Mutational analysis of the contribution of sequence motifs within the IgH enhancer to tissue specific transcriptional activation

Pérez-Mutul, José; Macchi, M.; Wasylyk, Bohdan

doi:10.1093/nar/16.13.6085

Nucleic Acids Research

1988

DOI: 10.1093/nar/16.13.6085

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Mutational analysis of the contribution of sequence motifs within the IgH enhancer to tissue specific transcriptional activation

José Pérez-Mutul¹,

M. Macchi²,

Bohdan Wasylyk³

Abstract: We have investigated the role of sequence motifs in the immunoglobulin heavy chain (IgH) enhancer on its activity in myeloma and fibroblast cell-lines. In transient transfection assays the transcription stimulatory activity of the enhancer is decreased in myeloma cells by mutating the E motifs 1, 2 and 3, the core motifs C1, C2, C3 and the octamer motif (OC) and in fibroblasts by mutating E2, E3, and C2. Our results suggest that transcription factors binding to E1, C1, C3 and OC contribute in a positive manner… Show more

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Cited by 48 publications

(30 citation statements)

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“…The E2 and E5 motifs are important E-protein binding sites found in the regulatory regions of many immune function genes, including those encoding the Igs (17)(18)(19)(20). Although knockout studies in mice indicate that E2A is an essential factor for lymphocyte development, other E-proteins (e.g., HEB) are able to compensate to a substantial degree for the lack of E2A when expressed in an appropriate context, i.e., as knockin mutants under the control of the E2A promoter (21).…”

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confidence: 99%

Evolution of Transcriptional Control of theIgHLocus: Characterization, Expression, and Function of TF12/HEB Homologs of the Catfish

Hikima

Cioffi

Middleton

et al. 2004

The Journal of Immunology

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The transcriptional enhancer (Eμ3′) of the IgH locus of the channel catfish, Ictalurus punctatus, differs from enhancers of the mammalian IgH locus in terms of its position, structure, and function. Transcription factors binding to multiple octamer motifs and a single μE5 motif (an E-box site, consensus CANNTG) interact for its function. E-box binding transcription factors of the class I basic helix-loop-helix family were cloned from a catfish B cell cDNA library in this study, and homologs of TF12/HEB were identified as the most highly represented E-proteins. Two alternatively spliced forms of catfish TF12 (termed CFEB1 and -2) were identified and contained regions homologous to the basic helix-loop-helix and activation domains of other vertebrate E-proteins. CFEB message is widely expressed, with CFEB1 message predominating over that of CFEB2. Both CFEB1 and -2 strongly activated transcription from a μE5-dependent artificial promoter. In catfish B cells, CFEB1 and -2 also activated transcription from the core region of the catfish IgH enhancer (Eμ3′) in a manner dependent on the presence of the μE5 site. Both CFEB1 and -2 bound the μE5 motif, and formed both homo- and heterodimers. CFEB1 and -2 were weakly active or inactive (in a promoter-dependent fashion) in mammalian B-lineage cells. Although E-proteins have been highly conserved in vertebrate evolution, the present results indicate that, at the phylogenetic level of a teleost fish, the TF12/HEB homolog differs from that of mammals in terms of 1) its high level of expression and 2) the presence of isoforms generated by alternative RNA processing.

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confidence: 99%

Evolution of Transcriptional Control of theIgHLocus: Characterization, Expression, and Function of TF12/HEB Homologs of the Catfish

Hikima

Cioffi

Middleton

et al. 2004

The Journal of Immunology

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“…The E2 to E5 motifs contain a consensus CANNTG motif that binds the basic helix-loop-helix (bHLH) family of transcription factors (12,17). Mutational analysis of E motifs has shown that loss of single elements does not significantly affect enhancer activity (13,15,23,35). This observation has been interpreted to indicate that there is redundancy among the E elements; that is, absence of one E element can be functionally compensated for by other E elements present in the enhancer.…”

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

Dang

Sun

Sen

1998

Molecular and Cellular Biology

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The E motifs of the immunoglobulin 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 E2 contributed to transcription activation in B cells only when the E3 site was intact, providing the first evidence for functional interactions between bHLH proteins. In vitro assays showed that bHLH zipper proteins binding to E3 enhanced Ets-1 binding to A. One of the consequences of this protein-protein interaction was to facilitate binding of a second bHLH protein, E47, to the E2 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.The immunoglobulin (Ig) heavy-chain gene enhancer is a cell-specific transcription regulatory sequence. Located in the J H -C intron, it is necessary for the expression of a rearranged IgH gene in transfection as well as transgenic assays. Moreover, when taken out of its normal context, the enhancer is sufficient to target a heterologous transgene (1,14,18,27) for expression at the appropriate differentiation stage in the Blymphocyte lineage. In addition to activating transcription from a V H gene promoter that has recombined into the C locus, this enhancer has also been implicated in the initiation of V(D)J recombination at the IgH locus. This is based on two experimental observations: transgenic recombination substrates were shown to be activated by the enhancer (7), and genetic deletion of the endogenous enhancer was shown to suppress IgH recombination (4, 31). At present, it is not clear whether the recombination activation and transcription activation properties of the enhancer are directly related; however, it is likely that enhancer-mediated chromatin reorganization resulting in increased accessibility of the IgH locus to polymerases and recombinases is important for both processes.B-cell-specific enhancer function is determined by multiple trans-acting nuclear proteins that bind to specific sites within the enhancer (6,19). Proteins that bind to the enhancer can be broadly classified into two categories: those that are more restricted in their tissue distribution, such as A, B, and octamer binding proteins, and those that are ubiquitously expressed in most cell types, such as the E1, E2, E3, and E5 binding proteins. However, no enhancer binding protein identified to date has an expression pattern that correlates perfectly with the cells in which the IgH gene is expressed. Based on the expression pattern of proteins binding to the A and B sites, we have previ...

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“…Two others, octamer (referring to the conserved octanucleotide ATGCAAAT; Singh et al 1986;Staudt et al 1986) and ~EBP-E , were defined first by in vitro binding assays. Deletions or mutations of these sites generally reduce overall enhancer activity (Lenardo et al 1987;Kiledjian et al 1988;Perez-Mutul et al 1988;Tsao et al 1988). Even though activity of the enhancer is restricted to B cells, only one of these sites (octamer) is known to bind B-cell-specific proteins (OTF-2/oct-2, OTF-2b; Landolfi et al 1986;Staudt et al 1986;Gerster IPresent address: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11 ;'24 USA.…”

mentioning

confidence: 99%

“…Studies of mutant enhancers have indicated that this site alone may be responsible for as much as 30% of the enhancer's activity in B cells (Lenardo et al 1987;Kiledjian et al 1988;Perez-Mutul et al 1988;Tsao et al 1988). The site was also shown to contribute to the low level of enhancer activity observed in non-B cells (Kiledjian et al 1988).…”

mentioning

confidence: 99%

TFE3: a helix-loop-helix protein that activates transcription through the immunoglobulin enhancer muE3 motif.

Beckmann¹,

Su²,

Kadesch³

1990

Genes Dev.

461

283

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The ttE3 motif within the immunoglobulin heavy-chain enhancer is required for full enhancer activity and is known to bind one, or perhaps a family, of related ubiquitous nuclear proteins. Here, we present the isolation of a cDNA that encodes an apparently novel ttE3-binding protein designated TFE3. The major open reading frame of the cDNA predicts a protein of 59 kD, with a leucine zipper situated adjacent to an myc-related motif that has been proposed to assume a helix-loop-helix structure. Both of these motifs have been shown (for other proteins) to facilitate protein-protein interactions and DNA binding. Expression of the cDNA in 3T3 cells stimulates transcription from an artificial promoter consisting of four I~E3 sites linked to a TATA box and also augments transcription of a reporter gene when it is linked to multiple copies of a particular heavy-chain enhancer subfragment but not when it is linked to the intact enhancer. Using GAL4 fusion proteins, we mapped a strong transcription activation domain within TFE3 that is distinct from the leucine zipper and helix-loophelix motifs and includes a potential negative amphipathic helix. Like the other ttE3-binding proteins detected in nuclear extracts, in vitro-synthesized TFE3 also binds to the USF/MLTF site found in the adenovirus major late promoter.[Key Words: ~E3; DNA-binding proteins; helix-loop-helix motif; immunoglobulin heavy-chain enhancer]

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Mutational analysis of the contribution of sequence motifs within the IgH enhancer to tissue specific transcriptional activation

Cited by 48 publications

References 40 publications

Evolution of Transcriptional Control of theIgHLocus: Characterization, Expression, and Function of TF12/HEB Homologs of the Catfish

Evolution of Transcriptional Control of theIgHLocus: Characterization, Expression, and Function of TF12/HEB Homologs of the Catfish

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

TFE3: a helix-loop-helix protein that activates transcription through the immunoglobulin enhancer muE3 motif.

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