The PAX6 gene is activated by the basic helix–loop–helix transcription factor NeuroD/BETA2

PAX6 is a transcription factor that plays an important role during pancreatic morphogenesis. The aim of the present study is to identify the upstream activator(s) of the PAX6 gene possibly involved in the early stages of pancreatic differentiation. Recently, individual elements regulating PAX6 gene activity in the pancreas have been identified in a 1100 bp Spe/HincII fragment 4.6 kb upstream of exon 0. Preliminary sequence analysis of this region revealed some potential DNA-binding sites (E boxes) specific for the binding of basic helix–loop–helix transcription factors. By using electrophoretic mobility shift assays, we demonstrated that both nuclear protein extracts from insulin-secreting RINm5F cells and in vitro-translated NeuroD/BETA2 can bind specifically to these E boxes. Furthermore, by transient transfection experiments we demonstrated that the expression of basic helix–loop–helix transcription factor NeuroD/BETA2 can induce activation of the PAX6 promoter in the NIH-3T3 cell line. Thus we show that NeuroD/BETA2 is involved in the activation of the expression of PAX6 through E boxes in the PAX6 promoter localized in a 1.1 kb sequence within the 4.6 kb untranslated region upstream of exon 0.

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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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Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.

Molecular and Cellular Biology ◽

10.1128/mcb.15.7.3813 ◽

1995 ◽

Vol 15 (7) ◽

pp. 3813-3822 ◽

Cited By ~ 443

Author(s):

S M Hollenberg ◽

R Sternglanz ◽

P F Cheng ◽

H Weintraub

Keyword(s):

Bhlh Protein ◽

Pharyngeal Arches ◽

Tissue Specific ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

New Family ◽

Specific Distribution ◽

Nih 3T3 ◽

Two Hybrid

With modified two-hybrid technology, we have isolated a member of a new family of basic helix-loop-helix (bHLH) transcription factors. Thing1 (Th1) was identified in a screen of a mouse embryo cDNA library as a partner for the Drosophila E protein daughterless. RNA in situ hybridization and reverse transcriptase-PCR demonstrate a stage- and tissue-specific distribution for the expression of Th1. Although tissue specific, the expression pattern of Th1 is fairly complex. During development, Th1 mRNA is widely expressed in extraembryonic tissues, portions of the heart, autonomic ganglia, the gut, and pharyngeal arches. At embryonic day 7.5 (E7.5), extraembryonic derivatives show robust Th1 expression. By E8.5, expression in the embryonic heart becomes detectable. During the next 2 days of development, the signal also includes gut and pharyngeal arches. Predominant expression at E13.5 is in neural crest derivatives, especially the autonomic nervous system and adrenal medulla. Expression of Th1 persists in the adult, in which it is localized to the smooth muscle cells of the gut. In vitro, Th1 protein recognizes a set of DNA sites that are more degenerate than has been determined for other bHLH factors, indicating a reduced binding specificity. Transient transfection of NIH 3T3 cells with GAL4-Th1 fusions reveals a repression activity mediated by the Th1 bHLH domain. In combination, these properties define Th1 as a new bHLH protein with a unique set of properties.

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Variants in a cis-regulatory element of TBX1 in conotruncal heart defect patients impair GATA6-mediated transactivation

Orphanet Journal of Rare Diseases ◽

10.1186/s13023-021-01981-4 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Xuechao Jiang ◽

Tingting Li ◽

Sijie Liu ◽

Qihua Fu ◽

Fen Li ◽

...

Keyword(s):

Transcription Factor ◽

Regulatory Element ◽

Expression Patterns ◽

Human Embryos ◽

Mobility Shift ◽

T Box ◽

Conotruncal Heart Defect ◽

Velo Cardio Facial Syndrome ◽

Electrophoretic Mobility Shift Assays

Abstract Background TBX1 (T-box transcription factor 1) is a major candidate gene that likely contributes to the etiology of velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). Although the haploinsufficiency of TBX1 in both mice and humans results in congenital cardiac malformations, little has been elucidated about its upstream regulation. We aimed to explore the transcriptional regulation and dysregulation of TBX1. Methods Different TBX1 promoter reporters were constructed. Luciferase assays and electrophoretic mobility shift assays (EMSAs) were used to identify a cis-regulatory element within the TBX1 promoter region and its trans-acting factor. The expression of proteins was identified by immunohistochemistry and immunofluorescence. Variants in the cis-regulatory element were screened in conotruncal defect (CTD) patients. In vitro functional assays were performed to show the effects of the variants found in CTD patients on the transactivation of TBX1. Results We identified a cis-regulatory element within intron 1 of TBX1 that was found to be responsive to GATA6 (GATA binding protein 6), a transcription factor crucial for cardiogenesis. The expression patterns of GATA6 and TBX1 overlapped in the pharyngeal arches of human embryos. Transfection experiments and EMSA indicated that GATA6 could activate the transcription of TBX1 by directly binding with its GATA cis-regulatory element in vitro. Furthermore, sequencing analyses of 195 sporadic CTD patients without the 22q11.2 deletion or duplication identified 3 variants (NC_000022.11:g.19756832C > G, NC_000022.11:g.19756845C > T, and NC_000022.11:g. 19756902G > T) in the non-coding cis-regulatory element of TBX1. Luciferase assays showed that all 3 variants led to reduced transcription of TBX1 when incubated with GATA6. Conclusions Our findings showed that TBX1 might be a direct transcriptional target of GATA6, and variants in the non-coding cis-regulatory element of TBX1 disrupted GATA6-mediated transactivation.

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Transcription factor AP-4 is a ligand for immunoglobulin-κ promoter E-box elements

Biochemical Journal ◽

10.1042/bj3540431 ◽

2001 ◽

Vol 354 (2) ◽

pp. 431-438 ◽

Cited By ~ 8

Author(s):

Alaitz ARANBURU ◽

Robert CARLSSON ◽

Christine PERSSON ◽

Tomas LEANDERSON

Keyword(s):

Transcription Factor ◽

Gene Families ◽

Sequence Motifs ◽

Mobility Shift ◽

Conserved Sequence ◽

Nuclear Extracts ◽

E Box ◽

Variable Gene ◽

E Boxes ◽

Electrophoretic Mobility Shift Assays

Immunoglobulin (Ig)-κ promoters from humans and mice share conserved sequences. The octamer element is common to all Ig promoters and pivotal for their function. However, other conserved sequence motifs, that differ between Ig variable gene families, are required for normal promoter function. These conserved motifs do not stimulate transcription in the absence of an octamer. One example is an E-box of the E47/E12 type (5′-CAGCTG-3′), which is found in all promoters of the human and murine Ig-κ gene subgroups/families, with the exception of subgroups II and VI and their related murine families. In the present study we show that the ubiquitously expressed transcription factor AP-4, and not E47, interacts specifically with the κ promoter E-boxes when tested in electrophoretic mobility-shift assays using nuclear extracts derived from human and murine B-cell lines. Furthermore, AP-4, unlike E47, did not act as a transactivator, which is in agreement with previous studies on intact κ promoters, showing that transcription is absent when the octamer element has been mutated. Based on these data, and the conservation of the 5′-CAGCTG-3′ motif among human and murine κ promoters, we propose that AP-4 is the major ligand for Ig-κ promoter E-boxes.

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Control of Directionality in Bacteriophage mv4 Site-Specific Recombination: Functional Analysis of the Xis Factor

Journal of Bacteriology ◽

10.1128/jb.00986-09 ◽

2009 ◽

Vol 192 (3) ◽

pp. 624-635 ◽

Cited By ~ 8

Author(s):

Michèle Coddeville ◽

Paul Ritzenthaler

Keyword(s):

Binding Sites ◽

Basic Protein ◽

Lactobacillus Delbrueckii ◽

Mobility Shift ◽

Site Specific ◽

Site Specific Recombination ◽

Dna Binding Sites ◽

Electrophoretic Mobility Shift Assays

ABSTRACT The integrase of the temperate bacteriophage mv4 catalyzes site-specific recombination between the phage attP site and the host attB site during Lactobacillus delbrueckii lysogenization. The mv4 prophage is excised during the induction of lytic growth. Excisive site-specific recombination between the attR and attL sites is also catalyzed by the phage-encoded recombinase, but the directionality of the recombination is determined by a second phage-encoded protein, the recombination directionality factor (RDF). We have identified and functionally characterized the RDF involved in site-specific excision of the prophage genome. The mv4 RDF, mv4Xis, is encoded by the second gene of the early lytic operon. It is a basic protein of 56 amino acids. Electrophoretic mobility shift assays demonstrated that mv4Xis binds specifically to the attP and attR sites via two DNA-binding sites, introducing a bend into the DNA. In vitro experiments and in vivo recombination assays with plasmids in Escherichia coli and Lactobacillus plantarum demonstrated that mv4Xis is absolutely required for inter- or intramolecular recombination between the attR and attL sites. In contrast to the well-known phage site-specific recombination systems, the integrative recombination between the attP and attB sites seems not to be inhibited by the presence of mv4Xis.

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Orphan Nuclear Receptor Small Heterodimer Partner, a Novel Corepressor for a Basic Helix-Loop-Helix Transcription Factor BETA2/NeuroD

Molecular Endocrinology ◽

10.1210/me.2003-0311 ◽

2004 ◽

Vol 18 (4) ◽

pp. 776-790 ◽

Cited By ~ 37

Author(s):

Joon-Young Kim ◽

Khoi Chu ◽

Han-Jong Kim ◽

Hyun-A Seong ◽

Ki-Cheol Park ◽

...

Keyword(s):

Transcription Factor ◽

Nuclear Receptor ◽

Transcriptional Activity ◽

Orphan Nuclear Receptor ◽

Small Heterodimer Partner ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

E Box ◽

Coactivator P300

Abstract Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA binding domain (DBD) and represses the transcriptional activity of various nuclear receptors. In this study, we examined the novel cross talk between SHP and BETA2/NeuroD, a basic helix-loop-helix transcription factor. In vitro and in vivo protein interaction studies showed that SHP physically interacts with BETA2/NeuroD, but not its heterodimer partner E47. Moreover, confocal microscopic study and immunostaining results demonstrated that SHP colocalized with BETA2 in islets of mouse pancreas. SHP inhibited BETA2/NeuroD-dependent transactivation of an E-box reporter, whereas SHP was unable to repress the E47-mediated transactivation and the E-box mutant reporter activity. In addition, SHP repressed the BETA2-dependent activity of glucokinase and cyclin-dependent kinase inhibitor p21 gene promoters. Gel shift and in vitro protein competition assays indicated that SHP inhibits neither dimerization nor DNA binding of BETA2 and E47. Rather, SHP directly repressed BETA2 transcriptional activity and p300-enhanced BETA2/NeuroD transcriptional activity by inhibiting interaction between BETA2 and coactivator p300. We also showed that C-terminal repression domain within SHP is also required for BETA2 repression. However, inhibition of BETA2 activity was not observed by naturally occurring human SHP mutants that cannot interact with BETA2/NeuroD. Taken together, these results suggest that SHP acts as a novel corepressor for basic helix-loop-helix transcription factor BETA2/NeuroD by competing with coactivator p300 for binding to BETA2/NeuroD and by its direct transcriptional repression function.

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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 ◽

1992 ◽

Vol 12 (8) ◽

pp. 3449-3459 ◽

Cited By ~ 31

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

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Targeting the Microphthalmia Basic Helix-Loop-Helix–Leucine Zipper Transcription Factor to a Subset of E-Box Elements In Vitro and In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.6930 ◽

1998 ◽

Vol 18 (12) ◽

pp. 6930-6938 ◽

Cited By ~ 136

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.

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Human Hand1 basic helix-loop-helix (bHLH) protein: extra-embryonic expression pattern, interaction partners and identification of its transcriptional repressor domains

Biochemical Journal ◽

10.1042/bj3610641 ◽

2002 ◽

Vol 361 (3) ◽

pp. 641-651 ◽

Cited By ~ 23

Author(s):

Martin KNÖFLER ◽

Gudrun MEINHARDT ◽

Sandra BAUER ◽

Thomas LOREGGER ◽

Richard VASICEK ◽

...

Keyword(s):

Transcriptional Activity ◽

Transcriptional Repressor ◽

Bhlh Protein ◽

Bhlh Transcription Factor ◽

Basic Helix Loop Helix ◽

Class A ◽

Helix Loop Helix ◽

Bhlh Factors ◽

E Boxes

The basic helix-loop-helix (bHLH) transcription factor, Hand1, plays an important role in the development of the murine extra-embryonic trophoblast cell lineage. In the present study, we have analysed the expression of Hand1 in human extra-embryonic cell types and determined its binding specificity and transcriptional activity upon interaction with different class A bHLH factors. Northern blotting and in situ hybridization showed that Hand1 mRNA is specifically expressed in amnion cells at different stages of gestation. Accordingly, we demonstrate that the protein is exclusively produced in the amniotic epithelium in vivo and in purified amnion cells in vitro using a novel polyclonal Hand1 antiserum. Reverse transcriptase-PCR and immunohistochemical staining of blastocysts revealed the production of Hand1 mRNA and polypeptide in the trophectodermal cell layer. In the presence of E12/E47, Hand1 stimulated the transcription of luciferase reporters harbouring degenerate E-boxes, suggesting that E-proteins are potential dimerization partners in trophoblastic tumour and amnion cells. In contrast, Hand1 diminished E12/E47-dependent transcription of reporters containing perfect E-boxes by inhibiting the interaction of Hand1/E-protein heterodimers with the palindromic cognate sequence. Furthermore, we show that Hand1 down-regulated GAL—E12-dependent reporter expression, indicating that the protein can also act directly as a transcriptional repressor. Mutational analyses of GAL-Hand1 suggested that two protein regions located within its N-terminal portion mainly confer the repressing activity. In conclusion, human Hand1 may play an important role in the differentiation of the amniotic membrane and the pre-implanting trophoblast. Furthermore, the data suggest that Hand1 can act as a repressor by two independent mechanisms; sequestration of class A bHLH factors from E-boxes and inhibition of their transcriptional activity.

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P/CAF rescues the Bhlhe40-mediated repression of MyoD transactivation

Biochemical Journal ◽

10.1042/bj20090072 ◽

2009 ◽

Vol 422 (2) ◽

pp. 343-352 ◽

Cited By ~ 17

Author(s):

Sheng P. Hsiao ◽

Kai M. Huang ◽

Hsin Y. Chang ◽

Shen L. Chen

Keyword(s):

Transcription Initiation ◽

Myogenic Differentiation ◽

Binding Protein ◽

Core Promoter ◽

Peroxisome Proliferator ◽

Dependent Manner ◽

Mobility Shift ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

E Boxes

Previously, we found that MRFs (myogenic regulatory factors) regulated the expression of PGC-1α (peroxisome-proliferator-activated receptor γ co-activator 1α) by targeting a short region, from nt −49 to +2 adjacent to the transcription initiation site, that contained two E-boxes. However, only the E2-box had significant affinity for MRFs, and the E1-box was predicted to be the target of Bhlhe40 (basic helix-loop-helix family, member e40, also known as Stra13, Bhlhb2, DEC1 and Sharp2), a transcriptional repressor implicated in the regulation of several physiological processes. In the present study, by using EMSA (electrophoresis mobility-shift assay), we confirmed that Bhlhe40 targeted the E1-box and formed a complex with the basic helix-loop-helix transcription factor MyoD (myogenic differentiation factor D) on the PGC-1α core promoter. We demonstrate that Bhlhe40 binds to the promoters of PGC-1α and myogenic genes in vivo and that Bhlhe40 represses the MyoD-mediated transactivation of these promoters. Furthermore, we found that this repression could be relieved by P/CAF (p300/CBP-associated factor) in a dose-dependent manner, but not by CBP [CREB (cAMP-response-element-binding protein)-binding protein]. Bhlhe40 interacted with P/CAF and this interaction disrupted the interaction between P/CAF and MyoD. These results suggest that Bhlhe40 functions as a repressor of MyoD by binding to adjacent E-boxes and sequestering P/CAF from MyoD.

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