Anti-Helix-Loop-Helix Domain Antibodies: Discovery of Autoantibodies That Inhibit DNA Binding Activity of Human Centromere Protein B (CENP-B)1

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

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Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein.

Molecular and Cellular Biology ◽

10.1128/mcb.17.8.4536 ◽

1997 ◽

Vol 17 (8) ◽

pp. 4536-4543 ◽

Cited By ~ 67

Author(s):

V Bailly ◽

S Prakash ◽

L Prakash

Keyword(s):

Dna Binding ◽

Protein Degradation ◽

Binding Activity ◽

Helix Loop Helix ◽

Dna Binding Activity ◽

C Terminus ◽

Rad6 Gene ◽

Ubiquitin Conjugating Enzyme ◽

Dependent Protein ◽

Conjugating Enzyme

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme required for postreplicational repair of UV-damaged DNA and for damage-induced mutagenesis. In addition, Rad6 functions in the N end rule pathway of protein degradation. Rad6 mediates its DNA repair role via its association with Rad18, whose DNA binding activity may target the Rad6-Rad18 complex to damaged sites in DNA. In its role in N end-dependent protein degradation, Rad6 interacts with the UBR1-encoded ubiquitin protein ligase (E3) enzyme. Previous studies have indicated the involvement of N-terminal and C-terminal regions of Rad6 in interactions with Ubr1. Here, we identify the regions of Rad6 and Rad18 that are involved in the dimerization of these two proteins. We show that a region of 40 amino acids towards the C terminus of Rad18 (residues 371 to 410) is sufficient for interaction with Rad6. This region of Rad18 contains a number of nonpolar residues that have been conserved in helix-loop-helix motifs of other proteins. Our studies indicate the requirement for residues 141 to 149 at the C terminus, and suggest the involvement of residues 10 to 22 at the N terminus of Rad6, in the interaction with Rad18. Each of these regions of Rad6 is indicated to form an amphipathic helix.

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Preferred sequences for DNA recognition by the TAL1 helix-loop-helix proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1256 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1256-1265 ◽

Cited By ~ 104

Author(s):

H L Hsu ◽

L Huang ◽

J T Tsan ◽

W Funk ◽

W E Wright ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Dna Binding ◽

Lymphoblastic Leukemia ◽

Dna Recognition ◽

Binding Activity ◽

Class A ◽

Helix Loop Helix ◽

Dna Binding Activity ◽

Cell Acute Lymphoblastic Leukemia

Tumor-specific activation of the TAL1 gene is the most common genetic alteration seen in patients with T-cell acute lymphoblastic leukemia. The TAL1 gene products contain the basic helix-loop-helix (bHLH) domain, a protein dimerization and DNA-binding motif common to several known transcription factors. A binding-site selection procedure has now been used to evaluate the DNA recognition properties of TAL1. These studies demonstrate that TAL1 polypeptides do not have intrinsic DNA-binding activity, presumably because of their inability to form bHLH homodimers. However, TAL1 readily interacts with any of the known class A bHLH proteins (E12, E47, E2-2, and HEB) to form heterodimers that bind DNA in a sequence-specific manner. The TAL1 heterodimers preferentially recognize a subset of E-box elements (CANNTG) that can be represented by the consensus sequence AACAGATGGT. This consensus is composed of half-sites for recognition by the participating class A bHLH polypeptide (AACAG) and the TAL1 polypeptide (ATGGT). TAL1 heterodimers with DNA-binding activity are readily detected in nuclear extracts of Jurkat, a leukemic cell line derived from a patient with T-cell acute lymphoblastic leukemia. Hence, TAL1 is likely to bind and regulate the transcription of a unique subset of subordinate target genes, some of which may mediate the malignant function of TAL1 during T-cell leukemogenesis.

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HERP, a Novel Heterodimer Partner of HES/E(spl) in Notch Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.21.17.6080-6089.2001 ◽

2001 ◽

Vol 21 (17) ◽

pp. 6080-6089 ◽

Cited By ~ 141

Author(s):

Tatsuya Iso ◽

Vittorio Sartorelli ◽

Coralie Poizat ◽

Simona Iezzi ◽

Hung-Yi Wu ◽

...

Keyword(s):

Dna Binding ◽

Notch Signaling ◽

Dna Sequences ◽

Transcriptional Repression ◽

De Novo ◽

Single Cells ◽

Binding Activity ◽

Bhlh Protein ◽

Helix Loop Helix ◽

Dna Binding Activity

ABSTRACT HERP1 and -2 are members of a new basic helix-loop-helix (bHLH) protein family closely related to HES/E(spl), the only previously known Notch effector. Like that of HES, HERP mRNA expression is directly up-regulated by Notch ligand binding without de novo protein synthesis. HES and HERP are individually expressed in certain cells, but they are also coexpressed within single cells after Notch stimulation. Here, we show that HERP has intrinsic transcriptional repression activity. Transcriptional repression by HES/E(spl) entails the recruitment of the corepressor TLE/Groucho via a conserved WRPW motif, whereas unexpectedly the corresponding—but modified—tetrapeptide motif in HERP confers marginal repression. Rather, HERP uses its bHLH domain to recruit the mSin3 complex containing histone deacetylase HDAC1 and an additional corepressor, N-CoR, to mediate repression. HES and HERP homodimers bind similar DNA sequences, but with distinct sequence preferences, and they repress transcription from specific DNA binding sites. Importantly, HES and HERP associate with each other in solution and form a stable HES-HERP heterodimer upon DNA binding. HES-HERP heterodimers have both a greater DNA binding activity and a stronger repression activity than do the respective homodimers. Thus, Notch signaling relies on cooperation between HES and HERP, two transcriptional repressors with distinctive repression mechanisms which, either as homo- or as heterodimers, regulate target gene expression.

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Silencing of the inhibitor of DNA binding protein 4 (ID4) contributes to the pathogenesis of mouse and human CLL

Blood ◽

10.1182/blood-2010-05-284638 ◽

2011 ◽

Vol 117 (3) ◽

pp. 862-871 ◽

Cited By ~ 50

Author(s):

Shih-Shih Chen ◽

Rainer Claus ◽

David M. Lucas ◽

Lianbo Yu ◽

Jiang Qian ◽

...

Keyword(s):

Dna Binding ◽

Promoter Methylation ◽

Binding Protein ◽

Dna Binding Protein ◽

Binding Activity ◽

Lymphocytic Leukemia ◽

Dominant Negative ◽

Helix Loop Helix ◽

Dna Binding Activity ◽

Inhibitor Of Dna Binding

Abstract Inhibitor of DNA binding protein 4 (ID4) is a member of the dominant-negative basic helix-loop-helix transcription factor family that lacks DNA binding activity and has tumor suppressor function. ID4 promoter methylation has been reported in acute myeloid leukemia and chronic lymphocytic leukemia (CLL), although the expression, function, and clinical relevance of this gene have not been characterized in either disease. We demonstrate that the promoter of ID4 is consistently methylated to various degrees in CLL cells, and increased promoter methylation in a univariable analysis correlates with shortened patient survival. However, ID4 mRNA and protein expression is uniformly silenced in CLL cells irrespective of the degree of promoter methylation. The crossing of ID4+/− mice with Eμ-TCL1 mice triggers a more aggressive murine CLL as measured by lymphocyte count and inferior survival. Hemizygous loss of ID4 in nontransformed TCL1-positive B cells enhances cell proliferation triggered by CpG oligonucleotides and decreases sensitivity to dexamethasone-mediated apoptosis. Collectively, this study confirms the importance of the silencing of ID4 in murine and human CLL pathogenesis.

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Preferred sequences for DNA recognition by the TAL1 helix-loop-helix proteins

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1256-1265.1994 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1256-1265

Author(s):

H L Hsu ◽

L Huang ◽

J T Tsan ◽

W Funk ◽

W E Wright ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Dna Binding ◽

Lymphoblastic Leukemia ◽

Dna Recognition ◽

Binding Activity ◽

Class A ◽

Helix Loop Helix ◽

Dna Binding Activity ◽

Cell Acute Lymphoblastic Leukemia

Tumor-specific activation of the TAL1 gene is the most common genetic alteration seen in patients with T-cell acute lymphoblastic leukemia. The TAL1 gene products contain the basic helix-loop-helix (bHLH) domain, a protein dimerization and DNA-binding motif common to several known transcription factors. A binding-site selection procedure has now been used to evaluate the DNA recognition properties of TAL1. These studies demonstrate that TAL1 polypeptides do not have intrinsic DNA-binding activity, presumably because of their inability to form bHLH homodimers. However, TAL1 readily interacts with any of the known class A bHLH proteins (E12, E47, E2-2, and HEB) to form heterodimers that bind DNA in a sequence-specific manner. The TAL1 heterodimers preferentially recognize a subset of E-box elements (CANNTG) that can be represented by the consensus sequence AACAGATGGT. This consensus is composed of half-sites for recognition by the participating class A bHLH polypeptide (AACAG) and the TAL1 polypeptide (ATGGT). TAL1 heterodimers with DNA-binding activity are readily detected in nuclear extracts of Jurkat, a leukemic cell line derived from a patient with T-cell acute lymphoblastic leukemia. Hence, TAL1 is likely to bind and regulate the transcription of a unique subset of subordinate target genes, some of which may mediate the malignant function of TAL1 during T-cell leukemogenesis.

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PER and TIM Inhibit the DNA Binding Activity of aDrosophila CLOCK-CYC/dBMAL1 Heterodimer without Disrupting Formation of the Heterodimer: a Basis for Circadian Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.19.8.5316 ◽

1999 ◽

Vol 19 (8) ◽

pp. 5316-5325 ◽

Cited By ~ 167

Author(s):

Choogon Lee ◽

Kiho Bae ◽

Isaac Edery

Keyword(s):

Dna Binding ◽

Clock Genes ◽

Binding Activity ◽

Core Element ◽

Enhancer Element ◽

Rhythmic Expression ◽

Helix Loop Helix ◽

Dna Binding Activity ◽

Circadian Clock Genes ◽

High Level

ABSTRACT The Drosophila CLOCK (dCLOCK) and CYCLE (CYC) (also referred to as dBMAL1) proteins are members of the basic helix-loop-helix PAS (PER-ARNT-SIM) superfamily of transcription factors and are required for high-level expression of the circadian clock genes period (per) andtimeless (tim). Several lines of evidence indicate that PER, TIM, or a PER-TIM heterodimer somehow inhibit the transcriptional activity of a putative dCLOCK-CYC complex, generating a negative-feedback loop that is a core element of theDrosophila circadian oscillator. In this report we show that PER and/or TIM inhibits the binding of a dCLOCK-CYC heterodimer to an E-box-containing DNA fragment that is present in the 5′ nontranscribed region of per and acts as a circadian enhancer element. Surprisingly, inhibition of this DNA binding activity by PER, TIM, or both is not accompanied by disruption of the association between dCLOCK and CYC. The results suggest that the interaction of PER, TIM, or both with the dCLOCK-CYC heterodimer induces a conformational change or masks protein regions in the heterodimer, leading to a reduction in DNA binding activity. Together with other findings, our results strongly suggest that daily cycles in the association of PER and TIM with the dCLOCK-CYC complex probably contribute to rhythmic expression of per andtim.

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The CIB1 Transcription Factor Regulates Light- and Heat-inducible Cell Elongation via a Two-step HLH/bHLH System

Journal of Experimental Botany ◽

10.1093/jxb/eraa567 ◽

2020 ◽

Author(s):

Miho Ikeda ◽

Nobutaka Mitsuda ◽

Toru Ishizuka ◽

Mai Satoh ◽

Masaru Ohme-Takagi

Keyword(s):

Transcription Factor ◽

High Temperature ◽

Dna Binding ◽

Cell Elongation ◽

Binding Activity ◽

Bhlh Transcription Factor ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Dna Binding Activity ◽

Underlying Mechanisms

Abstract Light and high temperature promote plant cell elongation. PHYTOCHROME INTERACTING FACTOR4 (PIF4, a typical basic helix-loop-helix [bHLH] transcriptional activator) and the non-DNA-binding atypical HLH inhibitors PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and LONG HYPOCOTYL IN FAR-RED 1 (HFR1) competitively regulate cell elongation in response to light conditions and high temperature. However, the underlying mechanisms have not been fully clarified. Here, we show that in Arabidopsis thaliana, the bHLH transcription factor CRYPTOCHROME-INTERACTING BASIC HELIX-LOOP-HELIX 1 (CIB1) positively regulates cell elongation under the control of PIF4, PAR1, and HFR1. Furthermore, PIF4 directly regulates CIB1 expression by interacting with its promoter, and PAR1 and HFR1 interfere with PIF4 binding to the CIB1 promoter. CIB1 activates genes that function in cell elongation, and PAR1 interferes with the DNA binding activity of CIB1, thus suppressing cell elongation. Hence, two antagonistic HLH/bHLH systems, the PIF4–PAR1/HFR1 and CIB1–PAR1 systems, regulate cell elongation in response to light and high temperature. We thus demonstrate the important role of non-DNA-binding small HLH proteins in the transcriptional regulation of cell elongation in plants.

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