scholarly journals Intramolecular Regulation of MyoD Activation Domain Conformation and Function

1998 ◽  
Vol 18 (9) ◽  
pp. 5478-5484 ◽  
Author(s):  
Jing Huang ◽  
Hal Weintraub ◽  
Larry Kedes
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Helix Loop Helix ◽  
Dna Binding Specificity ◽  
E Box ◽  
And Function ◽  
Bhlh Proteins ◽  
Basic Region

ABSTRACT The MyoD family of basic helix-loop-helix (bHLH) proteins is required for myogenic determination and differentiation. The basic region carries the myogenic code and DNA binding specificity, while the N terminus contains a potent transcriptional activation domain. Myogenic activation is abolished when the basic region, bound to a myogenic E box, carries a mutation of Ala-114. It has been proposed that DNA binding of the MyoD basic region leads to recruitment of a recognition factor that unmasks the activation domain. Here we demonstrate that an A114N mutant exhibits an altered conformation in the basic region and that this local conformational difference can lead to a more global change affecting the conformation of the activation domain. This suggests that the deleterious effects of this class of mutations may result directly from defective conformation. Thus, the activation domain is unmasked only upon DNA binding by the correct basic region. Such a coupled conformational relationship may have evolved to restrict myogenic specificity to a small number of bHLH proteins among many with diverse functions yet with DNA binding specificities known to be similar.

Misexpression of dHAND induces ectopic digits in the developing limb bud in the absence of direct DNA binding

Development ◽  
2002 ◽  
Vol 129 (13) ◽  
pp. 3077-3088 ◽  
Author(s):  
David G. McFadden ◽  
John McAnally ◽  
James A. Richardson ◽  
Jeroen Charité ◽  
Eric N. Olson
Keyword(s):  
Dna Binding ◽  
Sonic Hedgehog ◽  
Transcriptional Activation ◽  
Function Analysis ◽  
Limb Bud ◽  
Activation Domain ◽  
Limb Patterning ◽  
Transcriptional Activation Domain ◽  
Wide Range ◽  
E Box

Basic helix-loop-helix (bHLH) transcription factors control developmental decisions in a wide range of embryonic cell types. The HLH motif mediates homo- and heterodimerization, which juxtaposes the basic regions within the dimeric complex to form a bipartite DNA binding domain that recognizes a DNA consensus sequence known as an E-box. eHAND and dHAND (also known as HAND1 and HAND2) are closely related bHLH proteins that control cardiac, craniofacial and limb development. Within the developing limb, dHAND expression encompasses the zone of polarizing activity in the posterior region, where it has been shown to be necessary and sufficient to induce the expression of the morphogen sonic hedgehog. Misexpression of dHAND in the anterior compartment of the limb bud induces ectopic expression of sonic hedgehog, with resulting preaxial polydactyly and mirror image duplications of posterior digits. To investigate the potential transcriptional mechanisms involved in limb patterning by dHAND, we have performed a structure-function analysis of the protein in cultured cells and ectopically expressed dHAND mutant proteins in the developing limbs of transgenic mice. We show that an N-terminal transcriptional activation domain, and the bHLH region, are required for E-box-dependent transcription in vitro. Remarkably, however, digit duplication by dHAND requires neither the transcriptional activation domain nor the basic region, but only the HLH motif. eHAND has a similar limb patterning activity to dHAND in these misexpression experiments, indicating a conserved function of the HLH regions of these proteins. These findings suggest that dHAND may act via novel transcriptional mechanisms mediated by protein-protein interactions independent of direct DNA binding.

scholarly journals The basic region of myogenin cooperates with two transcription activation domains to induce muscle-specific transcription.

1992 ◽  
Vol 12 (1) ◽  
pp. 266-275 ◽  
Author(s):  
J J Schwarz ◽  
T Chakraborty ◽  
J Martin ◽  
J M Zhou ◽  
E N Olson
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
E Box ◽  
Myogenic Program ◽  
Basic Region

Myogenin is a skeletal muscle-specific transcription factor that can activate myogenesis when introduced into a variety of nonmuscle cell types. Activation of the myogenic program by myogenin is dependent on its binding to a DNA sequence known as an E box, which is associated with numerous muscle-specific genes. Myogenin shares homology with MyoD and other myogenic regulatory factors within a basic region and a helix-loop-helix (HLH) motif that mediate DNA binding and dimerization, respectively. Here we show that the basic region-HLH motif of myogenin alone lacks transcriptional activity and is dependent on domains in the amino and carboxyl termini to activate transcription. Analysis of these N- and C-terminal domains through creation of chimeras with the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that they act as strong transcriptional activators. These transcription activation domains are dependent for activity on a specific amino acid sequence within the basic region, referred to as the myogenic recognition motif (MRM), when an E box is the target for DNA binding. However, the activation domains function independent of the MRM when DNA binding is mediated through a heterologous DNA-binding domain. The activation domain of the acidic coactivator VP16 can substitute for the myogenin activation domains and restore strong myogenic activity to the basic region-HLH motif. Within a myogenin-VP16 chimera, however, the VP16 activation domain also relies on the MRM for activation of the myogenic program. These findings reveal that DNA binding and transcriptional activation are separable functions, encoded by different domains of myogenin, but that the activity of the transcriptional activation domains is influenced by the DNA-binding domain. Activation of muscle-specific transcription requires collaboration between the DNA-binding and activation domains of myogenin and is dependent on events in addition to DNA binding.

scholarly journals A chimeric enhancer-of-split transcriptional activator drives neural development and achaete-scute expression.

1997 ◽  
Vol 17 (8) ◽  
pp. 4355-4362 ◽  
Author(s):  
G Jiménez ◽  
D Ish-Horowicz
Keyword(s):  
Neural Development ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Wild Type ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Helix Loop Helix ◽  
Inhibition Process ◽  
Bhlh Proteins ◽  
Enhancer Of Split

Drosophila melanogaster neurogenesis requires the opposing activities of two sets of basic helix-loop-helix (bHLH) proteins: proneural proteins, which confer on cells the ability to become neural precursors, and the Enhancer-of-split [E(spl)] proteins, which restrict such potential as part of the lateral inhibition process. Here, we test if E(spl) proteins function as promoter-bound repressors by examining the effects on neurogenesis of an E(spl) derivative containing a heterologous transcriptional activation domain [E(spl) m7Act (m7Act)]. In contrast to the wild-type E(spl) proteins, m7Act efficiently induces neural development, indicating that it binds to and activates target genes normally repressed by E(spl). Mutations in the basic domain disrupt m7Act activity, suggesting that its effects are mediated through direct DNA binding. m7Act causes ectopic transcription of the proneural achaete and scute genes. Our results support a model in which E(spl) proteins normally regulate neurogenesis by direct repression of genes at the top of the neural determination pathway.

The basic region of myogenin cooperates with two transcription activation domains to induce muscle-specific transcription

1992 ◽  
Vol 12 (1) ◽  
pp. 266-275
Author(s):  
J J Schwarz ◽  
T Chakraborty ◽  
J Martin ◽  
J M Zhou ◽  
E N Olson
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
E Box ◽  
Myogenic Program ◽  
Basic Region

Myogenin is a skeletal muscle-specific transcription factor that can activate myogenesis when introduced into a variety of nonmuscle cell types. Activation of the myogenic program by myogenin is dependent on its binding to a DNA sequence known as an E box, which is associated with numerous muscle-specific genes. Myogenin shares homology with MyoD and other myogenic regulatory factors within a basic region and a helix-loop-helix (HLH) motif that mediate DNA binding and dimerization, respectively. Here we show that the basic region-HLH motif of myogenin alone lacks transcriptional activity and is dependent on domains in the amino and carboxyl termini to activate transcription. Analysis of these N- and C-terminal domains through creation of chimeras with the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that they act as strong transcriptional activators. These transcription activation domains are dependent for activity on a specific amino acid sequence within the basic region, referred to as the myogenic recognition motif (MRM), when an E box is the target for DNA binding. However, the activation domains function independent of the MRM when DNA binding is mediated through a heterologous DNA-binding domain. The activation domain of the acidic coactivator VP16 can substitute for the myogenin activation domains and restore strong myogenic activity to the basic region-HLH motif. Within a myogenin-VP16 chimera, however, the VP16 activation domain also relies on the MRM for activation of the myogenic program. These findings reveal that DNA binding and transcriptional activation are separable functions, encoded by different domains of myogenin, but that the activity of the transcriptional activation domains is influenced by the DNA-binding domain. Activation of muscle-specific transcription requires collaboration between the DNA-binding and activation domains of myogenin and is dependent on events in addition to DNA binding.

scholarly journals Multiple Roles for the MyoD Basic Region in Transmission of Transcriptional Activation Signals and Interaction with MEF2

1998 ◽  
Vol 18 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Brian L. Black ◽  
Jeffery D. Molkentin ◽  
Eric N. Olson
Keyword(s):  
Transcription Factors ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Activation Domain ◽  
Basic Domain ◽  
Helix Loop Helix ◽  
Bhlh Proteins ◽  
Basic Region

ABSTRACT Establishment of skeletal muscle lineages is controlled by the MyoD family of basic helix-loop-helix (bHLH) transcription factors. The ability of these factors to initiate myogenesis is dependent on two conserved amino acid residues, alanine and threonine, in the basic domains of these factors. It has been postulated that these two residues may be responsible for the initiation of myogenesis via interaction with an essential myogenic cofactor. The myogenic bHLH proteins cooperatively activate transcription and myogenesis through protein-protein interactions with members of the myocyte enhancer factor 2 (MEF2) family of MADS domain transcription factors. MyoD-E12 heterodimers interact with MEF2 proteins to synergistically activate myogenesis, while homodimers of E12, which lack the conserved alanine and threonine residues in the basic domain, do not interact with MEF2. We have examined whether the myogenic alanine and threonine in the MyoD basic region are required for interaction with MEF2. Here, we show that substitution of the MyoD basic domain with that of E12 does not prevent interaction with MEF2. Instead, the inability of alanine-threonine mutants of MyoD to initiate myogenesis is due to a failure to transmit transcriptional activation signals provided either from the MyoD or the MEF2 activation domain. This defect in transcriptional transmission can be overcome by substitution of the MyoD or the MEF2 activation domain with the VP16 activation domain. These results demonstrate that myogenic bHLH-MEF2 interaction can be uncoupled from transcriptional activation and support the idea that the myogenic residues in myogenic bHLH proteins are essential for transmission of a transcriptional activation signal.

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

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.

scholarly journals Determination of the consensus DNA-binding sequence and a transcriptional activation domain for ESE-2

2006 ◽  
Vol 398 (3) ◽  
pp. 497-507 ◽  
Author(s):  
Yeon Sook Choi ◽  
Satrajit Sinha
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Regulatory Elements ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Ets Proteins ◽  
Flanking Sequences ◽  
Binding Sequence

The ESE (epithelium-specific Ets) subfamily of Ets transcription factors plays an important role in regulating gene expression in a variety of epithelial cell types. Although ESE proteins have been shown to bind to regulatory elements of some epithelial genes, the optimal DNA-binding sequence has not been experimentally ascertained for any member of the ESE subfamily of transcription factors. This has made the identification and validation of their targets difficult. We are studying ESE-2 (Elf5), which is highly expressed in epithelial cells of many tissues including skin keratinocytes. Here, we identify the preferred DNA-binding site of ESE-2 by performing CASTing (cyclic amplification and selection of targets) experiments. Our analysis shows that the optimal ESE-2 consensus motif consists of a GGA core and an AT-rich 5′- and 3′-flanking sequences. Mutational and competition experiments demonstrate that the flanking sequences that confer high DNA-binding affinity for ESE-2 show considerable differences from the known consensus DNA-binding sites of other Ets proteins, thus reinforcing the idea that the flanking sequences may impart recognition specificity for Ets proteins. In addition, we have identified a novel isoform of murine ESE-2, ESE-2L, that is generated by use of a hitherto unreported new exon and an alternate promoter. Interestingly, transient transfection assays with an optimal ESE-2 responsive reporter show that both ESE-2 and ESE-2L are weak transactivators. However, similar studies utilizing GAL4 chimaeras of ESE-2 demonstrate that while the DNA-binding ETS (E twenty-six) domain functions as a repressor, the PNT (pointed domain) of ESE-2 can act as a potent transcriptional activation domain. This novel transactivating property of PNT is also shared by ESE-3, another ESE family member. Identification of the ESE-2 consensus site and characterization of the transcriptional activation properties of ESE-2 shed new light on its potential as a regulator of target genes.

scholarly journals Stress-induced binding of the transcriptional factor CHOP to a novel DNA control element.

1996 ◽  
Vol 16 (4) ◽  
pp. 1479-1489 ◽  
Author(s):  
M Ubeda ◽  
X Z Wang ◽  
H Zinszner ◽  
I Wu ◽  
J F Habener ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Activity ◽  
Control Element ◽  
Core Element ◽  
Transcriptional Activation Domain ◽  
Dna Binding Activity ◽  
Basic Region

CHOP (GADD153) is a mammalian nuclear protein that dimerizes with members of the C/EBP family of transcriptional factors. Absent under normal conditions, CHOP is induced by the stress encountered during nutrient deprivation, the acute-phase response, and treatment of cells with certain toxins. The basic region of CHOP deviates considerably in sequence from that of other C/EBP proteins, and CHOP-C/EBP heterodimers are incapable of binding to a common class of C/EBP sites. With respect to such sites, CHOP serves as an inhibitor of the activity of C/EBP proteins. However, recent studies indicate that certain functions of CHOP, such as the induction of growth arrest by overexpression of the wild-type protein and oncogenic transformation by the TLS-CHOP fusion protein, require an intact basic region, suggesting that DNA binding by CHOP may be implicated in these activities. In this study an in vitro PCR-based selection assay was used to identify sequences bound by CHOP-C/EBP dimers. These sequences were found to contain a unique core element PuPuPuTGCAAT(A/C)CCC. Competition in DNA-binding assays, DNase 1 footprint analysis, and methylation interference demonstrate that the binding is sequence specific. Deletions in the basic region of CHOP lead to a loss of DNA binding, suggesting that CHOP participates in this process. Stress induction in NIH 3T3 cells leads to the appearance of CHOP-containing DNA-binding activity. CHOP is found to contain a transcriptional activation domain which is inducible by cellular stress, lending further support to the notion that the protein can function as a positively acting transcription factor. We conclude that CHOP may serve a dual role both as an inhibitor of the ability of C/EBP proteins to activate some target genes and as a direct activator of others.

scholarly journals Molecular Distinction between Specification and Differentiation in the Myogenic Basic Helix-Loop-Helix Transcription Factor Family

2001 ◽  
Vol 21 (7) ◽  
pp. 2404-2412 ◽  
Author(s):  
Donald A. Bergstrom ◽  
Stephen J. Tapscott
Keyword(s):  
Skeletal Muscle ◽  
Transcriptional Activation ◽  
Molecular Basis ◽  
Alpha Helix ◽  
Endogenous Gene ◽  
Transcriptional Activation Domain ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Muscle Genes ◽  
Bhlh Proteins

ABSTRACT The myogenic basic helix-loop-helix (bHLH) proteins regulate both skeletal muscle specification and differentiation: MyoD and Myf5 establish the muscle lineage, whereas myogenin mediates differentiation. Previously, we demonstrated that MyoD was more efficient than myogenin at initiating the expression of skeletal muscle genes, and in this study we present the molecular basis for this difference. A conserved amphipathic alpha-helix in the carboxy terminus of the myogenic bHLH proteins has distinct activities in MyoD and myogenin: the MyoD helix facilitates the initiation of endogenous gene expression, whereas the myogenin helix functions as a general transcriptional activation domain. Thus, the alternate use of a similar motif for gene initiation and activation provides a molecular basis for the distinction between specification and differentiation within the myogenic bHLH gene family.

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