scholarly journals Exploring Functional Redundancy in the Immunoglobulin μ Heavy-Chain Gene Enhancer

1998 ◽  
Vol 18 (11) ◽  
pp. 6870-6878 ◽  
Author(s):  
Wei Dang ◽  
Barbara S. Nikolajczyk ◽  
Ranjan Sen
Keyword(s):  
Protein Binding ◽  
Heavy Chain ◽  
Binding Sites ◽  
Bhlh Protein ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Enhancer Activity ◽  
Protein Binding Sites ◽  
Gene Enhancer ◽  
Heavy Chain Gene Enhancer

ABSTRACT Immunoglobulin (Ig) μ heavy-chain gene enhancer activity is mediated by multiple DNA binding proteins. Mutations of several protein binding sites in the enhancer do not affect enhancer activity significantly. This feature, termed redundancy, is thought to be due to functional compensation of the mutated sites by other elements within the enhancer. In this study, we identified the elements that make the basic helix-loop-helix (bHLH) protein binding sites, μE2 and μE3, redundant. The major compensatory element is a binding site for interferon regulatory factors (IRFs) and not one of several other bHLH protein binding sites. These studies also provide the first evidence for a role of IRF proteins in Ig heavy-chain gene expression. In addition, we reconstituted the activity of a monomeric μ enhancer in nonlymphoid cells and defined the domains of the ETS gene required for function.

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 Complex regulation of the immunoglobulin mu heavy-chain gene enhancer: microB, a new determinant of enhancer function.

1990 ◽  
Vol 10 (6) ◽  
pp. 3145-3154 ◽  
Author(s):  
B Nelsen ◽  
T Kadesch ◽  
R Sen
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Heavy Chain ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Enhancer Activity ◽  
Octamer Motif ◽  
Gene Enhancer ◽  
Heavy Chain Gene Enhancer ◽  
Immunoglobulin Mu

The B-lymphocyte-specific activity of the immunoglobulin mu heavy-chain gene enhancer has been attributed to the octamer motif (ATTTGCAT) present within the enhancer that binds a B-cell-specific factor designated NF-A2/OTF-2. However, significant residual enhancer activity even after deletion of this element has suggested the presence of a second critical functional determinant. We have used deletion and mutational analyses to define an element, microB (TTTGGGGAA), that is essential for B-cell-specific enhancer activity in S194 myeloma cells in the absence of the octamer. Transfection analysis in a panel of lymphoid cell lines suggests that the presence of either microB or octamer leads to considerable enhancer activity in cell lines representing later stages of B-cell differentiation, whereas both elements are needed for function in cell lines representing earlier stages. Furthermore, in contrast to the results in pre-B-cell lines, both microB and octamer elements function independently in certain T-cell lines in which the mu enhancer is active.

Complex regulation of the immunoglobulin mu heavy-chain gene enhancer: microB, a new determinant of enhancer function

1990 ◽  
Vol 10 (6) ◽  
pp. 3145-3154
Author(s):  
B Nelsen ◽  
T Kadesch ◽  
R Sen
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Heavy Chain ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Enhancer Activity ◽  
Octamer Motif ◽  
Gene Enhancer ◽  
Heavy Chain Gene Enhancer ◽  
Immunoglobulin Mu

The B-lymphocyte-specific activity of the immunoglobulin mu heavy-chain gene enhancer has been attributed to the octamer motif (ATTTGCAT) present within the enhancer that binds a B-cell-specific factor designated NF-A2/OTF-2. However, significant residual enhancer activity even after deletion of this element has suggested the presence of a second critical functional determinant. We have used deletion and mutational analyses to define an element, microB (TTTGGGGAA), that is essential for B-cell-specific enhancer activity in S194 myeloma cells in the absence of the octamer. Transfection analysis in a panel of lymphoid cell lines suggests that the presence of either microB or octamer leads to considerable enhancer activity in cell lines representing later stages of B-cell differentiation, whereas both elements are needed for function in cell lines representing earlier stages. Furthermore, in contrast to the results in pre-B-cell lines, both microB and octamer elements function independently in certain T-cell lines in which the mu enhancer is active.

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.

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.

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