bHLH Binding Sites Research Articles

N-Twist, an Evolutionarily Conserved bHLH Protein Expressed in the Developing CNS, Functions as a Transcriptional Inhibitor

Members of the basic helix–loop–helix (bHLH) transcription factor family play an essential role in multiple developmental processes. During neurogenesis, positive and negative regulation by bHLH proteins is essential for proper development. Here we report the identification and initial characterization of the bHLH gene, Neuronal twist (N-twist), named for its neural expression pattern and high sequence homology and physical linkage to the mesodermal inhibitor, M-twist. N-twist is expressed in the developing mouse central nervous system in the midbrain, hindbrain, and neural tube. This neural expression is conserved in invertebrates, as expression of the Drosophila ortholog of N-twist is also restricted to the central nervous system. Like other bHLH family members, N-Twist heterodimerizes with E protein and binds DNA at a consensus bHLH-binding site, the E box. We show that N-Twist inhibits MASH1-dependent transcriptional activation by sequestering E protein in a dominant negative fashion. Thus, these studies support the notion that N-Twist represents a novel negative regulator of neurogenesis.

Functional Characterization of an Inositol-sensitive Upstream Activation Sequence in Yeast: A cis-REGULATORY ELEMENT RESPONSIBLE FOR INOSITOL-CHOLINE MEDIATED REGULATION OF PHOSPHOLIPID BIOSYNTHESIS

A repeated element, the inositol-sensitive upstream activation sequence (UASINO), having the consensus sequence, 5'-CATGTGAAAT-3', is present in the promoters of genes encoding enzymes of phospholipid biosynthesis that are regulated in response to the phospholipid precursors, inositol and choline. None of the naturally occurring variants of the UASINO element exactly recapitulates the consensus (for review, see Carman, G. M., and Henry, S. A. (1989) Annu. Rev. Biochem. 58, 635-669 and Paltauf, F., Kolwhein, S., and Henry, S. A. (1992) in Molecular Biology of the Yeast Saccharomyces cerevisiae (Broach, J., Jones, E., and Pringle, J., eds) Vol. 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The first six bases of the UASINO element are homologous with canonical binding motif for proteins of the basic helix-loop-helix (bHLH) family. Two bHLH regulatory proteins, Ino2p and Ino4p from yeast, were previously shown to bind to promoter fragments containing this element. In the present study, an extensive analysis of UASINO function has been conducted. We report that any base substitution within the putative bHLH binding site resulted either in a dramatic reduction or in a complete obliteration of UASINO function as tested in an expression assay in vivo. Base substitutions in the 5' region that flanks the 10-base pair repeat, as well as sequences within the repeat itself at its 3' end outside the bHLH core, were also assessed. The two bases immediately flanking the 5' end of the element proved to be very important to its function as a UAS element as did the two bases immediately 3' of the bHLH core motif. Substitutions of the final two bases of the original ten base pair consensus (i.e. 5'-CATGTGAAAT-3') had less dramatic effects. We also tested a subset of the altered elements for their ability to serve as competitors in an assay of Ino2p x Ino4p binding. The strength of any given sequence as a UASINO element, as assayed in vivo, was strongly correlated with its strength as a competitor for Ino2p x Ino4p binding. We also tested a subset of the modified UASINO elements for their effects on expression in vivo in a strain carrying an opi1 mutation. The opi1 mutation renders the coregulated enzymes of phospholipid synthesis constitutive in the presence of phospholipid precursors. All elements that retained some residual UASINO activity when tested in the wild-type strain were constitutively expressed at a level comparable with the wild-type derepressed level when tested in the opi1 mutant.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple modes of dorsal-bHLH transcriptional synergy in the Drosophila embryo.

Synergistic interactions between the maternal regulatory factor dorsal (dl) and basic helix-loop-helix (bHLH) activators are essential for initiating differentiation of the mesoderm and neuroectoderm in the early Drosophila embryo. Here we present evidence that dl-bHLH interactions mediating gene expression in the neuroectoderm and mesoderm are fundamentally distinct. Close proximity of dl and bHLH binding sites is essential for the synergistic activation of gene expression in the lateral neuroectoderm, where there are diminishing levels of the dl regulatory gradient. In contrast, sharp on/off patterns of gene expression in the presumptive mesoderm do not require linkage of these sites. Analysis of minimal and synthetic promoter elements suggests that dl and bHLH activators, such as twist, might interact with different rate-limiting components of the transcription complex. These results are consistent with two distinct modes of dl-bHLH synergy: cooperative binding to DNA (requiring linkage of sites) and synergistic contact of basal transcription factors (not requiring linkage). Finally, the characterization of a 57 bp synthetic minimal stripe unit (MSU) provides evidence for a third tier of dl-bHLH synergy. Tandem copies of the MSU function as a bona fide enhancer and can mediate neuroectoderm expression in transgenic embryos even when placed 4.5 kb downstream of a test promoter. Multiple copies of the MSU function synergistically only when linked, but not when separated. We propose that this linkage requirement provides the basis for the evolution of modular promoters composed of discrete, non-overlapping enhancers.

Upstream stimulatory factor, a basic-helix-loop-helix-zipper protein, regulates the activity of the alpha-glycoprotein hormone subunit gene in pituitary cells.

In an effort to determine whether basic-helix-loop-helix (bHLH) proteins are important in pituitary-specific expression of the alpha-glycoprotein hormone subunit gene, we examined the effect of the dominant negative HLH protein, Id, on the activity of the alpha-subunit gene promoter in pituitary cells. Id over-expression reduces the expression of alpha-subunit reporter genes in either alpha T3-1 gonadotrope-derived or alpha TSH thyrotrope-derived cells. A deletion fragment containing nucleotides from -131 to +44 of the human alpha-subunit promoter is inhibited to a similar degree as a -244 to +44 fragment in alpha T3-1 cells. Nuclear proteins in alpha T3-1 cells bind two potential bHLH protein binding sites (E-boxes, alpha EB1 and alpha EB2) present in this fragment but not to mutations that specifically alter only these sequences. An antibody-specific for upstream stimulatory factor, a widely expressed bHLH-leucine zipper protein, is able to inhibit factor binding to the alpha EB2 sequences but not the alpha EB1 site. Mutating the alpha EB1 element of the alpha-subunit promoter decreases basal activity of this promoter to about 42% of control levels in alpha T3-1 cells. A mutation that abolishes upstream stimulatory factor binding, either alone or in combination with the alpha EB1 mutation, reduces basal activity of the promoter to approximately 21% of control levels in alpha T3-1 cells and abolishes the decrease in promoter activity seen when Id is overexpressed. These results demonstrate that the bHLH family of proteins are important regulators of alpha-subunit gene expression in pituitary cells.

Human T-cell leukemia virus type I Tax protein represses gene expression through the basic helix-loop-helix family of transcription factors.

The human T-cell leukemia virus type I (HTLV-I) oncoprotein Tax is a potent activator of viral and cellular gene transcription. Tax does not bind DNA directly but utilizes cellular transcription factors to mediate activation. In this report, we examine the role of the basic helix-loop-helix (bHLH) proteins in Tax deregulation of gene expression, as these proteins play a critical role in progression through the cell cycle and have been implicated in neoplastic disease. We show that the bHLH proteins do not mediate activation, but instead mediate repression of gene expression in the presence of Tax. We further show that a consensus bHLH binding site in the promoter of the beta-polymerase gene, which encodes an enzyme involved in DNA repair, mediates the previously reported repression of beta-polymerase gene expression by Tax. Together, these results suggest that Tax may induce malignant transformation, at least in part, through bHLH-mediated repression of key cellular regulatory genes.

A human DNA replication origin: localization and transcriptional characterization.

A single-copy 13.7 kb human DNA region (L30E) located on Ch. 19 p13.3 contains an origin of DNA replication in myeloid HL-60 cells. The origin was localized, by means of quantitative PCR within approximately 3000 bp, in a highly transcribed region containing at least two closely spaced genes with the same polarity of transcription, one encoding lamin B2 and the other an unidentified protein. The origin region overlaps an undermethylated "CpG island" at the 5'-end of the second transcription unit. A binding site (CACGTG) for basic helix-loop-helix (bHLH) DNA binding proteins such as USF/MLTF or MYC-MAX was located by DNase I footprinting analysis in the promoter of the second gene. DMSO differentiation of HL-60 cells, that completely shuts off replication, also drastically reduces the transcription of L30E region. On the other hand such treatment does not modify the methylation pattern of the CpG island and does not abolish the DNase I protection of the bHLH binding site.

Discrimination between related DNA sites by a single amino acid residue of Myc-related basic-helix-loop-helix proteins.

A yeast genetic system was developed to study how the basic regions of basic-helix-loop-helix (bHLH) proteins distinguish between related consensus bHLH binding sites, with nucleotide sequence CANNTG. The yeast bHLH protein CBF1 binds to the sequence CAC(A/G)TG found in the yeast centromere element CDE1 and in promoter regions of several yeast genes involved in methionine biosynthesis. Using a functional assay to rescue a mutant cbf1 yeast strain from methionine auxotrophy, we determined that the basic region of CBF1 could be replaced by the homologous region of either the vertebrate USF transcription factor or c-Myc, both of which bind CACGTG. The homologous region of the AP4 transcription factor, which recognizes the sequence CAGCTG, could not functionally replace the CBF1 basic region. However, only a single substitution, Met----Arg, in the AP4 basic region of the inactive chimera CBF-AP4 was sufficient to restore CBF1 function. In randomization experiments, only arginine or lysine provided functional substitutions at the AP4 methionine position. The results suggest that this conserved arginine residue in the basic regions of Myc-related bHLH proteins discriminates between CAC(A/G)TG and related sites.

Integrative genomic analyses on GLI2: Mechanism of Hedgehog priming through basal GLI2 expression, and interaction map of stem cell signaling network with P53

Hedgehog-binding to Patched family receptors results in Smoothened-mediated activation of MAP3K10 (MST) and inactivation of SUFU. MAP3K10-induced DYRK2 phosphorylation combined with SUFU inhibition results in the stabilization and nuclear accumulation of GLI2 for transcriptional activation of GLI1, CCND1, CCND2, FOXA2, FOXC2, FOXP3, FOXQ1, RUNX2, and JAG2. Here, integrative genomic analyses on GLI2 orthologs were carried out. Rat Gli2 complete coding sequence was determined by assembling nucleotide sequences of exons 1, 2, and 5'-truncated rat Gli2 RefSeq (NM_001107169.1). GLI2 orthologs were more related to GLI3 orthologs than to GLI1 orthologs lacking the N-terminal repressor domain. betaTRCP1 (FBXW1)-binding DSYxxxS motif was conserved in GLI2 and GLI3 orthologs, while betaTRCP2 (FBXW11)-binding DSGxxxxxxxxxS motif in GLI2 and GLI1 orthologs. Human GLI2 mRNA was expressed in ES cells, NT2 cells, fetal lung, fetal heart, regenerating liver, gastric cancer, and other tumors. Mouse Gli2 mRNA was expressed in unfertilized egg, ES cells, and EG cells. Tandem RRRCWWGYYY motifs for P53, P63 or P73, and also four conserved bHLH-binding sites were identified within GLI2 proximal promoter region. Interaction map of P53 and stem cell signaling network were then constructed. P53-induced NOTCH1 upregulation leads to HES1, HES5, HEY1, HEY2 or HEYL upregulation for the repression of tissue specific bHLH transcriptional activators. DYRK2 functions as a positive regulator of P53-mediated apoptosis, and also as a negative regulator of the Hedgehog signaling cascade. GLI2 expression is regulated based on the balance of P53, Notch, and TGF-beta signaling, and Hedgehog signaling activation results in cell survival and proliferation due to transcriptional activation of Hedgehog-target genes, and also partly due to perturbation of P53-mediated transcriptional regulation.