Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs.

The binding of serum response factor (SRF) to the c-fos serum response element has been shown to be essential for serum and growth factor activation of c-Fos. Since SRF is ubiquitously expressed, it has been difficult to measure the activity of SRF introduced into cells. To assay for functions of SRF in cells, we have changed its DNA binding specificity by fusing it to the DNA binding domain of GAL4. Transfection of GAL4-SRF constructs into cells has allowed us to identify SRF's transcriptional activation domain as well as domains which inhibit this activity. First, we found that the transcriptional activation domain maps to between amino acids 339 and 508 in HeLa cells and to between amino acids 414 and 508 in NIH 3T3 cells. Second, we show that in the context of GAL4-SRF constructs, there are two separate domains of SRF that can inhibit its activation domain. Although these domains overlap the DNA binding and dimerization domains of SRF, these functions were not required for inhibition. Finally, we show that one of the inhibitory domains is modular in that it can also inhibit activation when it is moved amino terminal to GAL4's DNA binding domain in an SRF-GAL4-SRF construct. The implications of these inhibitory domains for SRF regulation are discussed.

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Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6056-6067.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6056-6067

Author(s):

M Tanaka ◽

W Herr

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Binding Domain ◽

Dna Binding Domain ◽

Activation Domain ◽

Activation Domains ◽

Transcriptional Activation Domain ◽

Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

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MAL and Ternary Complex Factor Use Different Mechanisms To Contact a Common Surface on the Serum Response Factor DNA-Binding Domain

Molecular and Cellular Biology ◽

10.1128/mcb.01902-05 ◽

2006 ◽

Vol 26 (11) ◽

pp. 4134-4148 ◽

Cited By ~ 61

Author(s):

Alexia-Ileana Zaromytidou ◽

Francesc Miralles ◽

Richard Treisman

Keyword(s):

Dna Binding ◽

Complex Formation ◽

Ternary Complex ◽

Serum Response Factor ◽

Dna Bending ◽

Binding Domain ◽

Response Factor ◽

Dna Binding Domain ◽

Serum Response ◽

Ternary Complex Factor

ABSTRACT The transcription factor serum response factor (SRF) interacts with its cofactor, MAL/MKL1, a member of the myocardin-related transcription factor (MRTF) family, through its DNA-binding domain. We define a seven-residue sequence within the conserved MAL B1 region essential and sufficient for complex formation. The neighboring Q-box sequence facilitates this interaction. The B1 and Q-box regions also have antagonistic effects on MAL nuclear import, but the residues involved are largely distinct. Both MAL and the ternary complex factor (TCF) family of SRF cofactors interact with a hydrophobic groove and pocket on the SRF DNA-binding domain. Unlike the TCFs, however, interaction of MAL with SRF is impaired by SRF αI-helix mutations that reduce DNA bending in the SRF-DNA complex. A clustered SRF αI-helix mutation strongly impairs MAL-SRF complex formation but does not affect DNA distortion in the MAL-SRF complex. MAL-SRF complex formation is facilitated by DNA binding. DNase I footprinting indicates that in the SRF-MAL complex MAL directly contacts DNA. These contacts, which flank the DNA sequences protected from DNase I by SRF, are required for effective MAL-SRF complex formation in gel mobility shift assays. We propose a model of MAL-SRF complex formation in which MAL interacts with SRF by the addition of a β-strand to the SRF DNA-binding domain β-sheet region, while SRF-induced DNA bending facilitates MAL-DNA contact.

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Analysis of DNA binding by the adenovirus type 5 E1A oncoprotein

Journal of General Virology ◽

10.1099/0022-1317-83-3-517 ◽

2002 ◽

Vol 83 (3) ◽

pp. 517-524 ◽

Cited By ~ 13

Author(s):

Nikita Avvakumov ◽

Majdina Sahbegovic ◽

Zhiying Zhang ◽

Michael Shuen ◽

Joe S. Mymryk

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Transcriptional Activation ◽

Adenovirus Type ◽

Dna Binding Domain ◽

Activation Domain ◽

Transcriptional Activation Domain ◽

Adenovirus Type 5

Adenovirus type 5 E1A proteins interact with cellular regulators of transcription to reprogram gene expression in the infected or transformed cell. Although E1A also interacts with DNA directly in vitro, it is not clear how this relates to its function in vivo. The N-terminal conserved regions 1, 2 and 3 and the C-terminal portions of E1A were prepared as purified recombinant proteins and analyses showed that only the C-terminal region bound DNA in vitro. Deletion of E1A amino acids 201–220 inhibited binding and a minimal fragment encompassing amino acids 201–218 of E1A was sufficient for binding single- and double-stranded DNA. This portion of E1A also bound the cation-exchange resins cellulose phosphate and carboxymethyl Sepharose. As this region contains six basic amino acids, in vitro binding of E1A to DNA probably results from an ionic interaction with the phosphodiester backbone of DNA. Studies in Saccharomyces cerevisiae have shown that expression of a strong transcriptional activation domain fused to a DNA-binding domain can inhibit growth. Although fusion of the C-terminal region of E1A to a strong transcriptional activation domain inhibited growth when expressed in yeast, this was not mediated by the DNA-binding domain identified in vitro. These data suggest that E1A does not bind DNA in vivo.

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The carboxyl-terminal transactivation domain of heat shock factor 1 is negatively regulated and stress responsive.

Molecular and Cellular Biology ◽

10.1128/mcb.15.8.4309 ◽

1995 ◽

Vol 15 (8) ◽

pp. 4309-4318 ◽

Cited By ~ 79

Author(s):

Y Shi ◽

P E Kroeger ◽

R I Morimoto

Keyword(s):

Heat Shock ◽

Dna Binding ◽

Transcriptional Activation ◽

Heat Shock Factor ◽

Binding Domain ◽

Heat Shock Factor 1 ◽

Dna Binding Domain ◽

Activation Domain ◽

Transcriptional Activation Domain ◽

Carboxyl Terminal

We have characterized a stress-responsive transcriptional activation domain of mouse heat shock factor 1 (HSF1) by using chimeric GAL4-HSF1 fusion proteins. Fusion of the GAL4 DNA-binding domain to residues 124 to 503 of HSF1 results in a chimeric factor that binds DNA yet lacks any transcriptional activity. Transactivation is acquired upon exposure to heat shock or by deletion of a negative regulatory domain including part of the DNA-binding-domain-proximal leucine zippers. Analysis of a collection of GAL4-HSF1 deletion mutants revealed the minimal region for the constitutive transcriptional activator to map within the extreme carboxyl-terminal 108 amino acids, corresponding to a region rich in acidic and hydrophobic residues. Loss of residues 395 to 425 or 451 to 503, which are located at either end of this activation domain, severely diminished activity, indicating that the entire domain is required for transactivation. The minimal activation domain of HSF1 also confers enhanced transcriptional response to heat shock or cadmium treatment. These results demonstrate that the transcriptional activation domain of HSF1 is negatively regulated and that the signal for stress induction is mediated by interactions between the amino-terminal negative regulator and the carboxyl-terminal transcriptional activation domain.

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Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain.

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6056 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6056-6067 ◽

Cited By ~ 28

Author(s):

M Tanaka ◽

W Herr

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Binding Domain ◽

Dna Binding Domain ◽

Activation Domain ◽

Activation Domains ◽

Transcriptional Activation Domain ◽

Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3652-3659.1991 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3652 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3652-3659 ◽

Cited By ~ 50

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.

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