scholarly journals Heterodimers of myogenic helix-loop-helix regulatory factors and E12 bind a complex element governing myogenic induction of the avian cardiac alpha-actin promoter.

1991 ◽  
Vol 11 (5) ◽  
pp. 2439-2450 ◽  
Author(s):  
B A French ◽  
K L Chow ◽  
E N Olson ◽  
R J Schwartz
Keyword(s):  
Dna Binding ◽  
Muscle Development ◽  
Specific Expression ◽  
Cis Acting ◽  
Helix Loop Helix ◽  
E Box ◽  
Carg Box ◽  
Actin Promoter ◽  
E Boxes ◽  
Alpha Actin

Recent studies have shown that two genes regulating myogenesis (MyoD and myogenin) are coexpressed with cardiac alpha-actin during early stages of skeletal muscle development. Myogenin and MyoD are members of a family of regulatory proteins which share a helix-loop-helix (HLH) motif required for dimerization and DNA binding. Myogenin and MyoD form heterodimers with the ubiquitous HLH protein E12 which bind cis-acting DNA elements that have an E box (CANNTG) at their core. E boxes are present in the control regions of numerous muscle-specific genes, although their functional importance in regulating many of these genes has not yet been evaluated. In this report we examine the possibility that myogenin (or MyoD) directly transactivates the cardiac alpha-actin promoter. Heterodimers of myogenin and E12 (or MyoD and E12) specifically bound a restriction fragment extending from -200 to -103 relative to the start of cardiac alpha-actin transcription. Methylation interference footprints pinpointed the site of interaction to an E box immediately adjacent to a previously identified CArG box (CArG3). Site-directed mutations to the DNA-binding site revealed that either an intact E box or an intact CArG3 is required for induction of the cardiac alpha-actin promoter in myoblasts and for transactivation by myogenin in cotransfected fibroblasts. However, deletion and substitution experiments indicate that the complex E box/CArG3 element alone does not confer muscle-specific expression to a minimal promoter. These results suggest that direct and indirect pathways involving multiple cis-acting elements mediate the induction of the cardiac alpha-actin promoter by myogenin and MyoD.

Heterodimers of myogenic helix-loop-helix regulatory factors and E12 bind a complex element governing myogenic induction of the avian cardiac alpha-actin promoter

1991 ◽  
Vol 11 (5) ◽  
pp. 2439-2450
Author(s):  
B A French ◽  
K L Chow ◽  
E N Olson ◽  
R J Schwartz
Keyword(s):  
Dna Binding ◽  
Muscle Development ◽  
Specific Expression ◽  
Cis Acting ◽  
Helix Loop Helix ◽  
E Box ◽  
Carg Box ◽  
Actin Promoter ◽  
E Boxes ◽  
Alpha Actin

Recent studies have shown that two genes regulating myogenesis (MyoD and myogenin) are coexpressed with cardiac alpha-actin during early stages of skeletal muscle development. Myogenin and MyoD are members of a family of regulatory proteins which share a helix-loop-helix (HLH) motif required for dimerization and DNA binding. Myogenin and MyoD form heterodimers with the ubiquitous HLH protein E12 which bind cis-acting DNA elements that have an E box (CANNTG) at their core. E boxes are present in the control regions of numerous muscle-specific genes, although their functional importance in regulating many of these genes has not yet been evaluated. In this report we examine the possibility that myogenin (or MyoD) directly transactivates the cardiac alpha-actin promoter. Heterodimers of myogenin and E12 (or MyoD and E12) specifically bound a restriction fragment extending from -200 to -103 relative to the start of cardiac alpha-actin transcription. Methylation interference footprints pinpointed the site of interaction to an E box immediately adjacent to a previously identified CArG box (CArG3). Site-directed mutations to the DNA-binding site revealed that either an intact E box or an intact CArG3 is required for induction of the cardiac alpha-actin promoter in myoblasts and for transactivation by myogenin in cotransfected fibroblasts. However, deletion and substitution experiments indicate that the complex E box/CArG3 element alone does not confer muscle-specific expression to a minimal promoter. These results suggest that direct and indirect pathways involving multiple cis-acting elements mediate the induction of the cardiac alpha-actin promoter by myogenin and MyoD.

scholarly journals Myogenin and MEF2 function synergistically to activate the MRF4 promoter during myogenesis.

1995 ◽  
Vol 15 (5) ◽  
pp. 2707-2718 ◽  
Author(s):  
P S Naidu ◽  
D C Ludolph ◽  
R Q To ◽  
T J Hinterberger ◽  
S F Konieczny
Keyword(s):  
Molecular Mechanisms ◽  
Muscle Development ◽  
Expression Patterns ◽  
Proximal Promoter ◽  
Sufficient Information ◽  
Specific Response ◽  
Specific Expression ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Box

The basic helix-loop-helix muscle regulatory factor (MRF) gene family encodes four distinct muscle-specific transcription factors known as MyoD, myogenin, Myf-5, and MRF4. These proteins represent key regulatory factors that control many aspects of skeletal myogenesis. Although the MRFs often exhibit overlapping functional activities, their distinct expression patterns during embryogenesis suggest that each protein plays a unique role in controlling aspects of muscle development. As a first step in determining how MRF4 gene expression is developmentally regulated, we examined the ability of the MRF4 gene to be expressed in a muscle-specific fashion in vitro. Our studies show that the proximal MRF4 promoter contains sufficient information to direct muscle-specific expression. Located within the proximal promoter are a single MEF2 site and E box that are required for maximum MRF4 expression. Mutation of the MEF2 site or E box severely impairs the ability of this promoter to produce a muscle-specific response. In addition, the MEF2 site and E box function in concert to synergistically activate the MRF4 gene in nonmuscle cells coexpressing MEF2 and myogenin proteins. Thus, the MRF4 promoter is regulated by the MEF2 and basic helix-loop-helix MRF protein family through a cross-regulatory circuitry. Surprisingly, the MRF4 promoter itself is not transactivated by MRF4, suggesting that this MRF gene is not subject to an autoregulatory pathway as previously implied by other studies. Understanding the molecular mechanisms regulating expression of each MRF gene is central to fully understanding how these factors control developmental events.

scholarly journals Cell-specific helix-loop-helix factor required for pituitary expression of the pro-opiomelanocortin gene.

1993 ◽  
Vol 13 (4) ◽  
pp. 2342-2353 ◽  
Author(s):  
M Therrien ◽  
J Drouin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Proteins ◽  
Specific Activity ◽  
Pituitary Cells ◽  
Immunoglobulin Gene ◽  
Helix Loop Helix ◽  
E Box ◽  
E Boxes ◽  
Hlh Transcription Factors

Pro-opiomelanocortin (POMC)-expressing cells appear to be the first pituitary cells committed to hormone production. In this work, we have identified an element of the POMC promoter which confers cell-specific activity. This element did not exhibit any activity on its own and required at least one other element of the promoter to manifest its cell-specific activity. Fine mutagenesis of this element indicated that a CANNTG motif is responsible for activity. This E-box motif is typical of binding sites for helix-loop-helix (HLH) transcription factors; however, the POMC cell-specific E box cannot be replaced by other E boxes like the kappa E2 site of the immunoglobulin gene or a muscle-specific E box. Similar E boxes which are present in the insulin gene promoter were shown to contribute to the pancreatic specificity of the insulin promoter. However, E-box-binding proteins found in nuclear extracts from POMC-expressing AtT-20 cells and from insulin-expressing cells have different electrophoretic mobilities. The AtT-20 proteins were named CUTE (for corticotroph upstream transcription element-binding) proteins, and they were not found in any other cells. CUTE proteins have DNA-binding properties characteristic of HLH transcription factors. Overexpression of the dominant negative HLH protein Id or of the ubiquitous positive HLH factor rat Pan-2 decreased or augmented POMC promoter activity, respectively. These observations are consistent with the hypothesis that CUTE factors might be heterodimers. This hypothesis was further supported by antibody shift experiments and by abrogation of DNA binding in the presence of bacterially expressed Id protein. Thus, the cell-specific CUTE proteins and their binding site in the POMC promoter appear to be important determinants for cell specificity of this promoter. The requirement for HLH factors in POMC transcription also presents the possibility that these factors are involved in differentiation of pituitary cells, in analogy with the role of HLH factors in muscle development.

scholarly journals Cell-specific helix-loop-helix factor required for pituitary expression of the pro-opiomelanocortin gene

1993 ◽  
Vol 13 (4) ◽  
pp. 2342-2353
Author(s):  
M Therrien ◽  
J Drouin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Proteins ◽  
Specific Activity ◽  
Pituitary Cells ◽  
Immunoglobulin Gene ◽  
Helix Loop Helix ◽  
E Box ◽  
E Boxes ◽  
Hlh Transcription Factors

Pro-opiomelanocortin (POMC)-expressing cells appear to be the first pituitary cells committed to hormone production. In this work, we have identified an element of the POMC promoter which confers cell-specific activity. This element did not exhibit any activity on its own and required at least one other element of the promoter to manifest its cell-specific activity. Fine mutagenesis of this element indicated that a CANNTG motif is responsible for activity. This E-box motif is typical of binding sites for helix-loop-helix (HLH) transcription factors; however, the POMC cell-specific E box cannot be replaced by other E boxes like the kappa E2 site of the immunoglobulin gene or a muscle-specific E box. Similar E boxes which are present in the insulin gene promoter were shown to contribute to the pancreatic specificity of the insulin promoter. However, E-box-binding proteins found in nuclear extracts from POMC-expressing AtT-20 cells and from insulin-expressing cells have different electrophoretic mobilities. The AtT-20 proteins were named CUTE (for corticotroph upstream transcription element-binding) proteins, and they were not found in any other cells. CUTE proteins have DNA-binding properties characteristic of HLH transcription factors. Overexpression of the dominant negative HLH protein Id or of the ubiquitous positive HLH factor rat Pan-2 decreased or augmented POMC promoter activity, respectively. These observations are consistent with the hypothesis that CUTE factors might be heterodimers. This hypothesis was further supported by antibody shift experiments and by abrogation of DNA binding in the presence of bacterially expressed Id protein. Thus, the cell-specific CUTE proteins and their binding site in the POMC promoter appear to be important determinants for cell specificity of this promoter. The requirement for HLH factors in POMC transcription also presents the possibility that these factors are involved in differentiation of pituitary cells, in analogy with the role of HLH factors in muscle development.

Pancreatic beta-cell-type-specific transcription of the insulin gene is mediated by basic helix-loop-helix DNA-binding proteins

1991 ◽  
Vol 11 (3) ◽  
pp. 1734-1738 ◽  
Author(s):  
S R Cordle ◽  
E Henderson ◽  
H Masuoka ◽  
P A Weil ◽  
R Stein
Keyword(s):  
Dna Binding ◽  
Beta Cell ◽  
Pancreatic Beta Cell ◽  
Insulin Gene ◽  
Cell Type ◽  
Specific Expression ◽  
Cis Acting ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Cell Type Specific

The pancreatic beta-cell-specific expression of the insulin gene is mediated, at least in part, by the interaction of unique trans-acting beta-cell factors with a cis-acting DNA element found within the insulin enhancer (5'-GC CATCTG-3'; referred to as the insulin control element [ICE]) present in the rat insulin II gene between positions -100 and -91. This sequence element contains the consensus binding site for a group of DNA-binding transcription factors called basic helix-loop-helix proteins (B-HLH). As a consequence of the similarity of the ICE with the DNA sequence motif associated with the cis-acting elements of the B-HLH class of binding proteins (CANNTG), the ability of this class of proteins to regulate cell-type-specific expression of the insulin gene was addressed. Cotransfection experiments indicated that overexpression of Id, a negative regulator of B-HLH protein function, inhibits ICE-mediated activity. Antibody to the E12/E47 B-HLH proteins attenuated the formation, in vitro, of a previously described (J. Whelan, S. R. Cordle, E. Henderson, P. A. Weil, and R. Stein, Mol. Cell. Biol. 10:1564-1572, 1990) beta-cell-specific activator factor(s)-ICE DNA complex. Both of these B-HLH proteins (E12 and E47) bound efficiently and specifically to the ICE sequences. The role of B-HLH proteins in mediating pancreatic beta-cell-specific transcription of the insulin gene is discussed.

scholarly journals Pancreatic beta-cell-type-specific transcription of the insulin gene is mediated by basic helix-loop-helix DNA-binding proteins.

1991 ◽  
Vol 11 (3) ◽  
pp. 1734-1738 ◽  
Author(s):  
S R Cordle ◽  
E Henderson ◽  
H Masuoka ◽  
P A Weil ◽  
R Stein
Keyword(s):  
Dna Binding ◽  
Beta Cell ◽  
Pancreatic Beta Cell ◽  
Insulin Gene ◽  
Cell Type ◽  
Specific Expression ◽  
Cis Acting ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Cell Type Specific

The pancreatic beta-cell-specific expression of the insulin gene is mediated, at least in part, by the interaction of unique trans-acting beta-cell factors with a cis-acting DNA element found within the insulin enhancer (5'-GC CATCTG-3'; referred to as the insulin control element [ICE]) present in the rat insulin II gene between positions -100 and -91. This sequence element contains the consensus binding site for a group of DNA-binding transcription factors called basic helix-loop-helix proteins (B-HLH). As a consequence of the similarity of the ICE with the DNA sequence motif associated with the cis-acting elements of the B-HLH class of binding proteins (CANNTG), the ability of this class of proteins to regulate cell-type-specific expression of the insulin gene was addressed. Cotransfection experiments indicated that overexpression of Id, a negative regulator of B-HLH protein function, inhibits ICE-mediated activity. Antibody to the E12/E47 B-HLH proteins attenuated the formation, in vitro, of a previously described (J. Whelan, S. R. Cordle, E. Henderson, P. A. Weil, and R. Stein, Mol. Cell. Biol. 10:1564-1572, 1990) beta-cell-specific activator factor(s)-ICE DNA complex. Both of these B-HLH proteins (E12 and E47) bound efficiently and specifically to the ICE sequences. The role of B-HLH proteins in mediating pancreatic beta-cell-specific transcription of the insulin gene is discussed.

scholarly journals A combination of closely associated positive and negative cis-acting promoter elements regulates transcription of the skeletal alpha-actin gene.

1990 ◽  
Vol 10 (2) ◽  
pp. 528-538 ◽  
Author(s):  
K L Chow ◽  
R J Schwartz
Keyword(s):  
Transcriptional Activity ◽  
Primary Cultures ◽  
Gene Promoter ◽  
Regulatory Sequence ◽  
Actin Gene ◽  
Cell Type ◽  
Specific Expression ◽  
Cis Acting ◽  
Cell Type Specific Expression ◽  
Alpha Actin

The chicken skeletal alpha-actin gene promoter region provides at least a 75-fold-greater transcriptional activity in muscle cells than in fibroblasts. The cis-acting sequences required for cell type-restricted expression within this 200-base-pair (bp) region were elucidated by chloramphenicol acetyltransferase assays of site-directed Bg/II linker-scanning mutations transiently transfected into primary cultures. Four positive cis-acting elements were identified and are required for efficient transcriptional activity in myogenic cells. These elements, conserved across vertebrate evolution, include the ATAAAA box (-24 bp), paired CCAAT-box-associated repeats (CBARs; at -83 bp and -127 bp), and the upstream T+A-rich regulatory sequence (at -176 bp). Basal transcriptional activity in fibroblasts was not as dependent on the upstream CBAR or regions of the upstream T+A-rich regulatory sequence. Transfection experiments provided evidence that positive regulatory factors required for alpha-actin expression in fibroblasts are limiting. In addition, negative cis-acting elements were detected and found closely associated with the G+C-rich sequences that surround the paired CBARs. Negative elements may have a role in restricting developmentally timed expression in myoblasts and appear to inhibit promoter activity in nonmyogenic cells. Cell type-specific expression of the skeletal alpha-actin gene promoter is regulated by combinatorial and possibly competitive interactions between multiple positive and negative cis-acting elements.

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.

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