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Cooperative Mesp activity is required for normal somitogenesis along the anterior-posterior axis

DEVELOPMENTAL BIOLOGY 2006年第2期300卷 687-698页

作者： Morimoto, Mitsuru Kiso, Makoto Sasaki, Nobuo Saga, Yumiko Natl Inst Genet Div Mammalian Dev Mishima Shizuoka 4118540 Japan SOKENDAI Mishima Shizuoka 4118540 Japan

Mesp2 is a bHLH-type transcription factor that plays a key role during somitogenesis. Mesp2 is transiently expressed and is quickly degraded once translated. In our current study, we find that Mesp2 contains a degradation domain, which acts as a target of proteasome-mediated proteolysis and appears to play this role in vivo. We have also defined the nuclear localization signals (NLS) and constructed a minimum Mesp2 protein (P2-HD) composed of the NLS, bHLH and the degradation domains. The ability of the P2-HD as a transcription factor in vivo was examined. Some of the defects that had been previously observed in the Mesp2-null mice were rescued in the knock-in mice but only in the posterior half of the body, indicating differential effects of P2-HD along the anterior-posterior (AP) axis. In addition, quantitative analysis of the expression along the AP axis revealed that the relative levels of Mesp2 increased, whereas Mesp1 is down-regulated in the later stages of development by the activities of Mesp2 in the wild-type embryo. Moreover, we have found that somitogenesis in the early stages is more susceptible to changes in the Mesp gene dosage.. indicating that a threshold level of Mesp activity must be required for the progression of normal somitogenesis. (c) 2006 Elsevier Inc. All rights reserved.

关键词： Mesp1 Mesp2 somitogenesis vertebra protein degradation proteasome knock-in mouse nuclear localization basic helix-loop-helix transcriptional factor

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The INDEHISCENT protein regulates unequal cell divisions in Arabidopsis fruit

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PLANTA 2006年第4期224卷 971-979页

作者： Wu, Hong Mori, Aiko Jiang, Xingshan Wang, Yixing Yang, Ming Oklahoma State Univ Dept Bot Stillwater OK 74078 USA S China Agr Univ Coll Life Sci Guangzhou 510642 Guangdong Peoples R China

INDEHISCENT (IND) is one of the major factors of a transcriptional network regulating fruit dehiscence in Arabidopsis thaliana. However, partially owing to the lack of knowledge on the morphogenic process at the junction between the valve margin and the replum, the cellular processes regulated by IND have not been elucidated. Here we report the identification of a zone of seven layers of cells relevant to silique dehiscence, and the isolation and characterization of a transposon-enhancer-trap-generated null allele of IND, ind-6. Light microscopy analysis revealed that the beta-glucuronidase from the enhancer trap was expressed in a seven-layer zone, encompassing the valve margin and the replum border. Electron and light microscopy analyses also revealed that unequal cell divisions actively occurred at stage 15 in the seven-layer zone in wild type (WT), which apparently produced the distinct cell types in the WT seven-layer zone. In contrast, siliques of ind-6 at stage 15 did not exhibit such cell divisions but fewer cells and defective cytokinesis, hence lacking the distinct cell types in the seven-layer zone. These results demonstrate a cellular mechanism for establishing the distinct cell types and a role for IND in regulating the unequal cell divisions in the seven-layer zone.

关键词： Arabidopsis thaliana basic helix-loop-helix fruit dehiscence morphogenesis beta-glucuronidase INDEHISCENT

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HEY2 mutations in malformed hearts.

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Human mutation 2006年第1期27卷 118-118页

作者： Reamon-Buettner, Stella Marie Borlak, Juergen Drug Research and Medical Biotechnology Fraunhofer Institute of Toxicology and Experimental Medicine Nikolai Fuchs Strasse 1 30625 Hannover Germany

The basic helix-loop-helix (bHLH) transcription factor Hey2 (gridlock) is an important determinant of mammalian heart development, but its role in human ventricular septal defects is unknown. Hey2 functions as a repressor through the bHLH domain. By direct sequencing, we analyzed the sequences encoding the bHLH domain of the human HEY2 in 52 explanted hearts of unrelated patients with complex cardiac malformations, notably ventricular (VSD) and atrioventricular septal defects (AVSD). We found three nonsynonymous mutations, namely, c.286A>G (***96Ala), c.293A>C (***98Ala), and c.299T>C (***100Ser) affecting the second helix of HEY2 in the diseased cardiac tissues of two patients with AVSD. This result suggests a possible role of HEY2 in the regulation of ventricular septation in humans. Since the two AVSD patients carried also binding domain mutations in other cardiac-specific transcription factors, e.g. NKX2-5, TBX5, and GATA4, breakdown of combinatorial interactions of transcription factors may have contributed to the complexity of their cardiac malformations. 2005 Wiley-Liss, Inc.

关键词： HEY2 mutations transcription factor basic helix-loop-helix congenital heart disease

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Nulp1, a novel basic helix-loop-helix protein expressed broadly during early embryonic organogenesis and prominently in developing dorsal root ganglia

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CELL AND TISSUE RESEARCH 2002年第3期308卷 361-370页

作者： Olsson, M Durbeej, M Ekblom, P Hjalt, T Lund Univ BMC Dept Cell & Mol Biol Sect Cell & Mol Biol S-22184 Lund Sweden

The basic helix-loop-helix (bHLH) proteins control differentiation and development of a variety of organs. We have isolated the complementary DNA (cDNA) of a novel class of bHLH transcription factors. The previously uncharacterized bHLH messenger RNA (mRNA) was identified by RNA fingerprinting by comC, paring embryonic and adult rnRNA. The reading frame sequence predicts a new class of bHLH family. Northern blotting of embryonic stages demonstrated a 3.2-kb transcript present in several embryonic tissues, including kidney, brain, heart, and lung, in a fashion confirmatory with the RNA-fingerprinting data. In situ hybridization of cryosections detected strong signals in the dorsal root ganglia of 14-day-old mouse embryos (E14). Transient transfection of human embryonic kidney cells with Nulp1-EGFP demonstrated nuclear localization. The complex expression pattern and unique protein sequence, including an acidic amino terminal and putative transcription activation domain, suggests that Nulp1 may have a distinct role in embryonic development of many organs, including the adult brain.

关键词： organogenesis gene expression development RNA fingerprinting basic helix-loop-helix nuclear localization dorsal root ganglia mouse (NMRIx129)

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The dosage of the neuroD2 transcription factor regulates amygdala development and emotional learning

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2005年第41期102卷 14877-14882页

作者： Lin, CH Hansen, S Wang, ZS Storm, DR Tapscott, SJ Olson, JM Fred Hutchinson Canc Res Ctr Div Clin Res Seattle WA 98109 USA Fred Hutchinson Canc Res Ctr Div Human Biol Seattle WA 98109 USA Univ Washington Dept Pharmacol Seattle WA 98105 USA Univ Washington Dept Neurol Seattle WA 98105 USA Univ Washington Dept Pathol Seattle WA 98105 USA Univ Washington Dept Pediat Seattle WA 98105 USA Univ Washington Program Neurobiol & Behav Seattle WA 98105 USA

The amygdala is centrally involved in formation of emotional memory and response to fear or risk. We have demonstrated that the lateral and basolateral amygdala nuclei fail to form in neuroD2 null mice and neuroD2 heterozygotes have fewer neurons in this region. NeuroD2 heterozygous mice show profound deficits in emotional learning as assessed by fear conditioning. Unconditioned fear was also diminished in neuroD2 heterozygotes compared to wild-type controls. Several key molecular regulators of emotional learning were diminished in the brains of neuroD2 heterozygotes including Ulip1, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, and GABA(A) receptor. Thus, neuroD2 is essential for amygdala development and genes involved in amygdala function are altered in neuroD2-deficient mice.

关键词： basic helix-loop-helix haploinsufficiency

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Regulation of inhibitor of differentiation gene 3 (Id3) expression by Sp2-motif binding factor in myogenic C2C12 cells: Downregulation of DNA binding activity following skeletal muscle differentiation

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BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION 2005年第1期1731卷 13-22页

作者： Wu, JM Lim, RW Univ Missouri Sch Med Dept Med Pharmacol & Physiol Columbia MO 65212 USA Univ Missouri Program Mol Biol Columbia MO 65212 USA

Id3 is a member of the Id family of transcriptional regulators that have been implicated in the development of multiple tissues. Altered expression of the Id genes and proteins contribute to carcinogenesis and atherosclerosis. Id3 is highly expressed in proliferating skeletal muscle cells but becomes downregulated upon terminal differentiation. We have identified several DNase I protected footprints within a proximal region of the mouse Id3 promoter that has been shown previously to support high levels of transcriptional activity in proliferating skeletal muscle cells. Two of these sites interacted, respectively, in vitro with Sp2 and Egr-1 proteins present in muscle cell nuclear extracts. Mutation analysis revealed that the Sp2 site accounted for a major part of the Id3 promoter activity in proliferating muscle cells whereas the Egr-1 site was dispensable. Consistent with the previously observed downregulation of the endogenous Id3 gene, protein binding to the Sp2 site was substantially reduced with extracts from differentiated muscle cells. Our results reveal Id3 as a potential target for Sp2 and raise the possibility that acute activation and the chronic and maintained expression of Id3 gene might be regulated by different mechanisms. (c) 2005 Elsevier B.V. All rights reserved.

关键词： myognesis basic helix-loop-helix Id gene Id3 promoter Egr-1 transcriptional regulation

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Solving the protein sequence metric problem

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2005年第18期102卷 6395-6400页

作者： Atchley, WR Zhao, JP Fernandes, AD Drüke, T N Carolina State Univ Dept Genet Raleigh NC 27695 USA N Carolina State Univ Bioinformat Res Ctr Raleigh NC 27695 USA N Carolina State Univ Grad Program Biomath Raleigh NC 27695 USA N Carolina State Univ Ctr Computat Biol Raleigh NC 27695 USA Univ Bielefeld Fac Technol D-33501 Bielefeld Germany

Biological sequences are composed of long strings of alphabetic letters rather than arrays of numerical values. Lack of a natural underlying metric for comparing such alphabetic data significantly inhibits sophisticated statistical analyses of sequences, modeling structural and functional aspects of proteins, and related problems. Herein, we use multivariate statistical analyses on almost 500 amino acid attributes to produce a small set of highly interpretable numeric patterns of amino acid variability. These high-dimensional attribute data are summarized by five multidimensional patterns of attribute covariation that reflect polarity, secondary structure, molecular volume, codon diversity, and electrostatic charge. Numerical scores for each amino acid then transform amino acid sequences for statistical analyses. Relationships between transformed data and amino acid substitution matrices show significant associations for polarity and codon diversity scores. Transformed alphabetic data are used in analysis of variance and discriminant analysis to study DNA binding in the basic helix-loop-helix proteins. The transformed scores offer a general solution for analyzing a wide variety of sequence analysis problems.

关键词： basic helix-loop-helix molecular evolution multivariate statistics amino acid attributes factor analysis

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Notch-mediated CBF-1/RBP-Jκ-dependent regulation of human vascular smooth muscle cell phenotype in vitro

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AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 2005年第5期289卷 C1188-C1196页

作者： Morrow, D Scheller, A Birney, YA Sweeney, C Guha, S Cummins, PM Murphy, R Walls, D Redmond, EM Cahill, PA Dublin City Univ Fac Sci & Hlth Vasc Hlth Res Ctr Dublin 9 Ireland Dublin City Univ Sch Biotechnol Dublin 9 Ireland Dublin City Univ Natl Ctr Sensor Res Dublin 9 Ireland Univ Rochester Med Ctr Dept Surg Rochester NY 14642 USA

Vascular smooth muscle cell (VSMC) phenotypic modulation is a key factor in vascular pathology. We have investigated the role of Notch receptor signaling in controlling human vascular smooth muscle cell (hVSMC) differentiation in vitro and established a role for cyclic strain-induced changes in Notch signaling in promoting this phenotypic response. The expression of alpha-actin, calponin, myosin, and smoothelin was examined by performing immunocytochemistry, Western blot analysis, and quantitative real-time PCR in hVSMCs cultured under static conditions after forced overexpression of constitutively active Notch 1 and 3 receptors, inhibition of endogenous Cp-binding factor 1 (CBF-1)/recombination signal sequence-binding protein-J kappa (RBP-J kappa) signaling, and exposure to cyclic strain using a Flexercell Tension Plus unit. Overexpression of constitutively active Notch intracellular (IC) receptors (Notch 1 IC and Notch 3 IC) resulted in a significant downregulation of alpha-actin, calponin, myosin, and smoothelin expression, an effect that was significantly attenuated after inhibition of Notch-mediated, CBF-1/RBP- J kappa-dependent signaling by coexpression of RPMS-1 (Epstein-Barr virus-encoded gene product) and selective knockdown of basic helix-loop-helix factors [hairy enhancer of split (HES) gene and Hes-related transcription (Hrt) factors Hrt-1, Hrt-2, and Hrt-3] using targeted small interfering RNA. Cells cultured under conditions of defined equibiaxial cyclic strain (10% strain, 60 cycles/min, 24 h) exhibited a significant reduction in Notch 1 IC and Notch 3 IC expression concomitant with a significant increase in VSMC differentiation marker expression. Moreover, this cyclic strain-induced increase was further enhanced after inhibition of CBF-1/RBP- J kappa-dependent signaling with RPMS-1. These findings suggest that Notch promotes changes in hVSMC phenotype via activation of CBF-1/RBP-J kappa-dependent pathways in vitro and contributes to the phenotypic re

关键词： basic helix-loop-helix cyclic strain myosin smoothelin

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ID1 and ID2 are retinoic acid responsive genes and induce a G0/G1 accumulation in acute promyelocytic leukemia cells

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LEUKEMIA 2005年第5期19卷 799-805页

作者： Nigten, J Breems-de Ridder, MC Erpelinck-Verschueren, CAJ Nikoloski, G van der Reijden, BA van Wageningen, S van Hennik, PB de Witte, T Löwenberg, B Jansen, JH Radboud Univ Nijmegen Med Ctr Cent Hematol Lab NL-6500 HB Nijmegen Netherlands Radboud Univ Nijmegen Med Ctr Dept Hematol NL-6500 HB Nijmegen Netherlands Erasmus Med Ctr Rotterdam Inst Hematol Rotterdam Netherlands

Acute promyelocytic leukemia (APL) is uniquely sensitive to treatment with all-trans retinoic acid ( ATRA), which results in the expression of genes that induce the terminal granulocytic differentiation of the leukemic blasts. Here we report the identification of two ATRA responsive genes in APL cells, ID1 and ID2. These proteins act as antagonists of basic helix - loop helix ( bHLH) transcription factors. ATRA induced a rapid increase in ID1 and ID2, both in the APL cell line NB4 as well as in primary patient cells. In addition, a strong downregulation of E2A was observed. E2A acts as a general heterodimerization partner for many bHLH proteins that are involved in differentiation control in various tissues. The simultaneous upregulation of ID1 and ID2, and the downregulation of E2A suggest a role for bHLH proteins in the induction of differentiation of APL cells following ATRA treatment. To test the relevance of this upregulation, ID1 and ID2 were overexpressed in NB4 cells. Overexpression inhibited proliferation and induced a G0/G1 accumulation. These results indicate that ID1 and ID2 are important retinoic acid responsive genes in APL, and suggest that the inhibition of specific bHLH transcription factor complexes may play a role in the therapeutic effect of ATRA in APL.

关键词： PML-RAR alpha acute promyelocytic leukemia ID proteins basic helix-loop-helix transcription factors

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Molecular distinction between specification and differentiation in the myogenic basic helix-loop-helix transcription factor family

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MOLECULAR AND CELLULAR BIOLOGY 2001年第7期21卷 2404-2412页

作者： Bergstrom, DA Tapscott, SJ Fred Hutchinson Canc Res Ctr Div Human Biol Program Dev Biol Seattle WA 98108 USA Univ Washington Sch Med Dept Pathol Seattle WA 98195 USA Univ Washington Sch Med Dept Neurol Seattle WA 98195 USA

The myogenic basic helix-loop-helix (bHLH) proteins regulate both skeletal muscle specification and differentiation: MyoD and Myf5 establish the muscle lineage, whereas myogenin mediates differentiation. Previously, we demonstrated that MyoD was more efficient than myogenin at initiating the expression of skeletal muscle genes, and in this study we present the molecular basis for this difference. A conserved amphipathic alpha-helix in the carboxy terminus of the myogenic bHLH proteins has distinct activities in MyoD and myogenin: the MyoD helix facilitates the initiation of endogenous gene expression, whereas the myogenin helix functions as a general transcriptional activation domain. Thus, the alternate use of a similar motif for gene initiation and activation provides a molecular basis for the distinction between specification and differentiation within the myogenic bHLH gene family.

关键词： .efficient distinction Myf basic helix-loop-helix myogenic bHLH Specification and Differentiation skeletal muscle genes MyoD and myogenin MyoD helix

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