scholarly journals E1A-mediated inhibition of myogenesis correlates with a direct physical interaction of E1A12S and basic helix-loop-helix proteins.

1993 ◽  
Vol 13 (8) ◽  
pp. 4714-4727 ◽  
Author(s):  
D A Taylor ◽  
V B Kraus ◽  
J J Schwarz ◽  
E N Olson ◽  
W E Kraus

The observation that adenovirus E1A gene products can inhibit differentiation of skeletal myocytes suggested that E1A may interfere with the activity of myogenic basic helix-loop-helix (bHLH) transcription factors. We have examined the ability of E1A to mediate repression of the muscle-specific creatine kinase (MCK) gene. Both the E1A12S and E1A13S products repressed MCK transcription in a concentration-dependent fashion. In contrast, amino-terminal deletion mutants (d2-36 and d15-35) of E1A12S were defective for repression. E1A12S also repressed expression of a promoter containing a multimer of the MCK high-affinity E box (the consensus site for myogenic bHLH protein binding) that was dependent, in C3H10T1/2 cells, on coexpression of a myogenin bHLH-VP16 fusion protein. A series of coprecipitation experiments with glutathione S-transferase fusion and in vitro-translated proteins demonstrated that E1A12S, but not amino-terminal E1A deletion mutants, could bind to full-length myogenin and E12 and to deletion mutants of myogenin and E12 that spare the bHLH domains. Thus, the bHLH domains of myogenin and E12, and the high-affinity E box, are targets for E1A-mediated repression of the MCK enhancer, and domains of E1A required for repression of muscle-specific gene transcription also mediate binding to bHLH proteins. We conclude that E1A mediates repression of muscle-specific gene transcription through its amino-terminal domain and propose that this may involve a direct physical interaction between E1A and the bHLH region of myogenic determination proteins.

1993 ◽  
Vol 13 (8) ◽  
pp. 4714-4727
Author(s):  
D A Taylor ◽  
V B Kraus ◽  
J J Schwarz ◽  
E N Olson ◽  
W E Kraus

The observation that adenovirus E1A gene products can inhibit differentiation of skeletal myocytes suggested that E1A may interfere with the activity of myogenic basic helix-loop-helix (bHLH) transcription factors. We have examined the ability of E1A to mediate repression of the muscle-specific creatine kinase (MCK) gene. Both the E1A12S and E1A13S products repressed MCK transcription in a concentration-dependent fashion. In contrast, amino-terminal deletion mutants (d2-36 and d15-35) of E1A12S were defective for repression. E1A12S also repressed expression of a promoter containing a multimer of the MCK high-affinity E box (the consensus site for myogenic bHLH protein binding) that was dependent, in C3H10T1/2 cells, on coexpression of a myogenin bHLH-VP16 fusion protein. A series of coprecipitation experiments with glutathione S-transferase fusion and in vitro-translated proteins demonstrated that E1A12S, but not amino-terminal E1A deletion mutants, could bind to full-length myogenin and E12 and to deletion mutants of myogenin and E12 that spare the bHLH domains. Thus, the bHLH domains of myogenin and E12, and the high-affinity E box, are targets for E1A-mediated repression of the MCK enhancer, and domains of E1A required for repression of muscle-specific gene transcription also mediate binding to bHLH proteins. We conclude that E1A mediates repression of muscle-specific gene transcription through its amino-terminal domain and propose that this may involve a direct physical interaction between E1A and the bHLH region of myogenic determination proteins.


Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 3984-3995 ◽  
Author(s):  
Andreas Himmelmann ◽  
Agostino Riva ◽  
Gaye Lynn Wilson ◽  
Brian P. Lucas ◽  
Claire Thevenin ◽  
...  

Abstract CD20 is a B-lineage–specific gene expressed at the pre–B-cell stage of B-cell development that disappears on differentiation to plasma cells. As such, it serves as an excellent paradigm for the study of lineage and developmental stage-specific gene expression. Using in vivo footprinting we identified two sites in the promoter at −45 and −160 that were occupied only in CD20+ B cells. The −45 site is an E box that binds basic helix-loop-helix-zipper proteins whereas the −160 site is a composite PU.1 and Pip binding site. Transfection studies with reporter constructs and various expression vectors verified the importance of these sites. The composite PU.1 and Pip site likely accounts for both lineage and stage-specific expression of CD20 whereas the CD20 E box binding proteins enhance overall promoter activity and may link the promoter to a distant enhancer.


1998 ◽  
Vol 18 (12) ◽  
pp. 6930-6938 ◽  
Author(s):  
I. Aksan ◽  
C. R. Goding

ABSTRACT The development of melanocytes, which are pigment-producing cells responsible for skin, hair, and eye color, is absolutely dependent on the action of the microphthalmia basic helix-loop-helix–leucine zipper (bHLH-LZ) transcription factor (Mi); mice lacking a functional Mi protein are entirely devoid of pigment cells. Mi has been shown to activate transcription of the tyrosinase,TRP-1, TRP-2, and QNR-71 genes through specific E-box elements, most notably the highly conserved M box. We investigated the mechanism which enables Mi to be recruited specifically to a restricted subset of E boxes in target promoters while being prevented from binding E-box elements in other promoters. We show both in vitro and in vivo that the presence of a T residue flanking a CATGTG E box is an essential determinant of the ability of Mi to bind DNA, and we successfully predict that the CATGTG E box from the P gene would not bind Mi. In contrast, no specific requirement for the sequences flanking a CACGTG E box was observed, and no binding to an atypical E box in the c-Kit promoter was detected. The relevance of these observations to the control of melanocyte-specific gene expression was highlighted by the fact that the E-box elements located in thetyrosinase, TRP-1, TRP-2, andQNR-71 promoters without exception possess a 5′ flanking T residue which is entirely conserved between species as diverse as man and turtle. The ability of Mi to discriminate between different E-box motifs provides a mechanism to restrict the repertoire of genes which are likely to be regulated by Mi and provides insight into the ability of bHLH-LZ transcription factors to achieve the specificity required for the precise coordination of transcription during development.


1997 ◽  
Vol 17 (7) ◽  
pp. 3924-3936 ◽  
Author(s):  
M P Gupta ◽  
C S Amin ◽  
M Gupta ◽  
N Hay ◽  
R Zak

The M-CAT binding factor transcription enhancer factor 1 (TEF-1) has been implicated in the regulation of several cardiac and skeletal muscle genes. Previously, we identified an E-box-M-CAT hybrid (EM) motif that is responsible for the basal and cyclic AMP-inducible expression of the rat cardiac alpha-myosin heavy chain (alpha-MHC) gene in cardiac myocytes. In this study, we report that two factors, TEF-1 and a basic helix-loop-helix leucine zipper protein, Max, bind to the alpha-MHC EM motif. We also found that Max was a part of the cardiac troponin T M-CAT-TEF-1 complex even when the DNA template did not contain an apparent E-box binding site. In the protein-protein interaction assay, a stable association of Max with TEF-1 was observed when glutathione S-transferase (GST)-TEF-1 or GST-Max was used to pull down in vitro-translated Max or TEF-1, respectively. In addition, Max was coimmunoprecipitated with TEF-1, thus documenting an in vivo TEF-1-Max interaction. In the transient transcription assay, overexpression of either Max or TEF-1 resulted a mild activation of the alpha-MHC-chloramphenicol acetyltransferase (CAT) reporter gene at lower concentrations and repression of this gene at higher concentrations. However, when Max and TEF-1 expression plasmids were transfected together, the repression mediated by a single expression plasmid was alleviated and a three- to fourfold transactivation of the alpha-MHC-CAT reporter gene was observed. This effect was abolished once the EM motif in the promoter-reporter construct was mutated, thus suggesting that the synergistic transactivation function of the TEF-1-Max heterotypic complex is mediated through binding of the complex to the EM motif. These results demonstrate a novel association between Max and TEF-1 and indicate a positive cooperation between these two factors in alpha-MHC gene regulation.


Development ◽  
1995 ◽  
Vol 121 (8) ◽  
pp. 2513-2523 ◽  
Author(s):  
J.C. Cross ◽  
M.L. Flannery ◽  
M.A. Blanar ◽  
E. Steingrimsson ◽  
N.A. Jenkins ◽  
...  

Trophoblast cells are the first lineage to form in the mammalian conceptus and mediate the process of implantation. We report the cloning of a basic helix-loop-helix (bHLH) transcription factor gene, Hxt, that is expressed in early trophoblast and in differentiated giant cells. A separate gene, Hed, encodes a related protein that is expressed in maternal deciduum surrounding the implantation site. Overexpression of Hxt in mouse blastomeres directed their development into trophoblast cells in blastocysts. In addition, overexpression of Hxt induced the differentiation of rat trophoblast (Rcho-1) stem cells as assayed by changes in cell adhesion and by activation of the placental lactogen-I gene promoter, a trophoblast giant cell-specific gene. In contrast, the negative HLH regulator, Id-1, inhibited Rcho-1 differentiation and placental lactogen-I transcription. These data demonstrate a role for HLH factors in regulating trophoblast development and indicate a positive role for Hxt in promoting the formation of trophoblast giant cells.


Development ◽  
1995 ◽  
Vol 121 (4) ◽  
pp. 1099-1110 ◽  
Author(s):  
P. Cserjesi ◽  
D. Brown ◽  
K.L. Ligon ◽  
G.E. Lyons ◽  
N.G. Copeland ◽  
...  

Members of the basic helix-loop-helix (bHLH) family of transcription factors have been shown to regulate growth and differentiation of numerous cell types. Cell-type-specific bHLH proteins typically form heterodimers with ubiquitous bHLH proteins, such as E12, and bind a DNA consensus sequence known as an E-box. We used the yeast two-hybrid system to screen mouse embryo cDNA libraries for cDNAs encoding novel cell-type-specific bHLH proteins that dimerize with E12. One of the cDNAs isolated encoded a novel bHLH protein, called scleraxis. During mouse embryogenesis, scleraxis transcripts were first detected between day 9.5 and 10.5 post coitum (p.c.) in the sclerotome of the somites and in mesenchymal cells in the body wall and limb buds. Subsequently, scleraxis was expressed at high levels within mesenchymal precursors of the axial and appendicular skeleton and in cranial mesenchyme in advance of chondrogenesis; its expression pattern in these cell types foreshadowed the developing skeleton. Prior to formation of the embryonic cartilaginous skeleton, scleraxis expression declined to low levels. As development proceeded, high levels of scleraxis expression became restricted to regions where cartilage and connective tissue formation take place. Scleraxis bound the E-box consensus sequence as a heterodimer with E12 and activated transcription of a reporter gene linked to its DNA-binding site. The expression pattern, DNA-binding properties and transcriptional activity of scleraxis suggest that it is a regulator of gene expression within mesenchymal cell lineages that give rise to cartilage and connective tissue.


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