scholarly journals Transcriptional Activation by ETS and Leucine Zipper-Containing Basic Helix-Loop-Helix Proteins

1999 ◽  
Vol 19 (4) ◽  
pp. 2946-2957 ◽  
Author(s):  
Gang Tian ◽  
Batu Erman ◽  
Haruhiko Ishii ◽  
Samudra S. Gangopadhyay ◽  
Ranjan Sen
Keyword(s):  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Transcription Activation ◽  
Chain Gene ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Gene Enhancer ◽  
Novel Interactions ◽  
Heavy Chain Gene Enhancer

ABSTRACT The immunoglobulin μ heavy-chain gene enhancer contains closely juxtaposed binding sites for ETS and leucine zipper-containing basic helix-loop-helix (bHLH-zip) proteins. To understand the μ enhancer function, we have investigated transcription activation by the combination of ETS and bHLH-zip proteins. The bHLH-zip protein TFE3, but not USF, cooperated with the ETS domain proteins PU.1 and Ets-1 to activate a tripartite domain of this enhancer. Deletion mutants were used to identify the domains of the proteins involved. Both TFE3 and USF enhanced Ets-1 DNA binding in vitro by relieving the influence of an autoinhibitory domain in Ets-1 by direct protein-protein associations. Several regions of Ets-1 were found to be necessary, whereas the bHLH-zip domain was sufficient for this effect. Our studies define novel interactions between ETS and bHLH-zip proteins that may regulate combinatorial transcription activation by these protein families.

scholarly journals ETS-Mediated Cooperation between Basic Helix-Loop-Helix Motifs of the Immunoglobulin μ Heavy-Chain Gene Enhancer

1998 ◽  
Vol 18 (3) ◽  
pp. 1477-1488 ◽  
Author(s):  
Wei Dang ◽  
Xiao-hong Sun ◽  
Ranjan Sen
Keyword(s):  
Heavy Chain ◽  
Transcription Activation ◽  
In Vitro Assays ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Enhancer Activity ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Gene Enhancer ◽  
Heavy Chain Gene Enhancer

ABSTRACT The μE motifs of the immunoglobulin μ heavy-chain gene enhancer bind ubiquitously expressed proteins of the basic helix-loop-helix (bHLH) family. These elements work together with other, more tissue-restricted elements to produce B-cell-specific enhancer activity by presently undefined combinatorial mechanisms. We found that μE2 contributed to transcription activation in B cells only when the μE3 site was intact, providing the first evidence for functional interactions between bHLH proteins. In vitro assays showed that bHLH zipper proteins binding to μE3 enhanced Ets-1 binding to μA. One of the consequences of this protein-protein interaction was to facilitate binding of a second bHLH protein, E47, to the μE2 site, thereby generating a three-protein–DNA complex. Furthermore, transcriptional synergy between bHLH and bHLH zipper factors also required an intermediate ETS protein, which may bridge the transcription activation domains of the bHLH factors. Our observations define an unusual form of cooperation between bHLH and ETS proteins and suggest mechanisms by which tissue-restricted and ubiquitous factors combine to generate tissue-specific enhancer activity.

scholarly journals TFEC, a basic helix-loop-helix protein, forms heterodimers with TFE3 and inhibits TFE3-dependent transcription activation.

1993 ◽  
Vol 13 (8) ◽  
pp. 4505-4512 ◽  
Author(s):  
G Q Zhao ◽  
Q Zhao ◽  
X Zhou ◽  
M G Mattei ◽  
B de Crombrugghe
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
In Vitro Translation ◽  
Chain Gene ◽  
Adult Rats ◽  
Sequence Identity ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Heavy Chain Gene Enhancer

We have identified a new basic helix-loop-helix (BHLH) DNA-binding protein, designated TFEC, which is closely related to TFE3 and TFEB. The basic domain of TFEC is identical to the basic DNA-binding domain of TFE3 and TFEB, whereas the helix-loop-helix motif of TFEC shows 88 and 85% identity with the same domains in TFE3 and TFEB, respectively. Like the other two proteins, TFEC contains a leucine zipper motif, which has a lower degree of sequence identity with homologous domains in TFE3 and TFEB than does the BHLH segment. Little sequence identity exists outside these motifs. Unlike the two other proteins, TFEC does not contain an acidic domain, which for TFE3 mediates the ability to activate transcription. Like the in vitro translation product of TFE3, the in vitro-translated TFEC binds to the mu E3 DNA sequence of the immunoglobulin heavy-chain gene enhancer. In addition, the product of cotranslation of TFEC RNA and TFE3 RNA forms a heteromeric protein-DNA complex with mu E3 DNA. In contrast to TFE3, TFEC is unable to transactivate a reporter gene linked to a promoter containing tandem copies of the immunoglobulin mu E3 enhancer motif. Cotransfection of TFEC DNA and TFE3 DNA strongly inhibits the transactivation caused by TFE3. TFEC RNA is found in many tissues of adult rats, but the relative concentrations of TFEC and TFE3 RNAs vary considerably in these different tissues. No TFEC RNA was detectable in several cell lines, including fibroblasts, myoblasts, chondrosarcoma cells, and myeloma cells, indicating that TFEC is not ubiquitously expressed.

TFEC, a basic helix-loop-helix protein, forms heterodimers with TFE3 and inhibits TFE3-dependent transcription activation

1993 ◽  
Vol 13 (8) ◽  
pp. 4505-4512
Author(s):  
G Q Zhao ◽  
Q Zhao ◽  
X Zhou ◽  
M G Mattei ◽  
B de Crombrugghe
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
In Vitro Translation ◽  
Chain Gene ◽  
Adult Rats ◽  
Sequence Identity ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Heavy Chain Gene Enhancer

We have identified a new basic helix-loop-helix (BHLH) DNA-binding protein, designated TFEC, which is closely related to TFE3 and TFEB. The basic domain of TFEC is identical to the basic DNA-binding domain of TFE3 and TFEB, whereas the helix-loop-helix motif of TFEC shows 88 and 85% identity with the same domains in TFE3 and TFEB, respectively. Like the other two proteins, TFEC contains a leucine zipper motif, which has a lower degree of sequence identity with homologous domains in TFE3 and TFEB than does the BHLH segment. Little sequence identity exists outside these motifs. Unlike the two other proteins, TFEC does not contain an acidic domain, which for TFE3 mediates the ability to activate transcription. Like the in vitro translation product of TFE3, the in vitro-translated TFEC binds to the mu E3 DNA sequence of the immunoglobulin heavy-chain gene enhancer. In addition, the product of cotranslation of TFEC RNA and TFE3 RNA forms a heteromeric protein-DNA complex with mu E3 DNA. In contrast to TFE3, TFEC is unable to transactivate a reporter gene linked to a promoter containing tandem copies of the immunoglobulin mu E3 enhancer motif. Cotransfection of TFEC DNA and TFE3 DNA strongly inhibits the transactivation caused by TFE3. TFEC RNA is found in many tissues of adult rats, but the relative concentrations of TFEC and TFE3 RNAs vary considerably in these different tissues. No TFEC RNA was detectable in several cell lines, including fibroblasts, myoblasts, chondrosarcoma cells, and myeloma cells, indicating that TFEC is not ubiquitously expressed.

scholarly journals Combinatorial determinants of tissue-specific transcription in B cells and macrophages.

1997 ◽  
Vol 17 (7) ◽  
pp. 3527-3535 ◽  
Author(s):  
B S Nikolajczyk ◽  
M Cortes ◽  
R Feinman ◽  
R Sen
Keyword(s):  
B Cells ◽  
Protein Binding ◽  
Binding Sites ◽  
Leucine Zipper ◽  
Chain Gene ◽  
Tissue Specific ◽  
Protein Binding Sites ◽  
Helix Loop Helix ◽  
Heavy Chain Gene Enhancer ◽  
Immunoglobulin Mu

A tripartite domain of the immunoglobulin mu heavy-chain gene enhancer that activates transcription in B cells contains binding sites for PU.1, Ets-1, and a leucine zipper-containing basic helix-loop-helix factor. Because PU.1 is expressed only in B cells and macrophages, we tested the activity of a minimal mu enhancer fragment in macrophages by transient transfections. The minimal mu enhancer activated transcription in macrophages, and the activity was dependent on all three sites. Analysis of mutated enhancers, in which spacing and orientation of the ETS protein binding sites had been changed, suggested that the mechanisms of enhancer activation were different in B cells and macrophages. Thus, ETS protein binding sites may be combined in different ways to generate tissue-specific transcription activators. Despite the activity of the minimal enhancer in macrophages, a larger mu enhancer fragment was inactive in these cells. We propose that formation of the nucleoprotein complex that is formed on the minimal enhancer in macrophages cannot be helped by the neighboring muE elements that are essential for activity of the monomeric enhancer.

scholarly journals MondoA, a Novel Basic Helix-Loop-Helix–Leucine Zipper Transcriptional Activator That Constitutes a Positive Branch of a Max-Like Network

2000 ◽  
Vol 20 (23) ◽  
pp. 8845-8854 ◽  
Author(s):  
Andrew N. Billin ◽  
Alanna L. Eilers ◽  
Kathryn L. Coulter ◽  
Jennifer S. Logan ◽  
Donald E. Ayer
Keyword(s):  
Transcriptional Activation ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Functional Characterization ◽  
Nuclear Accumulation ◽  
Cytoplasmic Localization ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix

ABSTRACT Max is a common dimerization partner for a family of transcription factors (Myc, Mad [or Mxi]), and Mnt [or Rox] proteins) that regulate cell growth, proliferation, and apoptosis. We recently characterized a novel Max-like protein, Mlx, which interacts with Mad1 and Mad4. Here we describe the cloning and functional characterization of a new family of basic helix-loop-helix–leucine zipper heterodimeric partners for Mlx termed the Mondo family. MondoA forms homodimers weakly and does not interact with Max or members of the Myc or Mad families. MondoA and Mlx associate in vivo, and surprisingly, they are localized primarily to the cytoplasm of cultured mammalian cells. Treatment of cells with the nuclear export inhibitor leptomycin B results in the nuclear accumulation of MondoA and Mlx, demonstrating that they shuttle between the cytoplasmic and nuclear compartments rather than having exclusively cytoplasmic localization. MondoA preferentially forms heterodimers with Mlx, and this heterocomplex can bind to, and activate transcription from, CACGTG E-boxes when targeted to the nucleus via a heterologous nuclear localization signal. The amino termini of the Mondo proteins are highly conserved among family members and contain separable and autonomous cytoplasmic localization and transcription activation domains. Therefore, Mlx can mediate transcriptional repression in conjunction with the Mad family and can mediate transcriptional activation via the Mondo family. We propose that Mlx, like Max, functions as the center of a transcription factor network.

Ets proteins: new factors that regulate immunoglobulin heavy-chain gene expression

1993 ◽  
Vol 13 (11) ◽  
pp. 7163-7169
Author(s):  
R R Rivera ◽  
M H Stuiver ◽  
R Steenbergen ◽  
C Murre
Keyword(s):  
Heavy Chain ◽  
Screening Method ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Helix Loop Helix ◽  
Ets Proteins ◽  
Gene Enhancer ◽  
Heavy Chain Gene Enhancer ◽  
A Site ◽  
Ig Heavy Chain

We used a DNA-protein interaction screening method to isolate a cDNA, Erg-3, whose product binds to a site, designated pi, present in the immunoglobulin (Ig) heavy-chain gene enhancer. Erg-3 is an alternatively spliced product of the erg gene and contains an Ets DNA-binding domain. Fli-1 and PU.1, related Ets proteins, also bind to the same site. In addition, PU.1 binds to a second site, designated microB, in the Ig heavy-chain enhancer. We demonstrate that the pi binding site is crucial for Ig heavy-chain gene enhancer function. In addition, we show that Erg-3 and Fli.1, but not PU.1, can activate a reporter construct containing a multimer of protein-binding sites, synergistically with helix-loop-helix protein E12. We discuss how combinatorial interactions between members of the helix-loop-helix and Ets families may account for the tissue specificity of these proteins.

scholarly journals Differences between MyoD DNA binding and activation site requirements revealed by functional random sequence selection.

1996 ◽  
Vol 16 (7) ◽  
pp. 3893-3900 ◽  
Author(s):  
J Huang ◽  
T K Blackwell ◽  
L Kedes ◽  
H Weintraub
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Random Sequence ◽  
In Vitro Binding ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Vitro Binding

A method has been developed for selecting functional enhancer/promoter sites from random DNA sequences in higher eukaryotic cells. Of sequences that were thus selected for transcriptional activation by the muscle-specific basic helix-loop-helix protein MyoD, only a subset are similar to the preferred in vitro binding consensus, and in the same promoter context an optimal in vitro binding site was inactive. Other sequences with full transcriptional activity instead exhibit sequence preferences that, remarkably, are generally either identical or very similar to those found in naturally occurring muscle-specific promoters. This first systematic examination of the relation between DNA binding and transcriptional activation by basic helix-loop-helix proteins indicates that binding per se is necessary but not sufficient for transcriptional activation by MyoD and implies a requirement for other DNA sequence-dependent interactions or conformations at its binding site.

Definition of the transcriptional activation domain of recombinant 43-kilodalton USF

1992 ◽  
Vol 12 (11) ◽  
pp. 5094-5101
Author(s):  
B J Kirschbaum ◽  
P Pognonec ◽  
R G Roeder
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Small Region ◽  
Initial Structure ◽  
Transcriptional Activation Domain ◽  
Cellular Transcription Factor ◽  
Helix Loop Helix ◽  
Activation Potential

The cellular transcription factor USF is involved in the regulation of both cellular and viral genes and consists of 43- and 44-kDa polypeptides which independently show site-specific DNA binding. Cloning of the corresponding cDNA revealed that the 43-kDa polypeptide (USF43) is a member of the basic (B)-helix-loop-helix (HLH)-leucine zipper (LZ) family of proteins and provided a means for its functional dissection. Initial structure-function studies revealed that the HLH and LZ regions are both important for USF43 oligomerization and DNA binding. The studies presented here have focused on the determination of domains that contribute to transcriptional activation in vitro and show that (i) both a small region close to the N terminus and a region between residues 93 and 156 contribute strongly to transcriptional activation, (ii) full activation depends on the presence of both domains, (iii) the B-HLH-LZ region has no intrinsic activation potential but DNA binding is absolutely required for transcriptional activation, and (iv) the B-HLH-LZ region can be replaced by the Gal4 DNA binding domain without loss of activation potential.

scholarly journals Ets proteins: new factors that regulate immunoglobulin heavy-chain gene expression.

1993 ◽  
Vol 13 (11) ◽  
pp. 7163-7169 ◽  
Author(s):  
R R Rivera ◽  
M H Stuiver ◽  
R Steenbergen ◽  
C Murre
Keyword(s):  
Heavy Chain ◽  
Screening Method ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Helix Loop Helix ◽  
Ets Proteins ◽  
Gene Enhancer ◽  
Heavy Chain Gene Enhancer ◽  
A Site ◽  
Ig Heavy Chain

We used a DNA-protein interaction screening method to isolate a cDNA, Erg-3, whose product binds to a site, designated pi, present in the immunoglobulin (Ig) heavy-chain gene enhancer. Erg-3 is an alternatively spliced product of the erg gene and contains an Ets DNA-binding domain. Fli-1 and PU.1, related Ets proteins, also bind to the same site. In addition, PU.1 binds to a second site, designated microB, in the Ig heavy-chain enhancer. We demonstrate that the pi binding site is crucial for Ig heavy-chain gene enhancer function. In addition, we show that Erg-3 and Fli.1, but not PU.1, can activate a reporter construct containing a multimer of protein-binding sites, synergistically with helix-loop-helix protein E12. We discuss how combinatorial interactions between members of the helix-loop-helix and Ets families may account for the tissue specificity of these proteins.

scholarly journals Targeting the Microphthalmia Basic Helix-Loop-Helix–Leucine Zipper Transcription Factor to a Subset of E-Box Elements In Vitro and In Vivo

1998 ◽  
Vol 18 (12) ◽  
pp. 6930-6938 ◽  
Author(s):  
I. Aksan ◽  
C. R. Goding
Keyword(s):  
Transcription Factor ◽  
Leucine Zipper ◽  
Pigment Cells ◽  
Specific Gene ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
P Gene ◽  
E Box

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.

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