Contribution of the Per/Arnt/Sim (PAS) Domains to DNA Binding by the Basic Helix-Loop-Helix PAS Transcriptional Regulators

Gene regulation by dioxins is mediated via the dioxin receptor, a ligand-dependent basic helix-loop-helix (bHLH)/PAS transcription factor. The latent dioxin receptor responds to dioxin signalling by forming an activated heterodimeric complex with a specific bHLH partner, Arnt, an essential process for target DNA recognition. We have analyzed the transactivating potential within this heterodimeric complex by dissecting it into individual subunits, replacing the dimerization and DNA-binding bHLH motifs with heterologous zinc finger DNA-binding domains. The uncoupled Arnt chimera, maintaining 84% of Arnt residues, forms a potent and constitutive transcription factor. Chimeric proteins show that the dioxin receptor also harbors a strong transactivation domain in the C terminus, although this activity was silenced by inclusion of 82 amino acids from the central ligand-binding portion of the dioxin receptor. This central repression region conferred binding of the molecular chaperone hsp90 upon otherwise constitutive chimeras in vitro, indicating that hsp90 has the ability to mediate a cis-repressive function on distant transactivation domains. Importantly, when the ligand-binding domain of the dioxin receptor remained intact, the ability of this hsp90-binding activity to confer repression became conditional rather than irreversible. Our data are consistent with a model in which crucial activities of the dioxin receptor, such as dimerization with Arnt and transactivation, are conditionally repressed by the central ligand- and-hsp90-binding region of the receptor. In contrast, the Arnt protein appears to be free from any repressive activity. Moreover, within the context of the dioxin response element (xenobiotic response element), the C terminus of Arnt conferred a potent, dominating transactivation function onto the native bHLH heterodimeric complex. Finally, the relative transactivation potencies of the individual dioxin receptor and Arnt chimeras varied with cell type and promoter architecture, indicating that the mechanisms for transcriptional activation may differ between these two subunits and that in the native complex the transactivation pathway may be dependent upon cell-specific and promoter contexts.

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Ras p21Val inhibits myogenesis without altering the DNA binding or transcriptional activities of the myogenic basic helix-loop-helix factors.

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5205 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5205-5213 ◽

Cited By ~ 47

Author(s):

Y Kong ◽

S E Johnson ◽

E J Taparowsky ◽

S F Konieczny

Keyword(s):

Growth Factor ◽

Dna Binding ◽

Intracellular Signaling ◽

Transcriptional Activity ◽

Molecular Mechanisms ◽

Terminal Differentiation ◽

Tgf Beta ◽

Regulatory Factors ◽

Basic Helix Loop Helix ◽

Helix Loop Helix

MRF4, MyoD, myogenin, and Myf-5 are muscle-specific basic helix-loop-helix transcription factors that share the ability to activate the expression of skeletal muscle genes such as those encoding alpha-actin, myosin heavy chain, and the acetylcholine receptor subunits. The muscle regulatory factors (MRFs) also exhibit the unique capacity to initiate the myogenic program when ectopically expressed in a variety of nonmuscle cell types, most notably C3H10T1/2 fibroblasts (10T1/2 cells). The commitment of myoblasts to terminal differentiation, although positively regulated by the MRFs, also is controlled negatively by a variety of agents, including several growth factors and oncoproteins such as fibroblast growth factor (FGF-2), transforming growth factor beta 1 (TGF-beta 1), and Ras p21Val. The molecular mechanisms by which these varied agents alter myogenic terminal differentiation events remain unclear. In an effort to establish whether Ras p21Val represses MRF activity by directly targeting the MRF proteins, we examined the DNA binding and transcription activation potentials of MRF4 and MyoD when expressed in 10T1/2 cells or in 10T1/2 cells expressing Ras p21Val. Our results demonstrate that Ras p21Val inhibits terminal differentiation events by targeting the basic domain of the MRFs, and yet the mechanism underlying this inhibition does not involve altering the DNA binding or the inherent transcriptional activity of these regulatory factors. In contrast, FGF-2 and TGF-beta 1 block terminal differentiation by repressing the transcriptional activity of the MRFs. We conclude that the Ras p21Val block in differentiation operates via an intracellular signaling pathway that is distinct from the FGF-2 and TGF-beta 1 pathways.

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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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DNA Binding Specificity of the Basic-Helix-Loop-Helix Protein MASH-1

Biochemistry ◽

10.1021/bi00035a008 ◽

1995 ◽

Vol 34 (35) ◽

pp. 11026-11036 ◽

Cited By ~ 28

Author(s):

Daniel Meierhans ◽

Chirine el-Ariss ◽

Margrit Neuenschwander ◽

Martin Sieber ◽

Joseph F. Stackhouse ◽

...

Keyword(s):

Dna Binding ◽

Binding Specificity ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Dna Binding Specificity

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Conformational activation of a basic helix-loop-helix protein (MyoD1) by the C-terminal region of murine HSP90 (HSP84).

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.5059 ◽

1992 ◽

Vol 12 (11) ◽

pp. 5059-5068 ◽

Cited By ~ 108

Author(s):

R Shaknovich ◽

G Shue ◽

D S Kohtz

Keyword(s):

Dna Binding ◽

Direct Evidence ◽

Conformational Structure ◽

Amphipathic Helix ◽

Expression Library ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Transient Interaction ◽

Dose Dependent Fashion ◽

Dose Dependent

A murine cardiac lambda gt11 expression library was screened with an amphipathic helix antibody, and a recombinant representing the C-terminal 194 residues of murine HSP90 (HSP84) was cloned. Both recombinant and native HSP90s were then found to rapidly convert a basic helix-loop-helix protein (MyoD1) from an inactive to an active conformation, as assayed by sequence-specific DNA binding. The conversion process involves a transient interaction between HSP90 and MyoD1 and does not result in the formation of a stable tertiary complex. Conversion does not require ATP and occurs stoichiometrically in a dose-dependent fashion. HSP90 is an abundant, ubiquitous, and highly conserved protein present in most eukaryotic cells. These results provide direct evidence that HSP90 can affect the conformational structure of a DNA-binding protein.

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Myc phosphorylation in its basic helix–loop–helix region destabilizes transient α-helical structures, disrupting Max and DNA binding

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.002709 ◽

2018 ◽

Vol 293 (24) ◽

pp. 9301-9310 ◽

Cited By ~ 8

Author(s):

Pavel Macek ◽

Matthew J. Cliff ◽

Kevin J. Embrey ◽

Geoffrey A. Holdgate ◽

J. Willem M. Nissink ◽

...

Keyword(s):

Dna Binding ◽

Helical Structures ◽

Basic Helix Loop Helix ◽

Helix Loop Helix

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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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Synthesis of the basic-helix-loop-helix region of the immunoglobulin enhancer binding protein E47 and evaluation of its structural and DNA binding properties

International journal of peptide & protein research ◽

10.1111/j.1399-3011.1995.tb01330.x ◽

2009 ◽

Vol 46 (2) ◽

pp. 149-154 ◽

Cited By ~ 5

Author(s):

PATRICIA BISHOP ◽

CORY JONES ◽

INDRANEEL GHOSH ◽

JEAN CHMIELEWSKI

Keyword(s):

Dna Binding ◽

Binding Protein ◽

Binding Properties ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Enhancer Binding Protein ◽

Dna Binding Properties

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Murine helix-loop-helix transcriptional activator proteins binding to the E-box motif of the Akv murine leukemia virus enhancer identified by cDNA cloning

Molecular and Cellular Biology ◽

10.1128/mcb.12.8.3449-3459.1992 ◽

1992 ◽

Vol 12 (8) ◽

pp. 3449-3459

Author(s):

A L Nielsen ◽

N Pallisgaard ◽

F S Pedersen ◽

P Jørgensen

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Transcriptional Activator ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

E Box ◽

Nih 3T3 ◽

Murine Leukemia

The enhancer region of Akv murine leukemia virus contains the sequence motif ACAGATGG. This sequence is homologous to the E-box motif originally defined as a regulatory element in the enhancers of immunoglobulin mu and kappa genes. We have used double-stranded oligonucleotide probes, corresponding to the E box of the murine leukemia virus Akv, to screen a randomly primed lambda gt11 cDNA expression library made from mouse NIH 3T3 fibroblast RNA. We have identified seven lambda clones expressing DNA-binding proteins representing two different genes termed ALF1 and ALF2. The results of sequencing ALF2 cDNA suggests that we have recovered the gene for the basic-helix-loop-helix transcription factor A1, the murine analog of the human transcription factor E47. The cDNA sequence of ALF1 codes for a new member of the basic-helix-loop-helix protein family. Two splice variants of ALF1 cDNA have been found, differing by a 72-bp insertion, coding for putative proteins of 682 and 706 amino acids. The two ALF1 mRNAs are expressed at various levels in mouse tissues. In vitro DNA binding assays, using prokaryotically expressed ALF1 proteins, demonstrated specific binding of the ALF1 proteins to the Akv murine leukemia virus E-box motif ACAGATGG. Expression in NIH 3T3 fibroblasts of GAL4-ALF1 chimeric protein stimulated expression from a minimal promoter linked to a GAL4 binding site, indicating the existence of a transcriptional activator domain in ALF1.

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Conformational activation of a basic helix-loop-helix protein (MyoD1) by the C-terminal region of murine HSP90 (HSP84)

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.5059-5068.1992 ◽

1992 ◽

Vol 12 (11) ◽

pp. 5059-5068

Author(s):

R Shaknovich ◽

G Shue ◽

D S Kohtz

Keyword(s):

Dna Binding ◽

Direct Evidence ◽

Conformational Structure ◽

Amphipathic Helix ◽

Expression Library ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Transient Interaction ◽

Dose Dependent Fashion ◽

Dose Dependent

A murine cardiac lambda gt11 expression library was screened with an amphipathic helix antibody, and a recombinant representing the C-terminal 194 residues of murine HSP90 (HSP84) was cloned. Both recombinant and native HSP90s were then found to rapidly convert a basic helix-loop-helix protein (MyoD1) from an inactive to an active conformation, as assayed by sequence-specific DNA binding. The conversion process involves a transient interaction between HSP90 and MyoD1 and does not result in the formation of a stable tertiary complex. Conversion does not require ATP and occurs stoichiometrically in a dose-dependent fashion. HSP90 is an abundant, ubiquitous, and highly conserved protein present in most eukaryotic cells. These results provide direct evidence that HSP90 can affect the conformational structure of a DNA-binding protein.

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