scholarly journals The sarcomeric actin CArG-binding factor is indistinguishable from the c-fos serum response factor.

1989 ◽  
Vol 9 (2) ◽  
pp. 515-522 ◽  
Author(s):  
L M Boxer ◽  
R Prywes ◽  
R G Roeder ◽  
L Kedes
Keyword(s):  
Transcriptional Control ◽  
Serum Response Factor ◽  
Specific Binding ◽  
Immune Serum ◽  
Response Factor ◽  
Mobility Shift ◽  
Binding Factor ◽  
Carg Box ◽  
Zinc Addition ◽  
Serum Response

The c-fos serum response element (SRE) and a sarcomeric actin promoter element (CArG box) are similar in sequence and are recognized, respectively, by the serum response factor (SRF) and the CArG-binding factor (CBF). Although the transcriptional controls for the c-fos and sarcomeric actin genes are rather different, SRF and CBF have been found to be indistinguishable by all criteria tested. They exhibited similar chromatographic properties, sedimentation rates, and temperature stabilities. In mobility shift assays, the SRE competed more strongly than the actin CArG box for formation of either the SRF-SRE or the CBF-CArG complex. The symmetric inverted repeat of the left side of the Xenopus cytoskeletal actin SRE also competed, even more strongly, for each complex. The site-specific binding of each protein was inhibited both by orthophenanthroline, whose effects were reversed by zinc addition, and by treatment with potato acid phosphatase. Furthermore, immune serum raised against the c-fos SRF also recognized the actin CBF. We discuss how transcriptional control of these diverse genes might be obtained with a single similar factor.

The sarcomeric actin CArG-binding factor is indistinguishable from the c-fos serum response factor

1989 ◽  
Vol 9 (2) ◽  
pp. 515-522
Author(s):  
L M Boxer ◽  
R Prywes ◽  
R G Roeder ◽  
L Kedes
Keyword(s):  
Transcriptional Control ◽  
Serum Response Factor ◽  
Specific Binding ◽  
Immune Serum ◽  
Response Factor ◽  
Mobility Shift ◽  
Binding Factor ◽  
Carg Box ◽  
Zinc Addition ◽  
Serum Response

The c-fos serum response element (SRE) and a sarcomeric actin promoter element (CArG box) are similar in sequence and are recognized, respectively, by the serum response factor (SRF) and the CArG-binding factor (CBF). Although the transcriptional controls for the c-fos and sarcomeric actin genes are rather different, SRF and CBF have been found to be indistinguishable by all criteria tested. They exhibited similar chromatographic properties, sedimentation rates, and temperature stabilities. In mobility shift assays, the SRE competed more strongly than the actin CArG box for formation of either the SRF-SRE or the CBF-CArG complex. The symmetric inverted repeat of the left side of the Xenopus cytoskeletal actin SRE also competed, even more strongly, for each complex. The site-specific binding of each protein was inhibited both by orthophenanthroline, whose effects were reversed by zinc addition, and by treatment with potato acid phosphatase. Furthermore, immune serum raised against the c-fos SRF also recognized the actin CBF. We discuss how transcriptional control of these diverse genes might be obtained with a single similar factor.

scholarly journals Serum response factor mRNA induction in the hypertrophying chicken patagialis muscle

1999 ◽  
Vol 86 (1) ◽  
pp. 377-382 ◽  
Author(s):  
James A. Carson ◽  
Frank W. Booth
Keyword(s):  
Serum Response Factor ◽  
Response Factor ◽  
Mobility Shift ◽  
Mrna Induction ◽  
Mrna Concentration ◽  
Nuclear Extracts ◽  
Actin Promoter ◽  
Gel Mobility Shift ◽  
Fast Twitch ◽  
Serum Response

Gene expression in the stretched chicken patagialis (Pat) muscle has not been extensively examined. This study’s purpose was to determine the Pat muscle’s expression pattern of serum response factor (SRF), skeletal α-actin, and MyoD mRNAs after 3 days (onset of stretch), 6 days (end of first week of rapid growth), and 14 days (slowed rate of stretch-induced growth) of stretch. SRF mRNA demonstrated two species (B1 and B2), with B2 being more prevalent in the predominantly fast-twitch Pat muscle, compared with the slow-tonic muscle. Stretch overload increased B1 and B2 SRF mRNA concentrations, and the increase in B1 SRF mRNA concentration was greater at day 6compared with days 3 or 14. MyoD mRNA concentration was greater in 3-day-stretched Pat muscles, compared with days 6 or 14 . Skeletal α-actin mRNA concentration was not changed during the study. Gel mobility shift assays demonstrated that SRF binding with serum response element 1 of the skeletal α-actin promoter had no altered binding patterns from 6-day-stretched Pat nuclear extracts. It appears that SRF and MyoD mRNAs are induced in the stretch-overloaded Pat muscle but at different time points.

scholarly journals Regulation of the murine alpha B-crystallin/small heat shock protein gene in cardiac muscle.

1995 ◽  
Vol 15 (12) ◽  
pp. 7081-7090 ◽  
Author(s):  
R Gopal-Srivastava ◽  
J I Haynes ◽  
J Piatigorsky
Keyword(s):  
Skeletal Muscle ◽  
Heat Shock ◽  
Serum Response Factor ◽  
Small Heat Shock Protein ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
E Box ◽  
Carg Box ◽  
Electrophoretic Mobility Shift Assays ◽  
Serum Response

The murine alpha B-crystallin/small heat shock protein gene is expressed at high levels in the lens and at lower levels in the heart, skeletal muscle, and numerous other tissues. Previously we have found a skeletal-muscle-preferred enhancer at positions -427 to -259 of the alpha B-crystallin gene containing at least four cis-acting regulatory elements (alpha BE-1, alpha BE-2, alpha BE-3, and MRF, which has an E box). Here we show that in transgenic mice, the alpha B-crystallin enhancer directs the chloramphenicol acetyltransferase reporter gene driven by the alpha B-crystallin promoter specifically to myocardiocytes of the heart. The alpha B-crystallin enhancer was active in conjugation with the herpes simplex virus thymidine kinase promoter/human growth hormone reporter gene in transfected rat myocardiocytes. DNase I footprinting and site-specific mutagenesis experiments showed that alpha BE-1, alpha BE-2, alpha BE-3, MRF, and a novel, heart-specific element called alpha BE-4 are required for alpha B-crystallin enhancer activity in transfected myocardiocytes. By contrast, alpha BE-4 is not utilized for enhancer activity in transfected lens or skeletal muscle cell lines. Alpha BE-4 contains an overlapping heat shock sequence and a reverse CArG box [5'-GG(A/T)6CC-3']. Electrophoretic mobility shift assays with an antibody to serum response factor and a CArG-box-competing sequence from the c-fos promoter indicated that a cardiac-specific protein with DNA-binding and antigenic similarities to serum response factor binds to alpha BE-4 via the reverse CArG box; electrophoretic mobility shift assays and antibody experiments with anti-USF antiserum and heart nuclear extract also raised the possibility that the MRF E box utilizes USF or an antigenically related protein. We conclude that the activity of the alpha B-crystallin enhancer in the heart utilizes a reverse CArG box and an E-box-dependent pathway.

Maximal serum stimulation of the c-fos serum response element requires both the serum response factor and a novel binding factor, SRE-binding protein

1992 ◽  
Vol 12 (10) ◽  
pp. 4769-4783
Author(s):  
A M Boulden ◽  
L J Sealy
Keyword(s):  
Long Terminal Repeat ◽  
Binding Site ◽  
Rous Sarcoma Virus ◽  
Serum Response Factor ◽  
Response Factor ◽  
High Affinity ◽  
Rous Sarcoma ◽  
Binding Factor ◽  
Sarcoma Virus ◽  
Serum Response

We have previously reported on the presence of a CArG motif at -100 in the Rous sarcoma virus long terminal repeat which binds an avian nuclear protein termed enhancer factor III (EFIII) (A. Boulden and L. Sealy, Virology 174:204-216, 1990). By all analyses, EFIII protein appears to be the avian homolog of the serum response factor (SRF). In this study, we identify a second CArG motif (EFIIIB) in the Rous sarcoma virus long terminal repeat enhancer at -162 and show only slightly lower binding affinity of the EFIII/SRF protein for this element in comparison with c-fos serum response element (SRE) and EFIII DNAs. Although all three elements bind the SRF with similar affinities, serum induction mediated by the c-fos SRE greatly exceeds that effected by the EFIII or EFIIIB sequence. We postulated that this difference in serum inducibility might result from binding of factors other than the SRF which occurs on the c-fos SRE but not on EFIII and EFIIIB sequences. Upon closer inspection of nuclear proteins which bind the c-fos SRE in chicken embryo fibroblast and NIH 3T3 nuclear extracts, we discovered another binding factor, SRE-binding protein (SRE BP), which fails to recognize EFIII DNA with high affinity. Competition analyses, methylation interference, and site-directed mutagenesis have determined that the SRE BP binding element overlaps and lies immediately 3' to the CArG box of the c-fos SRE. Mutation of the c-fos SRE so that it no longer binds SRE BP reduces serum inducibility to 33% of the wild-type level. Conversely, mutation of the EFIII sequence so that it binds SRE BP with high affinity results in a 400% increase in serum induction, with maximal stimulation equaling that of the c-fos SRE. We conclude that binding of both SRE BP and SRF is required for maximal serum induction. The SRE BP binding site coincides with the recently reported binding site for rNF-IL6 on the c-fos SRE. Nonetheless, we show that SRE BP is distinct from rNF-IL6, and identification of this novel factor is being pursued.

scholarly journals Transcriptional control of the chicken cardiac myosin light-chain gene is mediated by two AT-rich cis-acting DNA elements and binding of serum response factor.

1993 ◽  
Vol 13 (11) ◽  
pp. 6907-6918 ◽  
Author(s):  
N Papadopoulos ◽  
M T Crow
Keyword(s):  
Light Chain ◽  
Transcriptional Control ◽  
Myosin Light Chain ◽  
Nuclear Protein ◽  
Serum Response Factor ◽  
Protein Complexes ◽  
The Other ◽  
Response Factor ◽  
Cis Acting ◽  
Serum Response

Transcriptional control of the cardiac/slow skeletal alkali myosin light-chain (MLC1c/1s) gene is mediated, in part, by two highly conserved AT-rich cis-acting elements present in the immediate 5' flanking region. These elements cooperate to form an enhancer that can impart tissue specificity to heterologous promoters that are themselves not tissue specific in their pattern of expression. In the chicken, one of these elements matches the binding site for myocyte-specific enhancer-binding factor 2, while the other is a cis-acting element present in the transcriptional control regions of all striated alkali MLC genes (except MLC3f) and is referred to as the MLC box. The central decanucleotide core region of the MLC box closely resembles the CArG (CC[A/T]6GG) box of the serum response element, and the binding of muscle nuclear protein complexes to this element can be competed for with a synthetic serum response element. On the basis of their competition profiles and requirements for nonspecific competitor, two nuclear protein complexes, which compete for binding to the CArG-like region of the MLC box, have been identified. One of the complexes binds to a mutation of the CArG-like region that inactivates transcription of a linked reporter gene, while binding of the other complex is inhibited by this mutation. This latter complex reacts with an antibody to serum response factor (SRF) and exhibits the same binding characteristics as purified SRF. These results demonstrate that transcriptional control of the chicken MLC1c/1s gene resides in an upstream enhancer that is composed of two separate AT-rich elements, both of which are required to drive expression of a linked reporter gene. The binding of a nuclear protein complex containing SRF to one of these elements, the MLC box, is required for gene activation and apparently inhibited by other nuclear factors whose binding overlaps that of the SRF complex.

scholarly journals Serum response factor and protein-mediated DNA bending contribute to transcription of the dystrophin muscle-specific promoter.

1997 ◽  
Vol 17 (3) ◽  
pp. 1731-1743 ◽  
Author(s):  
F Galvagni ◽  
M Lestingi ◽  
E Cartocci ◽  
S Oliviero
Keyword(s):  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Dna Bending ◽  
Consensus Sequence ◽  
Response Factor ◽  
Flanking Sequence ◽  
Serum Stimulation ◽  
Carg Box ◽  
Transcriptional Complex ◽  
Serum Response

The minimal muscle-specific dystrophin promoter contains the consensus sequence CC(A/T)6GG, or the CArG element, which can be found in serum-inducible or muscle-specific promoters. The serum response factor (SRF), which mediates the transcriptional activation of the c-fos gene in response to serum stimulation, can bind to different CArG box elements, suggesting that it could be involved in muscle-constitutive transcription. Here we show that SRF binds to the dystrophin promoter and regulates its muscle-specific transcription. In transient transfections, an altered-binding-specificity SRF mutant restores the muscle-constitutive transcription of a dystrophin promoter with a mutation in its CArG box element. The muscle-constitutive transcription of the dystrophin promoter also requires the sequence GAAACC immediately downstream of the CArG box. This sequence is recognized by a novel DNA bending factor which was named dystrophin promoter-bending factor (DPBF). Mutations of the CArG flanking sequence abolish both DPBF binding and the promoter activity in muscle cells. Its replacement with a p62/ternary complex factor binding site changes the promoter specificity from muscle constitutive to serum responsive. These results show that, on the dystrophin promoter, the transcriptional activation induced by SRF requires the DNA bending induced by DPBF. The bending, next to the CArG box, could promote interactions between SRF and other proteins in the transcriptional complex.

scholarly journals Increased actin polymerization reduces the inhibition of serum response factor activity by Yin Yang 1

2002 ◽  
Vol 364 (2) ◽  
pp. 547-554 ◽  
Author(s):  
Peter D. ELLIS ◽  
Karen M. MARTIN ◽  
Colin RICKMAN ◽  
James C. METCALFE ◽  
Paul R. KEMP
Keyword(s):  
Binding Sites ◽  
Serum Response Factor ◽  
C2c12 Cells ◽  
Response Factor ◽  
P19 Cells ◽  
Lim Kinase ◽  
Yin Yang 1 ◽  
Yin Yang ◽  
Carg Box ◽  
Serum Response

Recent evidence has implicated CC(A/TrichG)GG (CArG) boxes, binding sites for serum response factor (SRF), in the regulation of expression of a number of genes in response to changes in the actin cytoskeleton. In many cases, the activity of SRF at CArG boxes is modulated by transcription factors binding to overlapping (e.g. Yin Yang 1, YY1) or adjacent (e.g. ets) binding sites. However, the mechanisms by which SRF activity is regulated by the cytoskeleton have not been determined. To investigate these mechanisms, we screened for cells that did or did not increase the activity of a fragment of the promoter for a smooth-muscle (SM)-specific gene SM22α, in response to changes in actin cytoskeletal polymerization induced by LIM kinase. These experiments showed that vascular SM cells (VSMCs) and C2C12 cells increased the activity of promoters containing at least one of the SM22α CArG boxes (CArG near) in response to LIM kinase, whereas P19 cells did not. Bandshift assays using a probe to CArG near showed that P19 cells lacked detectable YY1 DNA binding to the CArG box in contrast with the other two cell types. Expression of YY1 in P19 cells inhibited SM22α promoter activity and conferred responsiveness to LIM kinase. Mutation of the CArG box to inhibit YY1 or SRF binding indicated that both factors were required for the LIM kinase response in VSMCs and C2C12 cells. The data indicate that changes in the actin cytoskeletal organization modify SRF activity at CArG boxes by modulating YY1-dependent inhibition.

scholarly journals Maximal serum stimulation of the c-fos serum response element requires both the serum response factor and a novel binding factor, SRE-binding protein.

1992 ◽  
Vol 12 (10) ◽  
pp. 4769-4783 ◽  
Author(s):  
A M Boulden ◽  
L J Sealy
Keyword(s):  
Long Terminal Repeat ◽  
Binding Site ◽  
Rous Sarcoma Virus ◽  
Serum Response Factor ◽  
Response Factor ◽  
High Affinity ◽  
Rous Sarcoma ◽  
Binding Factor ◽  
Sarcoma Virus ◽  
Serum Response

We have previously reported on the presence of a CArG motif at -100 in the Rous sarcoma virus long terminal repeat which binds an avian nuclear protein termed enhancer factor III (EFIII) (A. Boulden and L. Sealy, Virology 174:204-216, 1990). By all analyses, EFIII protein appears to be the avian homolog of the serum response factor (SRF). In this study, we identify a second CArG motif (EFIIIB) in the Rous sarcoma virus long terminal repeat enhancer at -162 and show only slightly lower binding affinity of the EFIII/SRF protein for this element in comparison with c-fos serum response element (SRE) and EFIII DNAs. Although all three elements bind the SRF with similar affinities, serum induction mediated by the c-fos SRE greatly exceeds that effected by the EFIII or EFIIIB sequence. We postulated that this difference in serum inducibility might result from binding of factors other than the SRF which occurs on the c-fos SRE but not on EFIII and EFIIIB sequences. Upon closer inspection of nuclear proteins which bind the c-fos SRE in chicken embryo fibroblast and NIH 3T3 nuclear extracts, we discovered another binding factor, SRE-binding protein (SRE BP), which fails to recognize EFIII DNA with high affinity. Competition analyses, methylation interference, and site-directed mutagenesis have determined that the SRE BP binding element overlaps and lies immediately 3' to the CArG box of the c-fos SRE. Mutation of the c-fos SRE so that it no longer binds SRE BP reduces serum inducibility to 33% of the wild-type level. Conversely, mutation of the EFIII sequence so that it binds SRE BP with high affinity results in a 400% increase in serum induction, with maximal stimulation equaling that of the c-fos SRE. We conclude that binding of both SRE BP and SRF is required for maximal serum induction. The SRE BP binding site coincides with the recently reported binding site for rNF-IL6 on the c-fos SRE. Nonetheless, we show that SRE BP is distinct from rNF-IL6, and identification of this novel factor is being pursued.

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