Displacement of an E-box-binding repressor by basic helix-loop-helix proteins: implications for B-cell specificity of the immunoglobulin heavy-chain enhancer.

The activity of the immunoglobulin heavy-chain (IgH) enhancer is restricted to B cells, although it binds both B-cell-restricted and ubiquitous transcription factors. Activation of the enhancer in non-B cells upon overexpression of the basic helix-loop-helix (bHLH) protein E2A appears to be mediated not only by the binding of E2A to its cognate E box but also by the resulting displacement of a repressor from that same site. We have identified a "two-handed" zinc finger protein, denoted ZEB, the DNA-binding specificity of which mimics that of the cellular repressor. By employing a derivative E box that binds ZEB but not E2A, we have shown that the repressor is active in B cells and the IgH enhancer is silenced in the absence of binding competition by bHLH proteins. Hence, we propose that a necessary prerequisite of enhancer activity is the B-cell-specific displacement of a ZEB-like repressor by bHLH proteins.

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mTFE3, an X-linked transcriptional activator containing basic helix-loop-helix and zipper domains, utilizes the zipper to stabilize both DNA binding and multimerization.

Molecular and Cellular Biology ◽

10.1128/mcb.12.2.817 ◽

1992 ◽

Vol 12 (2) ◽

pp. 817-827 ◽

Cited By ~ 40

Author(s):

C Roman ◽

A G Matera ◽

C Cooper ◽

S Artandi ◽

S Blain ◽

...

Keyword(s):

Dna Binding ◽

Heavy Chain ◽

Leucine Zipper ◽

Immunoglobulin Heavy Chain ◽

Functional Roles ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

The Individual ◽

Bhlh Proteins ◽

High Degree

Southwestern (DNA-protein) screening of a murine L-cell cDNA library by using a probe for the microE3 site in the immunoglobulin heavy-chain enhancer yielded a clone, mTFE3, which is a member of the subset of basic helix-loop-helix (BHLH) proteins that also contain a leucine zipper (ZIP). Since the individual contribution of these domains is not well understood for proteins which contain them both, mutational analyses were performed to assess the functional roles of the HLH and ZIP regions for DNA binding and multimerization. The HLH region is stringently required for DNA binding but not for multimerization. The ZIP region is not stringently required for binding or multimerization, but stabilizes both multimer formation and DNA binding. A high degree of conservation at both the amino acid and nucleotide levels between the human transcription factor TFE3 and mTFE3 suggests that mTFE3 is the murine homolog of human TFE3. By using fluorescent in situ hybridization, mTFE3 was mapped to mouse chromosome X in band A2, which is just below the centromere. We show that in addition to the immunoglobulin heavy-chain microE3 site, mTFE3 binds to transcriptional elements important for lymphoid-specific, muscle-specific, and ubiquitously expressed genes. Binding of mTFE3 to DNA induces DNA bending.

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mTFE3, an X-linked transcriptional activator containing basic helix-loop-helix and zipper domains, utilizes the zipper to stabilize both DNA binding and multimerization

Molecular and Cellular Biology ◽

10.1128/mcb.12.2.817-827.1992 ◽

1992 ◽

Vol 12 (2) ◽

pp. 817-827

Author(s):

C Roman ◽

A G Matera ◽

C Cooper ◽

S Artandi ◽

S Blain ◽

...

Keyword(s):

Dna Binding ◽

Heavy Chain ◽

Leucine Zipper ◽

Immunoglobulin Heavy Chain ◽

Functional Roles ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

The Individual ◽

Bhlh Proteins ◽

High Degree

Southwestern (DNA-protein) screening of a murine L-cell cDNA library by using a probe for the microE3 site in the immunoglobulin heavy-chain enhancer yielded a clone, mTFE3, which is a member of the subset of basic helix-loop-helix (BHLH) proteins that also contain a leucine zipper (ZIP). Since the individual contribution of these domains is not well understood for proteins which contain them both, mutational analyses were performed to assess the functional roles of the HLH and ZIP regions for DNA binding and multimerization. The HLH region is stringently required for DNA binding but not for multimerization. The ZIP region is not stringently required for binding or multimerization, but stabilizes both multimer formation and DNA binding. A high degree of conservation at both the amino acid and nucleotide levels between the human transcription factor TFE3 and mTFE3 suggests that mTFE3 is the murine homolog of human TFE3. By using fluorescent in situ hybridization, mTFE3 was mapped to mouse chromosome X in band A2, which is just below the centromere. We show that in addition to the immunoglobulin heavy-chain microE3 site, mTFE3 binds to transcriptional elements important for lymphoid-specific, muscle-specific, and ubiquitously expressed genes. Binding of mTFE3 to DNA induces DNA bending.

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Selective utilization of basic helix-loop-helix-leucine zipper proteins at the immunoglobulin heavy-chain enhancer.

Molecular and Cellular Biology ◽

10.1128/mcb.17.1.18 ◽

1997 ◽

Vol 17 (1) ◽

pp. 18-23 ◽

Cited By ~ 37

Author(s):

R S Carter ◽

P Ordentlich ◽

T Kadesch

Keyword(s):

Heavy Chain ◽

Leucine Zipper ◽

Physiological Role ◽

Immunoglobulin Heavy Chain ◽

Dominant Negative ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

E Box ◽

Derivatives Of

The microE3 E box within the immunoglobulin heavy-chain (IgH) enhancer binds several proteins of the basic helix-loop-helix-leucine zipper (bHLHzip) class, including TFE3, USF1, and Max. Both TFE3 and USF have been described as transcriptional activators, and so we investigated their possible roles in activating the IgH enhancer in vivo. Although TFE3 activated various enhancer-based reporters, both USF1 and Max effectively inhibited transcription. Inhibition by USF correlated with the lack of a strong activation domain and was the result of the protein neutralizing the microE3 site. The effects of dominant-negative derivatives of TFE3 and USF1 confirmed that TFE3, or a TFE3-like protein, is the primary cellular bHLHzip protein that activates the IgH enhancer. In addition to providing a physiological role for TFE3, our results call into question the traditional view of USF1 as an obligate transcriptional activator.

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Transcription enhancer factor 1 interacts with a basic helix-loop-helix zipper protein, Max, for positive regulation of cardiac alpha-myosin heavy-chain gene expression.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.3924 ◽

1997 ◽

Vol 17 (7) ◽

pp. 3924-3936 ◽

Cited By ~ 61

Author(s):

M P Gupta ◽

C S Amin ◽

M Gupta ◽

N Hay ◽

R Zak

Keyword(s):

Myosin Heavy Chain ◽

Reporter Gene ◽

Heavy Chain ◽

Troponin T ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

E Box ◽

Two Factors ◽

Transcription Enhancer ◽

Enhancer Factor

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.

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Scleraxis: a basic helix-loop-helix protein that prefigures skeletal formation during mouse embryogenesis

Development ◽

10.1242/dev.121.4.1099 ◽

1995 ◽

Vol 121 (4) ◽

pp. 1099-1110 ◽

Cited By ~ 19

Author(s):

P. Cserjesi ◽

D. Brown ◽

K.L. Ligon ◽

G.E. Lyons ◽

N.G. Copeland ◽

...

Keyword(s):

Connective Tissue ◽

Expression Pattern ◽

Consensus Sequence ◽

Cell Types ◽

Cell Type ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

E Box ◽

Cell Type Specific ◽

Bhlh Proteins

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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Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development

Journal of Experimental Medicine ◽

10.1084/jem.185.4.609 ◽

1997 ◽

Vol 185 (4) ◽

pp. 609-620 ◽

Cited By ~ 92

Author(s):

Andrei Constantinescu ◽

Mark S. Schlissel

Keyword(s):

B Cells ◽

B Cell ◽

Heavy Chain ◽

Light Chain ◽

Immunoglobulin Heavy Chain ◽

Cell Development ◽

B Cell Development ◽

Allelic Exclusion ◽

Chain Locus ◽

Heavy Chain Locus

The process of V(D)J recombination is crucial for regulating the development of B cells and for determining their eventual antigen specificity. Here we assess the developmental regulation of the V(D)J recombinase directly, by monitoring the double-stranded DNA breaks produced in the process of V(D)J recombination. This analysis provides a measure of recombinase activity at immunoglobulin heavy and light chain loci across defined developmental stages spanning the process of B cell development. We find that expression of a complete immunoglobulin heavy chain protein is accompanied by a drastic change in the targeting of V(D)J recombinase activity, from being predominantly active at the heavy chain locus in pro-B cells to being exclusively restricted to the light chain loci in pre-B cells. This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus. Allelic exclusion is maintained by a different mechanism at the light chain locus. We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele. Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an Eμ-myc transgene.

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B cell progenitors have different growth requirements before and after immunoglobulin heavy chain commitment.

Journal of Experimental Medicine ◽

10.1084/jem.168.4.1511 ◽

1988 ◽

Vol 168 (4) ◽

pp. 1511-1516 ◽

Cited By ~ 13

Author(s):

H Sauter ◽

C J Paige

Keyword(s):

B Cells ◽

B Cell ◽

Heavy Chain ◽

Fetal Liver ◽

Immunoglobulin Heavy Chain ◽

Growth Conditions ◽

Selective Growth ◽

Cell Assay ◽

Growth Requirements ◽

Before And After

Using the clonable pre-B cell assay, we have identified B cell progenitors that are not yet committed to the production of a particular H chain allele. These cells represent approximately 10-20% of clonable pre-B cells found in 15-d fetal liver. The clonable pre-B cell assay provides an environment adequate for the expansion and differentiation of these cells into mature, Ig-secreting, cells. Using the same methodology, we have also identified progenitors that are uncommitted to the production of a particular L chain isotype. Moreover, investigating the growth requirements for clonable pre-B cells has led to the discovery of selective growth conditions that distinguish cells before and after commitment to H chain allotype.

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Phosphorylation of E47 as a potential determinant of B-cell-specific activity.

Molecular and Cellular Biology ◽

10.1128/mcb.16.12.6900 ◽

1996 ◽

Vol 16 (12) ◽

pp. 6900-6908 ◽

Cited By ~ 80

Author(s):

S R Sloan ◽

C P Shen ◽

R McCarrick-Walmsley ◽

T Kadesch

Keyword(s):

B Cells ◽

Dna Binding ◽

B Cell ◽

B Lymphocyte ◽

Specific Activity ◽

Cell Types ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Potential Determinant

The E2A gene encodes two basic helix-loop-helix proteins designated E12 and E47. Although these proteins are widely expressed, they are required only for the B-lymphocyte lineage where DNA binding is mediated distinctively by E47 homodimers. By studying the properties of deltaE47, an N-terminal truncation of E47, we provide evidence that phosphorylation may contribute to B-cell-specific DNA binding by E47. Two serines N terminal to the deltaE47 basic helix-loop-helix domain were found to be phosphorylated in a variety of cell types but were hypophosphorylated in B cells. Phosphorylating these serines in vitro inhibited DNA binding by deltaE47 homodimers but not by deltaE47-containing heterodimers, such as deltaE47:MyoD. These results argue that hypophosphorylation may be a prerequisite for activity of E47 homodimers in B cells, suggesting the use of an inductive (nonstochastic) step in early B-cell development.

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PU.1/Pip and Basic Helix Loop Helix Zipper Transcription Factors Interact With Binding Sites in the CD20 Promoter to Help Confer Lineage- and Stage-Specific Expression of CD20 in B Lymphocytes

Blood ◽

10.1182/blood.v90.10.3984 ◽

1997 ◽

Vol 90 (10) ◽

pp. 3984-3995 ◽

Cited By ~ 60

Author(s):

Andreas Himmelmann ◽

Agostino Riva ◽

Gaye Lynn Wilson ◽

Brian P. Lucas ◽

Claire Thevenin ◽

...

Keyword(s):

B Cell ◽

Plasma Cells ◽

Expression Vectors ◽

Specific Gene ◽

Cell Stage ◽

Specific Expression ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

E Box

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.

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