properties with two distinct nuclear proteins, serum response factor (SRF) and another factor described here as F-ACT1. SRF is able to bind to all actin SREs with various affinities. This multisite interaction is marked by cooperative binding events in that the two high-affinity proximal and distal SREs facilitate the weak central-site interaction with SRF, leading to the formation of a higher-order SRF-promoter complex. Functional analyses reveal that undisrupted multiple SRF-DNA interactions are absolutely essential for promoter activity in myogenic cells. F-ACT1, present at higher levels in nonmyogenic cells and replicating myoblasts than in myotubes, binds solely to the proximal SRE, and its binding is mutually exclusive with that of SRF owing to their overlapping base contacts. The cooperative promoter binding by SRF, however, can effectively displace prebound F-ACT1. In addition, an intact F-ACT1 binding site acts as a negative promoter element by restricting developmentally timed expression in myoblasts. F-ACT1 may therefore act as a repressor of skeletal alpha-actin gene transcription. This interplay between F-ACT1 and SRF may constitute a developmental as well as a physiologically regulated mechanism which modulates sarcomeric actin gene expression.

scholarly journals Dominant Negative Murine Serum Response Factor: Alternative Splicing within the Activation Domain Inhibits Transactivation of Serum Response Factor Binding Targets

1999 ◽  
Vol 19 (7) ◽  
pp. 4582-4591 ◽  
Author(s):  
Narasimhaswamy S. Belaguli ◽  
Wei Zhou ◽  
Thuy-Hanh T. Trinh ◽  
Mark W. Majesky ◽  
Robert J. Schwartz
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Smooth Muscles ◽  
Dominant Negative ◽  
C2c12 Cells ◽  
Luciferase Reporter ◽  
Response Factor ◽  
Activation Domain ◽  
Alternatively Spliced ◽  
Serum Response

ABSTRACT Primary transcripts encoding the MADS box superfamily of proteins, such as MEF2 in animals and ZEMa in plants, are alternatively spliced, producing several isoformic species. We show here that murine serum response factor (SRF) primary RNA transcripts are alternatively spliced at the fifth exon, deleting approximately one-third of the C-terminal activation domain. Among the different muscle types examined, visceral smooth muscles have a very low ratio of SRFΔ5 to SRF. Increased levels of SRFΔ5 correlates well with reduced smooth muscle contractile gene activity within the elastic aortic arch, suggesting important biological roles for differential expression of SRFΔ5 variant relative to wild-type SRF. SRFΔ5 forms DNA binding-competent homodimers and heterodimers. SRFΔ5 acts as a naturally occurring dominant negative regulatory mutant that blocks SRF-dependent skeletal α-actin, cardiac α-actin, smooth α-actin, SM22α, and SRF promoter-luciferase reporter activities. Expression of SRFΔ5 interferes with differentiation of myogenic C2C12 cells and the appearance of skeletal α-actin and myogenin mRNAs. SRFΔ5 repressed the serum-induced activity of the c-fos serum response element. SRFΔ5 fused to the yeast Gal4 DNA binding domain displayed low transcriptional activity, which was complemented by overexpression of the coactivator ATF6. These results indicate that the absence of exon 5 might be bypassed through recruitment of transcription factors that interact with extra-exon 5 regions in the transcriptional activating domain. The novel alternatively spliced isoform of SRF, SRFΔ5, may play an important regulatory role in modulating SRF-dependent gene expression.

scholarly journals Functional antagonism between YY1 and the serum response factor.

1992 ◽  
Vol 12 (9) ◽  
pp. 4209-4214 ◽  
Author(s):  
A Gualberto ◽  
D LePage ◽  
G Pons ◽  
S L Mader ◽  
K Park ◽  
...  
Keyword(s):  
Serum Response Factor ◽  
Regulatory Element ◽  
Target Sequence ◽  
Response Factor ◽  
Basal Expression ◽  
Actin Promoter ◽  
Alpha Actin ◽  
Serum Response

The rapid, transient induction of the c-fos proto-oncogene by serum growth factors is mediated by the serum response element (SRE). The SRE shares homology with the muscle regulatory element (MRE) of the skeletal alpha-actin promoter. It is not known how these elements respond to proliferative and cell-type-specific signals, but the response appears to involve the binding of the serum response factor (SRF) and other proteins. Here, we report that YY1, a multifunctional transcription factor, binds to SRE and MRE sequences in vitro. The methylation interference footprint of YY1 overlaps with that of the SRF, and YY1 competes with the SRF for binding to these DNA elements. Overexpression of YY1 repressed serum-inducible and basal expression from the c-fos promoter and repressed basal expression from the skeletal alpha-actin promoter. YY1 also repressed expression from the individual SRE and MRE sequences upstream from a TATA element. Unlike that of YY1, SRF overexpression alone did not influence the transcriptional activity of the target sequence, but SRF overexpression could reverse YY1-mediated trans repression. These data suggest that YY1 and the SRF have antagonistic functions in vivo.

scholarly journals Activation of skeletal alpha-actin gene transcription: the cooperative formation of serum response factor-binding complexes over positive cis-acting promoter serum response elements displaces a negative-acting nuclear factor enriched in replicating myoblasts and nonmyogenic cells.

1991 ◽  
Vol 11 (10) ◽  
pp. 5090-5100 ◽  
Author(s):  
T C Lee ◽  
K L Chow ◽  
P Fang ◽  
R J Schwartz
Keyword(s):  
Gene Transcription ◽  
Inner Core ◽  
Serum Response Factor ◽  
Actin Gene ◽  
Response Factor ◽  
Cis Acting ◽  
Promoter Complex ◽  
Functional Analyses ◽  
Alpha Actin ◽  
Serum Response

Three upstream CBAR cis-acting promoter elements, containing the inner core CC(A/T)6GG of the serum response element (SRE), are required for myogenic cell type-restricted expression of the avian skeletal alpha-actin gene (K.L. Chow and R.J. Schwartz, Mol. Cell. Biol. 10:528-538, 1990). These actin SRE elements display differential binding properties with two distinct nuclear proteins, serum response factor (SRF) and another factor described here as F-ACT1. SRF is able to bind to all actin SREs with various affinities. This multisite interaction is marked by cooperative binding events in that the two high-affinity proximal and distal SREs facilitate the weak central-site interaction with SRF, leading to the formation of a higher-order SRF-promoter complex. Functional analyses reveal that undisrupted multiple SRF-DNA interactions are absolutely essential for promoter activity in myogenic cells. F-ACT1, present at higher levels in nonmyogenic cells and replicating myoblasts than in myotubes, binds solely to the proximal SRE, and its binding is mutually exclusive with that of SRF owing to their overlapping base contacts. The cooperative promoter binding by SRF, however, can effectively displace prebound F-ACT1. In addition, an intact F-ACT1 binding site acts as a negative promoter element by restricting developmentally timed expression in myoblasts. F-ACT1 may therefore act as a repressor of skeletal alpha-actin gene transcription. This interplay between F-ACT1 and SRF may constitute a developmental as well as a physiologically regulated mechanism which modulates sarcomeric actin gene expression.

Sign in / Sign up

Export Citation Format

Share Document