scholarly journals 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 ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 3984-3995 ◽  
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.

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.

scholarly journals B lymphocytes express and lose syndecan at specific stages of differentiation.

1989 ◽  
Vol 1 (1) ◽  
pp. 27-35 ◽  
Author(s):  
R D Sanderson ◽  
P Lalor ◽  
M Bernfield
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
B Lymphocyte ◽  
Plasma Cells ◽  
Receptor Expression ◽  
Cell Stage ◽  
Precursor B Cells

Lymphopoietic cells require interactions with bone marrow stroma for normal maturation and show changes in adhesion to matrix during their differentiation. Syndecan, a heparan sulfate-rich integral membrane proteoglycan, functions as a matrix receptor by binding cells to interstitial collagens, fibronectin, and thrombospondin. Therefore, we asked whether syndecan was present on the surface of lymphopoietic cells. In bone marrow, we find syndecan only on precursor B cells. Expression changes with pre-B cell maturation in the marrow and with B-lymphocyte differentiation to plasma cells in interstitial matrices. Syndecan on B cell precursors is more heterogeneous and slightly larger than on plasma cells. Syndecan 1) is lost immediately before maturation and release of B lymphocytes into the circulation, 2) is absent on circulating and peripheral B lymphocytes, and 3) is reexpressed upon their differentiation into immobilized plasma cells. Thus, syndecan is expressed only when and where B lymphocytes associate with extracellular matrix. These results indicate that B cells differentiating in vivo alter their matrix receptor expression and suggest a role for syndecan in B cell stage-specific adhesion.

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
Keyword(s):  
Gene Transcription ◽  
Specific Gene ◽  
Deletion Mutants ◽  
Physical Interaction ◽  
Direct Physical Interaction ◽  
Amino Terminal ◽  
High Affinity ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Box

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.

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

1994 ◽  
Vol 14 (9) ◽  
pp. 6153-6163 ◽  
Author(s):  
T Genetta ◽  
D Ruezinsky ◽  
T Kadesch
Keyword(s):  
B Cells ◽  
B Cell ◽  
Heavy Chain ◽  
Zinc Finger Protein ◽  
Immunoglobulin Heavy Chain ◽  
Bhlh Protein ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Box ◽  
Bhlh Proteins

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.

scholarly journals Myogenin and MEF2 function synergistically to activate the MRF4 promoter during myogenesis.

1995 ◽  
Vol 15 (5) ◽  
pp. 2707-2718 ◽  
Author(s):  
P S Naidu ◽  
D C Ludolph ◽  
R Q To ◽  
T J Hinterberger ◽  
S F Konieczny
Keyword(s):  
Molecular Mechanisms ◽  
Muscle Development ◽  
Expression Patterns ◽  
Proximal Promoter ◽  
Sufficient Information ◽  
Specific Response ◽  
Specific Expression ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Box

The basic helix-loop-helix muscle regulatory factor (MRF) gene family encodes four distinct muscle-specific transcription factors known as MyoD, myogenin, Myf-5, and MRF4. These proteins represent key regulatory factors that control many aspects of skeletal myogenesis. Although the MRFs often exhibit overlapping functional activities, their distinct expression patterns during embryogenesis suggest that each protein plays a unique role in controlling aspects of muscle development. As a first step in determining how MRF4 gene expression is developmentally regulated, we examined the ability of the MRF4 gene to be expressed in a muscle-specific fashion in vitro. Our studies show that the proximal MRF4 promoter contains sufficient information to direct muscle-specific expression. Located within the proximal promoter are a single MEF2 site and E box that are required for maximum MRF4 expression. Mutation of the MEF2 site or E box severely impairs the ability of this promoter to produce a muscle-specific response. In addition, the MEF2 site and E box function in concert to synergistically activate the MRF4 gene in nonmuscle cells coexpressing MEF2 and myogenin proteins. Thus, the MRF4 promoter is regulated by the MEF2 and basic helix-loop-helix MRF protein family through a cross-regulatory circuitry. Surprisingly, the MRF4 promoter itself is not transactivated by MRF4, suggesting that this MRF gene is not subject to an autoregulatory pathway as previously implied by other studies. Understanding the molecular mechanisms regulating expression of each MRF gene is central to fully understanding how these factors control developmental events.

scholarly journals Identification of a DNA sequence involved in osteoblast-specific gene expression via interaction with helix-loop-helix (HLH)-type transcription factors.

1994 ◽  
Vol 126 (3) ◽  
pp. 773-782 ◽  
Author(s):  
M Tamura ◽  
M Noda
Keyword(s):  
Transcription Factors ◽  
Promoter Region ◽  
Gene Promoter ◽  
Binding Activity ◽  
Specific Gene ◽  
Specific Expression ◽  
Helix Loop Helix ◽  
Nuclear Extracts ◽  
Osteocalcin Gene ◽  
E Box

To elucidate regulatory mechanism(s) underlying differentiation of osteoblasts, we examined involvement of helix-loop-helix (HLH)-type transcription factors in osteoblast-specific expression of a phenotypic marker gene which encodes osteocalcin, a major noncollagenous bone matrix protein, exclusively expressed in osteoblasts. Overexpression of a dominant negative HLH protein, Id-1, decreased the activity of the 1.1-kb osteocalcin gene promoter cotransfected into rat osteoblastic osteosarcoma ROS17/2.8 cells. Analysis of deletion mutants revealed that a 264-bp fragment of osteocalcin promoter (-198 to +66) was sufficient for the Id-1-dependent suppression. Furthermore, the activity of the same promoter fragment (-198 to +66) was enhanced when antisense Id-1 expression vector was cotransfected. This osteocalcin gene promoter region contains two sites of an E-box motif, a consensus binding site for HLH proteins, which we refer to as OCE1 (CACATG, at -102) and OCE2 (CAGCTG, at -149), respectively. Mutagenesis in OCE1 but not OCE2 led to greater than 50% reduction in transcriptional activity of the osteocalcin gene promoter. Electrophoresis mobility shift assay indicated that factors in nuclear extracts prepared from ROS17/2.8 cells bound to the 30-bp oligonucleotide probe containing the E-box motif of OCE1. This binding was competed out by OCE1 oligonucleotide but neither by OCmE1 oligonucleotide in which E-box motif was mutated nor by OCE2. The OCE1-binding activity in the nuclear extracts of ROS17/2.8 cells was reduced by 70% when bacterially expressed Id-1 protein was added to the reaction mixture, suggesting the involvement of HLH proteins in the DNA/protein complex formation. In contrast to the osteoblast-like cells, OCE1-binding activity in the nuclear extracts of C3H10T1/2 fibroblasts was very low. However, when these fibroblasts were treated with recombinant human bone morphogenetic protein-2 which induced expression of osteocalcin as well as other phenotypic markers of osteoblasts, OCE1-binding activity was increased approximately 40-fold, indicating that OCE1 would be involved in the tissue-specific expression of the osteocalcin gene. These findings indicated for the first time that osteoblast-specific gene transcription is regulated via the interaction between certain E-box binding transcription factor(s) in osteoblasts and the OCE1 sequence in the promoter region of the osteocalcin gene.

scholarly journals Target Specificities of DrosophilaEnhancer of split Basic Helix-Loop-Helix Proteins

1999 ◽  
Vol 19 (7) ◽  
pp. 4600-4610 ◽  
Author(s):  
Barbara H. Jennings ◽  
David M. Tyler ◽  
Sarah J. Bray
Keyword(s):  
Dna Sequences ◽  
Target Genes ◽  
Notch Signalling Pathway ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Dna Binding Site ◽  
E Box ◽  
The Individual ◽  
Enhancer Of Split

ABSTRACT Seven Enhancer of split genes in Drosophila melanogaster encode basic-helix-loop-helix transcription factors which are components of the Notch signalling pathway. They are expressed in response to Notch activation and mediate some effects of the pathway by regulating the expression of target genes. Here we have determined that the optimal DNA binding site for the Enhancer of split proteins is a palindromic 12-bp sequence, 5′-TGGCACGTG(C/T)(C/T)A-3′, which contains an E-box core (CACGTG). This site is recognized by all of the individual Enhancer of split basic helix-loop-helix proteins, consistent with their ability to regulate similar target genes in vivo. We demonstrate that the 3 bp flanking the E-box core are intrinsic to DNA recognition by these proteins and that the Enhancer of split and proneural proteins can compete for binding on specific DNA sequences. Furthermore, the regulation conferred on a reporter gene in Drosophila by three closely related sequences demonstrates that even subtle sequence changes within an E box or flanking bases have dramatic consequences on the overall repertoire of proteins that can bind in vivo.

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
Keyword(s):  
Gene Transcription ◽  
Specific Gene ◽  
Deletion Mutants ◽  
Physical Interaction ◽  
Direct Physical Interaction ◽  
Amino Terminal ◽  
High Affinity ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Box

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.

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

1994 ◽  
Vol 14 (9) ◽  
pp. 6153-6163
Author(s):  
T Genetta ◽  
D Ruezinsky ◽  
T Kadesch
Keyword(s):  
B Cells ◽  
B Cell ◽  
Heavy Chain ◽  
Zinc Finger Protein ◽  
Immunoglobulin Heavy Chain ◽  
Bhlh Protein ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Box ◽  
Bhlh Proteins

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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