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

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.

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Two distinct class A helix-loop-helix transcription factors, E2A and BETA1, form separate DNA binding complexes on the insulin gene E box.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)47336-x ◽

1994 ◽

Vol 269 (41) ◽

pp. 25936-25941

Author(s):

M Peyton ◽

L G Moss ◽

M J Tsai

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Insulin Gene ◽

Distinct Class ◽

Class A ◽

Helix Loop Helix ◽

E Box ◽

Dna Binding Complexes

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2439 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2439-2450 ◽

Cited By ~ 41

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.

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Determinants of Myogenic Specificity within MyoD Are Required for Noncanonical E Box Binding

Molecular and Cellular Biology ◽

10.1128/mcb.01700-06 ◽

2007 ◽

Vol 27 (16) ◽

pp. 5910-5920 ◽

Cited By ~ 37

Author(s):

Analeah B. Heidt ◽

Anabel Rojas ◽

Ian S. Harris ◽

Brian L. Black

Keyword(s):

Transcription Factors ◽

Promoter Regions ◽

Basic Domain ◽

Helix Loop Helix ◽

E Box ◽

Muscle Genes ◽

E Boxes ◽

Bhlh Transcription Factors

ABSTRACT The MyoD family of basic helix-loop-helix (bHLH) transcription factors has the remarkable ability to induce myogenesis in vitro and in vivo. This myogenic specificity has been mapped to two amino acids in the basic domain, an alanine and threonine, referred to as the myogenic code. These essential determinants of myogenic specificity are conserved in all MyoD family members from worms to humans, yet their function in myogenesis is unclear. Induction of the muscle transcriptional program requires that MyoD be able to locate and stably bind to sequences present in the promoter regions of critical muscle genes. Recent studies have shown that MyoD binds to noncanonical E boxes in the myogenin gene, a critical locus required for myogenesis, through interactions with resident heterodimers of the HOX-TALE transcription factors Pbx1A and Meis1. In the present study, we show that the myogenic code is required for MyoD to bind to noncanonical E boxes in the myogenin promoter and for the formation of a tetrameric complex with Pbx/Meis. We also show that these essential determinants of myogenesis are sufficient to confer noncanonical E box binding to the E12 basic domain. Thus, these data show that noncanonical E box binding correlates with myogenic potential, and we speculate that the myogenic code residues in MyoD function as myogenic determinants via their role in noncanonical E box binding and recognition.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2439-2450.1991 ◽

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.

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B-cell- and myocyte-specific E2-box-binding factors contain E12/E47-like subunits

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1156-1160.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1156-1160

Author(s):

C Murre ◽

A Voronova ◽

D Baltimore

Keyword(s):

Dna Binding ◽

B Cell ◽

Dna Sequence ◽

Binding Proteins ◽

Dna Binding Proteins ◽

Dimerization Domain ◽

Drosophila Development ◽

Helix Loop Helix ◽

Nuclear Factors ◽

Biochemical Function

Recent studies have identified a family of DNA-binding proteins that share a common DNA-binding and dimerization domain with the potential to form a helix-loop-helix (HLH) structure. Various HLH proteins can form heterodimers that bind to a common DNA sequence, termed the E2-box. We demonstrate here that E2-box-binding B-cell- and myocyte-specific nuclear factors contain subunits which are identical or closely related to ubiquitously expressed (E12/E47) HLH proteins. These biochemical function for E12/E47-like molecules in mammalian differentiation, similar to the genetically defined function of daughterless in Drosophila development.

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