Abnormal Heart Development and Lung Remodeling in Mice Lacking the Hypoxia-Inducible Factor-Related Basic Helix-Loop-Helix PAS Protein NEPAS

ABSTRACT Hypoxia-inducible factors (HIFs) are crucial for oxygen homeostasis during both embryonic development and postnatal life. Here we show that a novel HIF family basic helix-loop-helix (bHLH) PAS (Per-Arnt-Sim) protein, which is expressed predominantly during embryonic and neonatal stages and thereby designated NEPAS (neonatal and embryonic PAS), acts as a negative regulator of HIF-mediated gene expression. NEPAS mRNA is derived from the HIF-3α gene by alternative splicing, replacing the first exon of HIF-3α with that of inhibitory PAS. NEPAS can dimerize with Arnt and exhibits only low levels of transcriptional activity, similar to that of HIF-3α. NEPAS suppressed reporter gene expression driven by HIF-1α and HIF-2α. By generating mice with a targeted disruption of the NEPAS/HIF-3α locus, we found that homozygous mutant mice (NEPAS/HIF-3α− / −) were viable but displayed enlargement of the right ventricle and impaired lung remodeling. The expression of endothelin 1 and platelet-derived growth factor β was increased in the lung endothelial cells of NEPAS/HIF-3α-null mice. These results demonstrate a novel regulatory mechanism in which the activities of HIF-1α and HIF-2α are negatively regulated by NEPAS in endothelial cells, which is pertinent to lung and heart development during the embryonic and neonatal stages.

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Cardiomyopathy in Irx4-Deficient Mice Is Preceded by Abnormal Ventricular Gene Expression

Molecular and Cellular Biology ◽

10.1128/mcb.21.5.1730-1736.2001 ◽

2001 ◽

Vol 21 (5) ◽

pp. 1730-1736 ◽

Cited By ~ 100

Author(s):

Benoit G. Bruneau ◽

Zheng-Zheng Bao ◽

Diane Fatkin ◽

Jose Xavier-Neto ◽

Dimitrios Georgakopoulos ◽

...

Keyword(s):

Gene Expression ◽

Cardiac Hypertrophy ◽

Heart Development ◽

Contractile Function ◽

Specific Gene ◽

Postnatal Life ◽

Helix Loop Helix ◽

And Function ◽

Expression Program ◽

Deficient Mice

ABSTRACT To define the role of Irx4, a member of the Iroquoisfamily of homeobox transcription factors in mammalian heart development and function, we disrupted the murine Irx4 gene. Cardiac morphology in Irx4-deficient mice (designatedIrx4 Δex2/Δex2) was normal during embryogenesis and in early postnatal life. AdultIrx4 Δex2/Δex2 mice developed a cardiomyopathy characterized by cardiac hypertrophy and impaired contractile function. Prior to the development of cardiomyopathy,Irx4 Δex2/Δex2 hearts had abnormal ventricular gene expression: Irx4-deficient embryos exhibited reduced ventricular expression of the basic helix-loop-helix transcription factor eHand (Hand1), increasedIrx2 expression, and ventricular induction of an atrial chamber-specific transgene. In neonatal hearts, ventricular expression of atrial natriuretic factor and α-skeletal actin was markedly increased. Several weeks subsequent to these changes in embryonic and neonatal gene expression, increased expression of hypertrophic markers BNP and β-myosin heavy chain accompanied adult-onset cardiac hypertrophy. Cardiac expression of Irx1, Irx2, and Irx5 may partially compensate for loss of Irx4 function. We conclude that Irx4 is not sufficient for ventricular chamber formation but is required for the establishment of some components of a ventricle-specific gene expression program. In the absence of genes under the control of Irx4, ventricular function deteriorates and cardiomyopathy ensues.

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Control of the Hypoxic Response in Drosophila melanogaster by the Basic Helix-Loop-Helix PAS Protein Similar

Molecular and Cellular Biology ◽

10.1128/mcb.22.19.6842-6853.2002 ◽

2002 ◽

Vol 22 (19) ◽

pp. 6842-6853 ◽

Cited By ~ 157

Author(s):

Sofía Lavista-Llanos ◽

Lázaro Centanin ◽

Maximiliano Irisarri ◽

Daniela M. Russo ◽

Jonathan M. Gleadle ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Time Course ◽

Hypoxia Inducible Factor ◽

Ectopic Expression ◽

Prolyl Hydroxylase ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Oxygen Homeostasis ◽

Multicellular Organisms

ABSTRACT In mammalian systems, the heterodimeric basic helix-loop-helix (bHLH)-PAS transcription hypoxia-inducible factor (HIF) has emerged as the key regulator of responses to hypoxia. Here we define a homologous system in Drosophila melanogaster, and we characterize its activity in vivo during development. By using transcriptional reporters in developing transgenic flies, we show that hypoxia-inducible activity rises to a peak in late embryogenesis and is most pronounced in tracheal cells. We show that the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) function as HIF-α and HIF-β homologues, respectively, and demonstrate a conserved mode of regulation for Sima by oxygen. Sima protein, but not its mRNA, was upregulated in hypoxia. Time course experiments following pulsed ectopic expression demonstrated that Sima is stabilized in hypoxia and that degradation relies on a central domain encompassing amino acids 692 to 863. Continuous ectopic expression overrode Sima degradation, which remained cytoplasmic in normoxia, and translocated to the nucleus only in hypoxia, revealing a second oxygen-regulated activation step. Abrogation of the Drosophila Egl-9 prolyl hydroxylase homologue, CG1114, caused both stabilization and nuclear localization of Sima, indicating a central involvement in both processes. Tight conservation of the HIF/prolyl hydroxylase system in Drosophila provides a new focus for understanding oxygen homeostasis in intact multicellular organisms.

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Wingless blocks bristle formation and morphogenetic furrow progression in the eye through repression of Daughterless

Development ◽

10.1242/dev.129.14.3393 ◽

2002 ◽

Vol 129 (14) ◽

pp. 3393-3402 ◽

Cited By ~ 1

Author(s):

Kenneth M. Cadigan ◽

Austin D. Jou ◽

Roel Nusse

Keyword(s):

Gene Expression ◽

Ectopic Expression ◽

Dominant Negative ◽

Proneural Gene ◽

Morphogenetic Furrow ◽

Heterologous Promoter ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Dominant Negative Form ◽

Negative Form

In the developing eye, wingless activity represses proneural gene expression (and thus interommatidial bristle formation) and positions the morphogenetic furrow by blocking its initiation in the dorsal and ventral regions of the presumptive eye. We provide evidence that wingless mediates both effects, at least in part, through repression of the basic helix-loop-helix protein Daughterless. daughterless is required for high proneural gene expression and furrow progression. Ectopic expression of wingless blocks Daughterless expression in the proneural clusters. This repression, and that of furrow progression, can be mimicked by an activated form of armadillo and blocked by a dominant negative form of pangolin/TCF. Placing daughterless under the control of a heterologous promoter blocks the ability of ectopic wingless to inhibit bristle formation and furrow progression. hedgehog and decapentapleigic could not rescue the wingless furrow progression block, indicating that wingless acts downstream of these genes. In contrast, Atonal and Scute, which are thought to heterodimerize with Daughterless to promote furrow progression and bristle formation, respectively, can block ectopic wingless action. These results are summarized in a model where daughterless is a major, but probably not the only, target of wingless action in the eye.

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The Basic Helix-Loop-Helix TAL1/SCL and Its Partners E47 and LMO2 Act Upstream of the VE-Cadherin Gene in Endothelial Cells.

Blood ◽

10.1182/blood.v108.11.1795.1795 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1795-1795

Author(s):

Virginie Deleuze ◽

Elias Chalhoub ◽

Rawan El-Hajj ◽

Christiane Dohet ◽

Mikael Le Clech ◽

...

Keyword(s):

Endothelial Cells ◽

Ectopic Expression ◽

Cell Junctions ◽

Small Interfering Rnas ◽

Hek 293 ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Cell Cell

Abstract The basic helix-loop-helix protein TAL-1/SCL, essential for the formation of the hematopoietic system, is also required for vascular development and more particularly for embryonic angiogenesis. We previously reported that TAL-1 acts as a positive factor for post-natal angiogenesis by stimulating endothelial morphogenesis. To understand how TAL-1 modulates angiogenesis, we investigated the functional consequences of TAL-1 silencing, mediated by small-interfering RNAs, in human primary endothelial cells (ECs). We found that TAL-1 knockdown impaired in vitro EC tubulomorphogenesis (in 2-D on Matrigel or 3-D in collagen I gel), with the notable absence of cell-cell contacts, a prerequisite for morphogenesis initiation. This cellular deficiency was associated with a dramatic reduction in the vascular-endothelial (VE)-cadherin at intercellular junctions, the major component of endothelial adherens junctions. In contrast, PECAM (or CD31) was present at cell-cell junctions at the same levels as control cells. Importantly, silencing of two known TAL-1-partners in hematopoietic cells, E47 or LMO2, produce the same effects as TAL-1. Accordingly, silencing of TAL-1, as well as E47 and LMO2, provoked down-regulation of VE-cadherin at both the mRNA and protein levels. Transient transfection experiments in HUVECs showed that TAL-1 and E47 regulate the VE-cadherin promoter through a specialized E-box element. Finally, endogenous VE-cadherin transcription could be directly activated in non-endothelial HEK-293 cells that neither express TAL-1 or LMO2, by the sole concomitant ectopic expression of TAL-1, E47 and LMO2. Overall, our data demonstrate that a multiprotein complex containing at least TAL-1, LMO2 and E47 act upstream of the VE-cadherin gene. We are currently performing chromatin immunoprecipitation (ChIP) to investigate whether the TAL-1-containing complex binds in vivo the VE-cadherin promoter. This study identifies VE-cadherin as an upstream TAL-1-target gene in the endothelial lineage, and provides a first clue in TAL-1 function in the control of angiogenesis.

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A genome-wide identification and classification of basic helix-loop-helix genes in the jewel wasp, Nasonia vitripennis (Hymenoptera: Pteromalidae)

Genome ◽

10.1139/gen-2014-0171 ◽

2014 ◽

Vol 57 (10) ◽

pp. 525-536 ◽

Cited By ~ 9

Author(s):

Xiao-Ting Liu ◽

Yong Wang ◽

Xu-Hua Wang ◽

Xia-Fang Tao ◽

Qin Yao ◽

...

Keyword(s):

Heart Development ◽

Family Members ◽

Insect Species ◽

Structural Domain ◽

Nasonia Vitripennis ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

A Genome ◽

Bhlh Genes ◽

Bhlh Proteins

Basic helix-loop-helix (bHLH) proteins are highly conserved DNA-binding transcription factors of a large superfamily. Animal bHLH proteins play important regulatory roles in various developmental processes such as neurogenesis, myogenesis, heart development, and hematopoiesis. The jewel wasp (Nasonia vitripennis) is a good model organism of hymenoptera insects for studies of developmental and evolutionary genetics. In this study, we identified 48 bHLH genes in the genome of N. vitripennis. According to phylogenetic analysis, based on N. vitripennis bHLH (NvbHLH) motif sequences and structural domain distribution in their full-length protein sequences, the identified NvbHLH genes were classified into 36 bHLH families with 19, 12, 9, 1, 6, and 1 member(s) in groups A, B, C, D, E, and F, respectively. Our classification to the identified NvbHLH family members confirms GenBank annotations for 21 of the 48 NvbHLH proteins and provides useful information for further characterization and annotation of the remaining 27 NvbHLH proteins. Compared to other insect species, N. vitripennis has the lowest number of bHLH family members. No NvbHLH members have been found in the families Net, MyoRa, and PTFa, while all other insect species have at least one member in each of the families. These data constitute a solid basis for further investigations into the functions of bHLH proteins in developmental regulation of N. vitripennis.

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FoSTUA, Encoding a Basic Helix-Loop-Helix Protein, Differentially Regulates Development of Three Kinds of Asexual Spores, Macroconidia, Microconidia, and Chlamydospores, in the Fungal Plant Pathogen Fusarium oxysporum

Eukaryotic Cell ◽

10.1128/ec.3.6.1412-1422.2004 ◽

2004 ◽

Vol 3 (6) ◽

pp. 1412-1422 ◽

Cited By ~ 60

Author(s):

Toshiaki Ohara ◽

Takashi Tsuge

Keyword(s):

Fusarium Oxysporum ◽

Fluorescent Protein ◽

Negative Regulator ◽

Vascular Wilt ◽

Wild Type ◽

Low Frequencies ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Chlamydospore Formation ◽

Green Fluorescent

ABSTRACT The soil-borne fungus Fusarium oxysporum causes vascular wilt of a wide variety of plant species. F. oxysporum produces three kinds of asexual spores, macroconidia, microconidia, and chlamydospores. Falcate macroconidia are formed generally from terminal phialides on conidiophores and rarely from intercalary phialides on hyphae. Ellipsoidal microconidia are formed from intercalary phialides on hyphae. Globose chlamydospores with thick walls are developed by the modification of hyphal and conidial cells. Here we describe FoSTUA of F. oxysporum, which differentially regulates the development of macroconidia, microconidia, and chlamydospores. FoSTUA encodes a basic helix-loop-helix protein with similarity to Aspergillus nidulans StuA, which has been identified as a transcriptional regulator controlling conidiation. Nuclear localization of FoStuA was verified by using strains expressing FoStuA-green fluorescent protein fusions. The FoSTUA-targeted mutants exhibited normal microconidium formation in cultures. However, the mutants lacked conidiophores and produced macroconidia at low frequencies only from intercalary phialides. Thus, FoSTUA appears to be necessary to induce conidiophore differentiation. In contrast, chlamydospore formation was dramatically promoted in the mutants. These data demonstrate that FoStuA is a positive regulator and a negative regulator for the development of macroconidia and chlamydospores, respectively, and is dispensable for microconidium formation in cultures. The disease-causing ability of F. oxysporum was not affected by mutations in FoSTUA. However, the mutants produced markedly fewer macroconidia and microconidia in infected plants than the wild type. These results suggest that FoSTUA also has an important role for microconidium formation specifically in infected plants.

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Integrated Analysis of Basic Helix Loop Helix Transcription Factor Family and Targeted Terpenoids Reveals Candidate AarbHLH Genes Involved in Terpenoid Biosynthesis in Artemisia argyi

Frontiers in Plant Science ◽

10.3389/fpls.2021.811166 ◽

2022 ◽

Vol 12 ◽

Author(s):

Xiaozhe Yi ◽

Xingwen Wang ◽

Lan Wu ◽

Mengyue Wang ◽

Liu Yang ◽

...

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Well Being ◽

Treated Group ◽

Integrated Analysis ◽

Regulation Mechanism ◽

Terpenoid Biosynthesis ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Artemisia Argyi

Artemisia argyi is a valuable traditional medicinal plant in Asia. The essential oil from its leaves is rich in terpenoids and has been used to enhance health and well-being. In China, the market scale of industries related to A. argyi has attained tens of billions of Chinese Yuan. The basic helix-loop-helix (bHLH) family is one of the largest transcription factors families in plants that plays crucial roles in diverse biological processes and is an essential regulatory component of terpenoid biosynthesis. However, the bHLH TFs and their regulatory roles in A. argyi remain unknown. Here, 53 AarbHLH genes were identified from the transcriptome of A. argyi and were classified into 15 subfamilies based on the classification of bHLH proteins in Arabidopsis thaliana. The MEME analysis showed that the conserved motif 1 and motif 2 constituted the most conserved bHLH domain and distributed in most AarbHLH proteins. Additionally, integrated analysis of the expression profiles of AarbHLH genes and the contents of targeted terpenoids in different tissues group and JA-treated group were performed. Eleven up-regulated AarbHLHs and one down-regulated AarbHLH were screened as candidate genes that may participate in the regulation of terpenoid biosynthesis (TPS-AarbHLHs). Correlation analysis between gene expression and terpenoid contents indicated that the gene expression of these 12 TPS-AarbHLHs was significantly correlated with the content changes of 1,8-cineole or β-caryophyllene. Protein–protein interaction networks further illustrated that these TPS-AarbHLHs might be involved in terpenoid biosynthesis in A. argyi. This finding provides a basis to further investigate the regulation mechanism of AarbHLH genes in terpenoid biosynthesis, and will be helpful to improve the quality of A. argyi.

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The Basic Helix-Loop-Helix Transcription Factor SmbHLH1 Represses Anthocyanin Biosynthesis in Eggplant

Frontiers in Plant Science ◽

10.3389/fpls.2021.757936 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zhaofei Duan ◽

Shiyu Tian ◽

Guobin Yang ◽

Min Wei ◽

Jing Li ◽

...

Keyword(s):

Plant Species ◽

Anthocyanin Biosynthesis ◽

Solanum Melongena ◽

Amino Acid Sequences ◽

Negative Regulator ◽

Bimolecular Fluorescence Complementation ◽

Luciferase Assay ◽

Anthocyanin Accumulation ◽

Basic Helix Loop Helix ◽

Helix Loop Helix

Many basic helix-loop-helix transcription factors (TFs) have been reported to promote anthocyanin biosynthesis in numerous plant species, but little is known about bHLH TFs that inhibit anthocyanin accumulation. In this study, SmbHLH1 from Solanum melongena was identified as a negative regulator of anthocyanin biosynthesis. However, SmbHLH1 showed high identity with SmTT8, which acts as a SmMYB113-dependent positive regulator of anthocyanin-biosynthesis in plants. Overexpression of SmbHLH1 in eggplant caused a dramatic decrease in anthocyanin accumulation. Only the amino acid sequences at the N and C termini of SmbHLH1 differed from the SmTT8 sequence. Expression analysis revealed that the expression pattern of SmbHLH1 was opposite to that of anthocyanin accumulation. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that SmbHLH1 could not interact with SmMYB113. Dual-luciferase assay demonstrated that SmbHLH1 directly repressed the expression of SmDFR and SmANS. Our results demonstrate that the biological function of bHLHs in anthocyanin biosynthesis may have evolved and provide new insight into the molecular functions of orthologous genes from different plant species.

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Abstract 182: Inhibitor of differentiation (Id)3 Regulation of the Vasculature in Adipose Tissue

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.182 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Victoria Osinski ◽

Jennifer Kirby ◽

Swapnil Sonkusare ◽

Mete Civelek ◽

Coleen McNamara

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Adipose Tissue ◽

Blood Vessel ◽

Vascular Development ◽

Tissue Expansion ◽

Dependent Manner ◽

Blood Vessel Formation ◽

Vessel Formation ◽

Helix Loop Helix

Rationale: Over half of the U.S. is overweight or obese and excessive weight gain greatly increases one’s risk of cardiovascular disease. Adipose tissue requires new blood vessel formation to support transport of nutrients during expansion. Our lab has previously published that global loss of the helix-loop-helix transcription factor Inhibitor of differentiation 3 (Id3) results in attenuated increases in adipose depot size and microvascular blood volume in mice fed a high fat diet (HFD). Id3 has also been implicated in regulating tumor angiogenesis. Together, our results suggest that Id3 is regulating HFD-induced angiogenesis in adipose tissue. Methods and Results: To determine if Id3 is regulating angiogenesis, we performed genetic, cellular, and arterial experiments and analyses. Using bromodeoxyuridine (BrdU) treatments and flow cytometry, we found that endothelial cells within adipose tissue of mice with a global genetic knockout of Id3 (Id3 KO) had fewer proliferating (BrdU-positive) endothelial cells (0.0679% BrdU+CD31+ cells) than wild type (WT) mice (0.754% BrdU+CD31+). Using mouse adipose tissue gene expression data from the Gene Ontology Consortium database, we found that genes correlating most significantly with Id3 were functionally classified under categories of vascular development (p = 4.45E-8, correlation significance) and angiogenesis (p = 1.66E-6). Sox18, a known transcriptional regulator of blood vessel formation, shows higher relative gene expression in Id3 WT (1.42 ± 0.417) than Id3 KO (0.332 ± 0.126) adipose tissue. Finally, preliminary findings using pressure myography demonstrate that Id3 KO mice have a higher baseline arterial diameter than Id3 WT. Conclusion: Dysregulation of vascular development during adipose tissue expansion is altering arterial function in an Id3-dependent manner.

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Myostatin silencing effect on basic helix–loop–helix transcription factors in caprine foetal fibroblast cells

Indian Journal of Animal Research ◽

10.18805/ijar.v0iof.7828 ◽

2017 ◽

Author(s):

Biswajyoti Borah ◽

Ajit Pratap Singh ◽

Hamen Gogoi ◽

Amlan Jyoti Phukan ◽

Bikash Chandra Sarkhel

Keyword(s):

Transcription Factors ◽

Muscle Development ◽

Pearson Correlation ◽

Negative Regulator ◽

Fibroblast Cells ◽

Animal Cells ◽

Myostatin Gene ◽

Basic Helix Loop Helix ◽

Helix Loop Helix

Transgenic food animal production is one of the potential and need oriented research to mitigate the food crises of the world. In vitro gene silenced animal cells and making use of these cells for transgenesis one of the suitable way to produce productive animals. Myostatin is a negative regulator of muscle growth, has the potential to increase the muscle mass upon its silencing. Four Hush 29-mer anti- myostatin (MSTN) shRNA constructs were checked for myostatin gene silencing in caprine foetal fibroblast cells and its subsequent effect on basic helix– loop–helix (bHLH) transcription factors. These factors are necessary for the terminal differentiation, proliferation, and homeostasis of muscle development. Different shRNA constructs displayed 55.1 to 91.5% (p less than 0.01) of myostatin silencing in caprine foetal fibroblast cells and upregulation of myogenic gene. Upregulation of 7.97 to 111.67 % for MyoD, 77.0 % to 319.47 % for myogenin, 16.67 % to 138.0 % for Myf5 were observed . The Pearson correlation established a negative correlation between myostatin and genes under study. Result suggests that knockdown of MSTN a potential approach to improve caprine musculatures.

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