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Sequences of Circadian Clock Proteins in the Nudibranch Molluscs Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea

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BIOLOGICAL BULLETIN 2018年第3期234卷 207-218页

作者： Cook, Geoffrey M. Gruen, Anna E. Morris, John Pankey, M. Sabrina Senatore, Adriano Katz, Paul S. Watson, Winsor H., III Newcomb, James M. New England Coll Dept Biol & Hlth Sci Henniker NH 03242 USA Univ New Hampshire Dept Biol Sci Durham NH 03824 USA Univ New Hampshire Dept Mol Cellular & Biomed Sci Durham NH 03824 USA Univ Massachusetts Dept Biol Amherst MA 01003 USA Univ Toronto Dept Biol Mississauga ON L5L 1C6 Canada

While much is known about the genes and proteins that make up the circadian clocks in vertebrates and several arthropod species, much less is known about the clock genes in many other invertebrates, including nudibranchs. The goal of this project was to identify the RNA and protein products of putative clock genes in the central nervous system of three nudibranchs, Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea. Using previously published transcriptomes (Hermissenda and Tritonia) and a new transcriptome (Melibe), we identified nudibranch orthologs for the products of five canonical clock genes: brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1, circadian locomotor output cycles kaput, non-photoreceptive cryptochrome, period, and timeless. Additionally, orthologous sequences for the products of five related genes-aryl hydrocarbon receptor nuclear translocator like, photoreceptive cryptochrome, cryptochrome DASH, 6-4 photolyase, and timeout-were determined. Phylogenetic analyses confirmed that the nudibranch proteins were most closely related to known orthologs in related invertebrates, such as oysters and annelids. In general, the nudibranch clock proteins shared greater sequence similarity with Mus musculus orthologs than Drosophila melanogaster orthologs, which is consistent with the closer phylogenetic relationships recovered between lophotrochozoan and vertebrate orthologs. The suite of clock-related genes in nudibranchs includes both photoreceptive and non-photoreceptive cryptochromes, as well as timeout and possibly timeless. Therefore, the nudibranch clock may resemble the one exhibited in mammals, or possibly even in non-drosopholid insects and oysters. The latter would be evidence supporting this as the ancestral clock for bilaterians.

关键词： ARNTL aryl hydrocarbon receptor nuclear translocator like bHLH basic helix-loop-helix BMAL1 brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1 CLOCK circadian locomotor output cycles kaput CNS central nervous system CRY DASH cryptochrome DASH FAD flavin adenine dinucleotide G plus I gamma-distributed and invariant ML maximum likelihood MSA multiple sequence alignments NCBI National Center for Biotechnology Information NPCRY non-photoreceptive cryptochrome PAC Per-Arnt-Sim-associated C-terminal PAS Per-Arnt-Sim PCRY photoreceptive cryptochrome PHR 6-4 photolyase TSA transcriptome shotgun assembly

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APETALA2/ETHYLENE RESPONSE FACTOR and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis

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PLANT JOURNAL 2011年第6期66卷 1053-1065页

作者： De Boer, Kathleen Tilleman, Sofie Pauwels, Laurens Vanden Bossche, Robin De Sutter, Valerie Vanderhaeghen, Rudy Hilson, Pierre Hamill, John D. Goossens, Alain Univ Ghent VIB Dept Plant Syst Biol B-9052 Ghent Belgium Monash Univ Sch Biol Sci Melbourne Vic 3800 Australia Univ Ghent Dept Plant Biotechnol & Genet B-9052 Ghent Belgium

Transcription factors of the plant-specific apetala2/ethylene response factor (AP2/ERF) family control plant secondary metabolism, often as part of signalling cascades induced by jasmonate (JA) or other elicitors. Here, we functionally characterized the JA-inducible tobacco (Nicotiana tabacum) AP2/ERF factor ORC1, one of the members of the NIC2-locus ERFs that control nicotine biosynthesis and a close homologue of ORCA3, a transcriptional activator of alkaloid biosynthesis in Catharanthus roseus. ORC1 positively regulated the transcription of several structural genes coding for the enzymes involved in nicotine biosynthesis. Accordingly, overexpression of ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures. In contrast to ORCA3 in C. roseus, which needs only the GCC motif in the promoters of the alkaloid synthesis genes to induce their expression, ORC1 required the presence of both GCC-motif and G-box elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation. Correspondingly, combined application with the JA-inducible Nicotiana basic helix-loop-helix (bHLH) factors that bind the G-box element in these promoters enhanced ORC1 action. Conversely, overaccumulation of JAZ repressor proteins that block bHLH activity reduced ORC1 functionality. Finally, the activity of both ORC1 and bHLH proteins was post-translationally upregulated by a JA-modulated phosphorylation cascade, in which a specific mitogen-activated protein kinase kinase, JA-factor stimulating MAPKK1 (JAM1), was identified. This study highlights the complexity of the molecular machinery involved in the regulation of tobacco alkaloid biosynthesis and provides mechanistic insights about its transcriptional regulators.

关键词： Nicotiana basic helix-loop-helix ethylene response factor nicotine jasmonate mitogen-activated protein kinase

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Role and functioning of bHLH transcription factors in jasmonate signalling

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JOURNAL OF EXPERIMENTAL BOTANY 2017年第6期68卷 1333-1347页

作者： Goossens, Jonas Mertens, Jan Goossens, Alain VIB Dept Plant Syst Biol Technol Pk 927 B-9052 Ghent Belgium Univ Ghent Dept Plant Biotechnol & Bioinformat B-9052 Ghent Belgium

Plant growth, development and interaction with the environment involve the action of multiple phytohormones. Transcription factors (TFs) of diverse families play essential roles in the signalling cascades triggered by the perception of a particular hormone. TFs may act alone or in a combinatorial fashion with other TFs, and may act specifically in a single hormonal signalling cascade or as signalling hubs for multiple hormones. In the signalling cascades triggered by the phytohormone jasmonate (JA), which modulates a diverse, but specific, range of aspects of plant growth, development and defence, the TFs of the basic helix-loop-helix (bHLH) family play an essential and often conserved role in the plant kingdom. Here, we first discuss the bHLH TFs involved in all kinds of JA-modulated processes in the model plant Arabidopsis thaliana. Secondly, we elaborate on the identity and role of bHLH TFs in the conserved JA-mediated elicitation of specialized metabolism of medicinal and crop species. Finally, we discuss which directions future fundamental research on the functioning of bHLH TFs in JA signalling may head for and how this research can be translated from model plants into crop and medicinal plant species to engineer traits of agronomical and industrial interest.

关键词： Arabidopsis thaliana basic helix-loop-helix Catharanthus roseus jasmonate JAZ MYC2 specialized metabolism

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Crystal Structure of Tetrameric Arabidopsis MYC2 Reveals the Mechanism of Enhanced Interaction with DNA

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CELL REPORTS 2017年第7期19卷 1334-1342页

作者： Lian, Teng-fei Xu, Yong-ping Li, Lan-fen Su, Xiao-Dong Peking Univ Sch Life Sci State Key Lab Prot & Plant Gene Res Beijing 100871 Peoples R China Peking Univ Sch Life Sci Biodynam Opt Imaging Ctr BIOPIC Beijing 100871 Peoples R China

Jasmonates (JAs) are essential plant hormones that play important roles in the regulation of plant growth and the response to environmental stress. In the JA signaling pathway, the core transcription factors are a class of basic helix-loop-helix (bHLH) proteins, including MYC2, MYC3, and MYC4, that have different regulatory capacities. Here, we report the 2.7 angstrom crystal structure of the MYC2 bHLH domain complexed with G-box DNA, showing a cis-tetrameric structure. Biochemical assays confirmed that full-length MYC2 forms a stable homo-tetramer both in solution and in DNA-bound states, whereas MYC3 forms only a homodimer. Isothermal titration calorimetry (ITC) assays demonstrated that tetramerization enhanced DNA binding affinity, and fluorescence resonance energy transfer (FRET) assay indicated DNA looping potential of tetrameric MYC2. Luciferase assay further confirmed the importance of tetramerization in transcriptional regulation. Our studies provide a mechanistic explanation for the regulatory differences of MYC transcription factors.

关键词： transcription factor gene regulation MYC2 MYC3 jasmonates basic helix-loop-helix G-box binding sites DNA looping

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Interacting mechanism of ID3 HLH domain towards E2A/E12 transcription factor - An Insight through molecular dynamics and docking approach

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Biochemistry and Biophysics Reports 2016年 5卷 180-190页

作者： Topno, Nishith Saurav Kannan, Muthu Krishna, Ramadas Centre for Bioinformatics School of Life Sciences Pondicherry University Puducherry 605014 India

Inhibitor of DNA binding protein 3 (ID3) has long been characterized as an oncogene that implicates its functional role through its helix-loop-helix (HLH) domain upon protein-protein interaction. An insight into the dimerization brought by this domain helps in identifying the key residues that favor the mechanism behind it. Molecular dynamics (MD) simulations were performed for the HLH proteins ID3 and Transcription factor E2-alpha (E2A/E12) and their ensemble complex (ID3-E2A/E12) to gather information about the HLH domain region and its role in the interaction process. Further evaluation of the results by Principal Component Analysis (PCA) and Free Energy Landscape (FEL) helped in revealing residues of E2A/E12: Lys570, Ala595, Val598, and Ile599 and ID3: Glu53, Gln63, and Gln66 buried in their HLH motifs imparting key roles in dimerization process. Furthermore the T-pad analysis results helped in identifying the key fluctuations and conformational transitions using the intrinsic properties of the residues present in the domain region of the proteins thus specifying their crucial role towards molecular recognition. The study provides an insight into the interacting mechanism of the ID3-E2A/E12 complex and maps the structural transitions arising in the essential conformational space indicating the key structural changes within the helical regions of the motif. It thereby describes how the internal dynamics of the proteins might regulate their intrinsic structural features and its subsequent functionality. © 2015 The Authors.

关键词： basic helix-loop-helix Free Energy Landscape Inhibitor of DNA binding protein 3 Molecular dynamics Principal Component Analysis Transcription factor E2-alpha

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CjbHLH1 homologs regulate sanguinarine biosynthesis in Eschscholzia californica cells

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PLANT AND CELL PHYSIOLOGY 2015年第5期56卷 1019-1030页

作者： Yamada, Yasuyuki Motomura, Yukiya Sato, Fumihiko Kyoto Univ Grad Sch Biostudies Dept Plant Gene & Totipotency Sakyo Ku Kyoto 6068502 Japan

Isoquinoline alkaloids (IQAs), terpenoid indole alkaloid and nicotine are some of the most studied alkaloids. Recently, several groups have reported that the biosynthesis of these alkaloids is regulated by basic helix-loop-helix (bHLH) transcription factors. Whereas the biosyntheses of nicotine and terpenoid indole alkaloid in Nicotiana plants and Catharanthus roseus are directly or indirectly regulated by Arabidopsis thaliana MYC2 homologs, a non-MYC2-type bHLH transcription factor, CjbHLH1, comprehensively regulates berberine biosynthesis in Coptis japonica. Interestingly, CjbHLH1 homologous genes were found in many IQA-producing plant species, which suggests that non-MYC2-type CjbHLH homologs are specifically associated with IQA biosynthesis. To test whether CjbHLH1 homologs are involved in the biosynthesis of IQA in a plant other than C. japonica, we isolated two genes homologous to CjbHLH1, i.e. EcbHLH1-1 and EcbHLH1-2, from Eschscholzia californica (California poppy). Stable transformants in which the expression levels of EcbHLH1 genes were constitutively suppressed by RNA interference (RNAi) showed a reduced expression of some IQA biosynthetic enzyme genes. A metabolite analysis confirmed that the suppression of EcbHLH1, particularly EcbHLH1-2, caused a decrease in sanguinarine accumulation in transgenic cultured cells. These results indicate that non-MYC2-type EcbHLH1s regulate IQA biosynthesis in California poppy like CjbHLH1 in C. japonica.

关键词： basic helix-loop-helix Eschscholzia californica (California poppy) Isoquinoline alkaloids Transcriptional regulation

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Hes1: a key role in stemness, metastasis and multidrug resistance

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CANCER BIOLOGY & THERAPY 2015年第3期16卷 353-359页

作者： Liu, Zi-Hao Dai, Xiao-Meng Du, Bin Jinan Univ Sch Med Dept Pathol Guangzhou Guangdong Peoples R China

Hes1 is one mammalian counterpart of the Hairy and Enhancer of split proteins that play a critical role in many physiological processes including cellular differentiation, cell cycle arrest, apoptosis and self-renewal ability. Recent studies have shown that Hes1 functions in the maintenance of cancer stem cells (CSCs), metastasis and antagonizing drug-induced apoptosis. Pathways that are involved in the up-regulation of Hes1 level canonically or non-canonically, such as the Hedgehog, Wnt and hypoxia pathways are frequently aberrant in cancer cells. Here, we summarize the recent data supporting the idea that Hes1 may have an important function in the maintenance of cancer stem cells self-renewal, cancer metastasis, and epithelial-mesenchymal transition (EMT) process induction, as well as chemotherapy resistance, and conclude with the possible mechanisms by which Hes1 functions have their effect, as well as their crosstalk with other carcinogenic signaling pathways.

关键词： cancer stem cell chemotherapy resistance Hes1 metastasis Notch signaling pathway non-canonical Notch CSCs cancer stem cells EMT epithelial-mesenchymal transition MASH-1 Mammalian achaete-scute homolog-1 bHLH basic helix-loop-helix TLE transducin-like Enhancer of split HDACs histone deacetylases dnMAM dominant-negative mastermind Runx2 Runt-related protein 2 ABC ATP-binding cassette NICD Notch intracellular domain CSL CBF1 Suppressor of Hairless Lag1 MAML Mastermind-like protein family GSI -secretase inhibitor

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Reprogramming during epithelial to mesenchymal transition under the control of TGFβ

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CELL ADHESION & MIGRATION 2015年第3期9卷 233-246页

作者： Tan, E-Jean Olsson, Anna-Karin Moustakas, Aristidis Uppsala Univ Sci Life Lab Ludwig Inst Canc Res Uppsala Sweden Uppsala Univ Sci Life Lab Dept Med Biochem & Microbiol Uppsala Sweden

Epithelial-mesenchymal transition (EMT) refers to plastic changes in epithelial tissue architecture. Breast cancer stromal cells provide secreted molecules, such as transforming growth factor (TGF), that promote EMT on tumor cells to facilitate breast cancer cell invasion, stemness and metastasis. TGF signaling is considered to be abnormal in the context of cancer development;however, TGF acting on breast cancer EMT resembles physiological signaling during embryonic development, when EMT generates or patterns new tissues. Interestingly, while EMT promotes metastatic fate, successful metastatic colonization seems to require the inverse process of mesenchymal-epithelial transition (MET). EMT and MET are interconnected in a time-dependent and tissue context-dependent manner and are coordinated by TGF, other extracellular proteins, intracellular signaling cascades, non-coding RNAs and chromatin-based molecular alterations. Research on breast cancer EMT/MET aims at delivering biomolecules that can be used diagnostically in cancer pathology and possibly provide ideas for how to improve breast cancer therapy.

关键词： epithelial-mesenchymal transition signal transduction transforming growth factor tumor invasiveness bHLH basic helix-loop-helix BMP bone morphogenetic protein CSC cancer stem cell DNMT DNA methyltransferase EMT epithelial-mesenchymal transition FGF fibroblast growth factor HDAC histone deacetylase lncRNA long non-coding RNA mTORC mammalian target of rapamycin complex MET mesenchymal-epithelial transition miRNA micro-RNA MAPK mitogen activated protein kinase PDGF platelet derived growth factor PRC polycomb repressive complex TF transcription factor TGF transforming growth factor

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越橘转录组高通量测序及bHLH转录因子分析

越橘转录组高通量测序及bHLH转录因子分析

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第六届全国果树分子生物学学术研讨会

作者：宋杨刘红弟周强张红军张志东李亚东王海波刘凤之中国农业科学院果树研究所农业部园艺作物种质资源利用重点实验室辽宁兴城125100 吉林农业大学园艺学院吉林长春130118

以'Duke'越橘(Vaccinium corymbosum'Duke')为试材,构建De novo转录组测序文库并进行数据统计分析,利用Mega 4、CLC Sequence Viewer6等软件和定量PCR技术对测序文库中的BHLH转录因子进行生物信息学和表达分析.结果显... 详细信息

以'Duke'越橘(Vaccinium corymbosum'Duke')为试材,构建De novo转录组测序文库并进行数据统计分析,利用Mega 4、CLC Sequence Viewer6等软件和定量PCR技术对测序文库中的BHLH转录因子进行生物信息学和表达分析.结果显示,转录组测序共获得有效数据28.38 Gb,通过组装得到Unigene序列108 033个.功能注释结果表明,有32 244个Unigenes可注释到COG、GO等数据库上;75 789个Unigenes无匹配信息.利用COG和GO分类工具可将具有注释信息的序列分别划分为25个和52个类别,涉及转运与代谢、转录与调控和信号转导等功能.分析转录组文库中BHLH基因,结果表明共得到59个BHLH基因.序列结构分析显示,其中55个基因均含有完整的BHLH区域.进化树分析显示,越橘BHLH基因分为13个亚组.定量PCR结果表明,45个基因在芽、茎、叶、花和果实不同发育阶段均能表达,但表达模式不同,推测BHLH基因的功能涉及越橘不同组织器官发育的各个方面.本文为进一步研究越橘BHLH基因家族的分类及功能奠定了基础.

关键词：越橘生物信息学转录组测序 basic helix-loop-helix 转录因子

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Effects of overexpression of a bHLH transcription factor on biomass and lipid production in Nannochloropsis salina

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BIOTECHNOLOGY FOR BIOFUELS 2015年第1期8卷 200-200页

作者： Kang, Nam Kyu Jeon, Seungjib Kwon, Sohee Koh, Hyun Gi Shin, Sung-Eun Lee, Bongsoo Choi, Gang-Guk Yang, Ji-Won Jeong, Byeong-ryool Chang, Yong Keun Korea Adv Inst Sci & Technol Dept Chem & Biomol Engn Taejon 305701 South Korea Korea Adv Inst Sci & Technol Adv Biomass R&D Ctr Taejon 305701 South Korea

Background: Microalgae are considered promising alternative energy sources because they consume CO2 and accumulate large amounts of lipids that can be used as biofuel. Nannochloropsis is a particularly promising microalga due to its high growth rate and lipid content, and the availability of genomic information. Transcription factors (TFs) are global regulators of biological pathways by up- or down-regulation of related genes. Among these, basic helix-loop-helix (bHLH) TFs regulate growth, development, and stress responses in plants and animals, and have been identified in microalgae. We identified two bHLH TFs in the genome of N. salina CCMP1776, NsbHLH1, and NsbHLH2, and characterized functions of NsbHLH2 that may be involved in growth and nutrient uptake. Results: We obtained NsbHLH2 overexpressing transformants of N. salina CCMP1776 by particle bombardment and confirmed that these were stable transformants. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting using antibodies against the FLAG tag that was attached at the end of the coding sequence confirmed the expression of the NsbHLH2 protein under various culture conditions. The qRT-PCR results also indicated that the endogenous and transgenic expression of NsbHLH2 was reduced under stressed conditions. Overexpression of NsbHLH2 led to increased growth rate in the early growth period, and concomitantly higher nutrient uptake, than wild type (WT). These enhanced growth and nutrient uptake resulted in increased productivities of biomass and FAME. For example, one of the transformants, NsbHLH2 3-6, showed increased biomass productivity by 36 % under the normal condition, and FAME productivity by 33 % under nitrogen limitation condition. Conclusively, the improved growth in the transformants can be associated with the enhanced nutrient uptake. We are currently assessing their potential for scale-up cultivation with positive outcomes. Conclusion: Overexpression of NsbHLH2 led to enhanced

关键词： Microalgae Nannochloropsis salina Transcription factor basic helix-loop-helix Specific growth rate Fatty acid methyl ester

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