Loop Helix Transcription Factor Research Articles

Abstract 4922: NEUROD1 is highly expressed in extensive small cell lung cancer by promoting migration

Abstract Introduction:Small cell lung cancer (SCLC) exhibits high-grade neuroendocrine features and recent genomic analyses revealed that aberrant expression of bacic helix loop helix transcription factors such as neurogenic differentiation 1 (NEUROD1) as well as achaete-scute homolog 1(ASCL1) have also been observed in SCLC. NEUROD1 plays important role for the development, maturation in several nerve system and formation of distal lung of neuroendocrine morphology. Previous report showed that mRNA expression as well as target genes of NEUROD1 are largely distinct from those of ASCL1 in human SCLC cell lines, however, protein level and expression patterns of these transcription factors in SCLC clinical tumor samples as well as its relation to clinicopathological characteristics have not been fully elucidated. Method: We retrospectively analyzed specimens from 95 SCLC patients between June 1988 and December 2017, and immunohistochemical (IHC) staining of NEUROD1 as well as ASCL1 was performed. Furthermore, we examined the effect of gain or loss of NEUROD1 function on proliferation and migration by transfection in SCLC cell lines. Result:Dual high staining score of ASCL1 and NEUROD1 was present in SCLC samples (32/95: 34%) and IHC score of NEUROD1 was higher in extensive disease (ED) samples than those in limited disease (LD) samples (median score 160 vs 80 ; P = 0.0389).Overall survival did not significantly differ between SCLC patients with high or low score of NEUROD1 or ASCL1. Next, our vitro data showed that upregulation of NEUROD1 in SCLC cell lines promoted transwell migration whereas downregulation of NEUROD1 reduced cell migration. Conclusion: Our results from clinical and in vitro experimental data collectively indicate that NEUROD1 expression increases in extensive SCLC by promoting migration and metastasis of SCLC cells. Citation Format: Yuki Ikematsu, Kentaro Tanaka, Nobuhisa Ando, Kayo Ijichi, Yasuto Yoneshima, Hiroyuki Inoue, Goji Toyokawa, Tetsuzo Tagawa, Yoichi Nakanishi, Isamu Okamoto. NEUROD1 is highly expressed in extensive small cell lung cancer by promoting migration [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4922.

ASCL1 regulates neurodevelopmental transcription factors and cell cycle genes in brain tumors of glioma mouse models.

Glioblastomas (GBMs) are incurable brain tumors with a high degree of cellular heterogeneity and genetic mutations. Transcription factors that normally regulate neural progenitors and glial development are aberrantly coexpressed in GBM, conferring cancer stem‐like properties to drive tumor progression and therapeutic resistance. However, the functional role of individual transcription factors in GBMs in vivo remains elusive. Here, we demonstrate that the basic‐helix–loop–helix transcription factor ASCL1 regulates transcriptional targets that are central to GBM development, including neural stem cell and glial transcription factors, oncogenic signaling molecules, chromatin modifying genes, and cell cycle and mitotic genes. We also show that the loss of ASCL1 significantly reduces the proliferation of GBMs induced in the brain of a genetically relevant glioma mouse model, resulting in extended survival times. RNA‐seq analysis of mouse GBM tumors reveal that the loss of ASCL1 is associated with downregulation of cell cycle genes, illustrating an important role for ASCL1 in controlling the proliferation of GBM.

LDB1 Enforces Stability on Direct and Indirect Oncoprotein Partners in Leukemia.

The LMO2/LDB1 macromolecular complex is critical in hematopoietic stem and progenitor cell specification and in the development of acute leukemia. This complex is comprised of core subunits of LMO2 and LDB1 as well as single-stranded DNA-binding protein (SSBP) cofactors and DNA-binding basic helix-loop-helix (bHLH) and GATA transcription factors. We analyzed the steady-state abundance and kinetic stability of LMO2 and its partners via Halo protein tagging in conjunction with variant proteins deficient in binding their respective direct protein partners. We discovered a hierarchy of protein stabilities (with half-lives in descending order) as follows: LDB1 > SSBP > LMO2 > TAL1. Importantly, LDB1 is a remarkably stable protein that confers enhanced stability upon direct and indirect partners, thereby nucleating the formation of the multisubunit protein complex. The data imply that free subunits are more rapidly degraded than those incorporated within the LMO2/LDB1 complex. Our studies provided significant insights into LMO2/LDB1 macromolecular protein complex assembly and stability, which has implications for understanding its role in blood cell formation and for therapeutically targeting this complex in human leukemias.

Cyanidin based anthocyanin biosynthesis in orange carrot is restored by expression of AmRosea1 and AmDelila, MYB and bHLH transcription factors.

The simultaneous expression of AmRosea1 and AmDelila transcription factors from snapdragon can activate the anthocyanin pathway in orange carrots, leading to the synthesis and accumulation of anthocyanins in the taproots. Anthocyanins are phenolic compounds produced in various parts of plants. They are used as natural food dyes and are reported as beneficial antioxidants for humans. Black carrot is an important source for anthocyanins; however, the reason for the lack of anthocyanin production in the orange carrot is unknown. Anthocyanins are synthesized by a specific branch of the phenylpropanoid pathway that has previously been reported to be activated by a triad of R2R3-MYB, basic helix-loop helix (bHLH) and WD40 transcription factors (TFs). In the current study, orange carrots were turned purple by simultaneous expression of R2R3-MYB and bHLH TFs, i.e. AmRosea1 and AmDelila from snapdragon (Antirrhinum majus). Simultaneous transgenic expression of the TFs under a constitutive promoter in the orange carrot cultivar 'Danvers 126' lead to consistent upregulation of anthocyanin-related biosynthetic genes and significant accumulation of anthocyanins in leaves, stems and taproots. Highest overall content of soluble anthocyanins in the taproot among the transformants amounted to 44.38mgg-1 dry weight. The anthocyanin profile of the transformants were significantly different from the profile in the reference black carrot 'Deep Purple'. The main anthocyanins present in the transformed taproots were cyanidin 3-xylosyl(sinapoylglucosyl)galactoside, whereas the main anthocyanin present in Deep Purple was cyanidin 3-xylosyl(feruloylglucosyl)galactoside. This study confirms the presence of the necessary biosynthetic genes in orange carrots for production of anthocyanins and demonstrates the absence of suitable R2R3-MYB and bHLH TFs for stimulating anthocyanin biosynthesis in the orange carrot.

Overexpression of ThMYC4E Enhances Anthocyanin Biosynthesis in Common Wheat.

The basic helix-loop helix (bHLH) transcription factor has been inferred to play an important role in blue and purple grain traits in common wheat, but to date, its overexpression has not been reported. In this study, the bHLH transcription factor ThMYC4E, the candidate gene controlling the blue grain trait from Th. Ponticum, was transferred to the common wheat JW1. The positive transgenic lines displayed higher levels of purple anthocyanin pigments in their grains, leaves and glumes. Stripping the glumes (light treatment) caused white grains to become purple in transgenic lines. RNA-Seq and qRT-PCR analysis demonstrated that the transcript levels of structural genes associated with anthocyanin biosynthesis were higher in transgenic wheat than the wild-type (WT), which indicated that ThMYC4E activated anthocyanin biosynthesis in the transgenic lines. Correspondingly, the anthocyanin contents in grains, roots, stems, leaves and glumes of transgenic lines were higher than those in the WT. Metabolome analysis demonstrated that the anthocyanins were composed of cyanidin and delphinidin in the grains of the transgenic lines. Moreover, the transgenic lines showed higher antioxidant activity, in terms of scavenging DPPH radicals, in the ethanol extracts of their grains. The overexpression of ThMYC4E sheds light on the traits related to anthocyanin biosynthesis in common wheat and provide a new way to improve anthocyanin content.

Protein phosphatase 2A activation as a therapeutic strategy for managing MYC-driven cancers.

The tumor suppressor protein phosphatase 2A (PP2A) is a serine/threonine phosphatase whose activity is inhibited in most human cancers. One of the best-characterized PP2A substrates is MYC proto-oncogene basic helix-loop-helix transcription factor (MYC), whose overexpression is commonly associated with aggressive forms of this disease. PP2A directly dephosphorylates MYC, resulting in its degradation. To explore the therapeutic potential of direct PP2A activation in a diverse set of MYC-driven cancers, here we used biochemical assays, recombinant cell lines, gene expression analyses, and immunohistochemistry to evaluate a series of first-in-class small-molecule activators of PP2A (SMAPs) in Burkitt lymphoma, KRAS-driven non-small cell lung cancer, and triple-negative breast cancer. In all tested models of MYC-driven cancer, the SMAP treatment rapidly and persistently inhibited MYC expression through proteasome-mediated degradation, inhibition of MYC transcriptional activity, decreased cancer cell proliferation, and tumor growth inhibition. Importantly, we generated a series of cell lines expressing PP2A-dependent phosphodegron variants of MYC and demonstrated that the antitumorigenic activity of SMAPs depends on MYC degradation. Collectively, the findings presented here indicate a pharmacologically tractable approach to drive MYC degradation by using SMAPs for the management of a broad range of MYC-driven cancers.

CSIG-15. INHIBITION OF THE bHLH TRANSCRIPTIONAL NETWORKS BY A MUTATED E47 PROTEIN LEADS TO A STRONG ANTI-GLIOMA ACTIVITY IN VITRO AND IN VIVO

Abstract OBJECTIVE The transcription factor E47 heterodimerizes with helix-loop-helix (HLH) and basic helix-loop-helix transcription (bHLH) factors like ID-1 and Olig2 that are overexpressed in glioblastoma. A dominant-negative variant of the E47 (dnE47) lacking the nuclear translocation signal, leads to cytoplasmatic sequestration of HLH and bHLH transcription factors. Here, we investigated combinations of dnE47-mediated inhibition of the bHLH transcriptional network with temozolomide and irradiation and explored the underlying molecular mechanisms. METHODS Long-term and stem cell glioma lines were transduced with a Doxycycline-inducible dnE47 lentivirus. Functional characterizations included immunocytochemistry, immunoblots, cytotoxicity and clonogenicity assays in vitro and latency until the onset of symptoms in vivo. CAGE and RNASeq were conducted for analyzing the dnE47-induced molecular profile. RESULTS The induction of dnE47 led to cytoplasmatic sequestration of HLH/bHLH transcription, reduced proliferation, increased cytotoxicity and reduced clonogenic survival in vitro and a prolonged latency until the onset of neurological symptoms in vivo. CAGE and RNASeq data revealed alterations in several cancer-relevant pathways. CONCLUSIONS A dnE47-mediated inhibition of the bHLH transcription network induced actionable molecular alterations in glioma cells that could be exploited for the design of novel therapies.

Dec1 Deficiency Suppresses Cardiac Perivascular Fibrosis Induced by Transverse Aortic Constriction.

Cardiac fibrosis is a major cause of cardiac dysfunction in hypertrophic hearts. Differentiated embryonic chondrocyte gene 1 (Dec1), a basic helix–loop–helix transcription factor, has circadian expression in the heart; however, its role in cardiac diseases remains unknown. Therefore, using Dec1 knock-out (Dec1KO) and wild-type (WT) mice, we evaluated cardiac function and morphology at one and four weeks after transverse aortic constriction (TAC) or sham surgery. We found that Dec1KO mice retained cardiac function until four weeks after TAC. Dec1KO mice also revealed more severely hypertrophic hearts than WT mice at four weeks after TAC, whereas no significant change was observed at one week. An increase in Dec1 expression was found in myocardial and stromal cells of TAC-treated WT mice. In addition, Dec1 circadian expression was disrupted in the heart of TAC-treated WT mice. Cardiac perivascular fibrosis was suppressed in TAC-treated Dec1KO mice, with positive immunostaining of S100 calcium binding protein A4 (S100A4), alpha smooth muscle actin (αSMA), transforming growth factor beta 1 (TGFβ1), phosphorylation of Smad family member 3 (pSmad3), tumor necrosis factor alpha (TNFα), and cyclin-interacting protein 1 (p21). Furthermore, Dec1 expression was increased in myocardial hypertrophy and myocardial infarction of autopsy cases. Taken together, our results indicate that Dec1 deficiency suppresses cardiac fibrosis, preserving cardiac function in hypertrophic hearts. We suggest that Dec1 could be a new therapeutic target in cardiac fibrosis.

Variation in a Left Ventricle-Specific Hand1 Enhancer Impairs GATA Transcription Factor Binding and Disrupts Conduction System Development and Function.

The ventricular conduction system (VCS) rapidly propagates electrical impulses through the working myocardium of the ventricles to coordinate chamber contraction. GWAS (Genome-wide association studies) have associated nucleotide polymorphisms, most are located within regulatory intergenic or intronic sequences, with variation in VCS function. Two highly correlated polymorphisms (r2>0.99) associated with VCS functional variation (rs13165478 and rs13185595) occur 5' to the gene encoding the basic helix-loop-helix transcription factor HAND1 (heart- and neural crest derivatives-expressed protein 1). Here, we test the hypothesis that these polymorphisms influence HAND1 transcription thereby influencing VCS development and function. We employed transgenic mouse models to identify an enhancer that is sufficient for left ventricle (LV) cis-regulatory activity. Two evolutionarily conserved GATA transcription factor cis-binding elements within this enhancer are bound by GATA4 and are necessary for cis-regulatory activity, as shown by in vitro DNA binding assays. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated deletion of this enhancer dramatically reduces Hand1 expression solely within the LV but does not phenocopy previously published mouse models of cardiac Hand1 loss-of-function. Electrophysiological and morphological analyses reveals that mice homozygous for this deleted enhancer display a morphologically abnormal VCS and a conduction system phenotype consistent with right bundle branch block. Using 1000 Genomes Project data, we identify 3 additional single nucleotide polymorphisms (SNPs), located within the Hand1 LV enhancer, that compose a haplotype with rs13165478 and rs13185595. One of these SNPs, rs10054375, overlaps with a critical GATA cis-regulatory element within the Hand1 LV enhancer. This SNP, when tested in electrophoretic mobility shift assays, disrupts GATA4 DNA-binding. Modeling 2 of these SNPs in mice causes diminished Hand1 expression and mice present with abnormal VCS function. Together, these findings reveal that SNP rs10054375, which is located within a necessary and sufficient LV-specific Hand1 enhancer, exhibits reduces GATA DNA-binding in electrophoretic mobility shift assay, and this enhancer in total, is required for VCS development and function in mice and perhaps humans.

The plant Mediator complex and its role in jasmonate signaling.

The Mediator complex is an essential, multisubunit transcriptional coactivator that is highly conserved in eukaryotes. Mediator interacts with gene-specific transcription factors, the RNA polymerase II transcriptional machinery, as well as several other factors involved in transcription, and acts as an integral hub to regulate various aspects of transcription. Recent studies of the plant Mediator complex have established that it functions in diverse aspects of plant development and fitness. Jasmonate (JA) is an oxylipin-derived plant hormone that regulates plant immunity and development. The basic helix–loop–helix transcription factor MYC2, which is a master regulator of JA signaling, orchestrates genome-wide transcriptional reprogramming of plant cells to coordinate defense- and growth-related processes. Here, we review the function of the plant Mediator complex in regulating JA signaling. We focus on the multifunctional Mediator subunit MED25, which emerges as an integrative hub for the transcriptional regulation of jasmonate signaling.

Identification of the grape basic helix–loop–helix transcription factor family and characterization of expression patterns in response to different stresses

Basic helix–loop–helix (bHLH) proteins comprise one of the largest transcription factors (TFs) families in plants and members have been identified in model plant species, such as Arabidopsis thaliana and Oryza sativa. However, far less is known about the evolutionary history and expression patterns of bHLH proteins in woody species, such as grape (Vitis vinifera). In this study, we identified a total of 126 bHLH genes (VvbHLH) in grape genome, which were classified into 24 subfamilies based on the phylogenetic analysis included homologs from four plant species. The VvbHLH genes belonging to the same subfamilies have similar protein motifs and exon/intron structures and synteny analysis indicated that tandem and segmental duplication events have been major contributors to the expansion of the VvbHLH family. Synteny analysis between grape and A. thaliana suggested that some bHLH members shared a common ancestor. Expression analysis of 26 VvbHLH genes, representing members of the III and IV subfamilies, revealed profiles that were tissue-specific, hormone-responsive, and responsive to abiotic and biotic stresses. Taken together, the genome-wide identification and characterization of the grape bHLH TFs provide insights into their evolutionary history and represents a resource for further functional characterization in the context of crop improvement and stress tolerance.

Injury to hypothalamic Sim1 neurons is a common feature of obesity by exposure to high-fat diet in male and female mice.

The hypothalamus is essential for regulation of energy homeostasis and metabolism. Feeding hypercaloric, high-fat (HF) diet induces hypothalamic arcuate nucleus injury and alters metabolism more severely in male than in female mice. The site(s) and extent of hypothalamic injury in male and female mice are not completely understood. In the paraventricular nucleus (PVN) of the hypothalamus, single-minded family basic helix-loop helix transcription factor 1 (Sim1) neurons are essential to control energy homeostasis. We tested the hypothesis that exposure to HF diet induces injury to Sim1 neurons in the PVN of male and female mice. Mice expressing membrane-bound enhanced green fluorescent protein (mEGFP) in Sim1 neurons (Sim1-Cre:Rosa-mEGFP mice) were generated to visualize the effects of exposure to HF diet on these neurons. Male and female Sim1-Cre:Rosa-mEGFP mice exposed to HF diet had increased weight, hyperleptinemia, and developed hepatosteatosis. In male and female mice exposed to HF diet, expression of mEGFP was reduced by >40% in Sim1 neurons of the PVN, an effect paralleled by cell apoptosis and neuronal loss, but not by microgliosis. In the arcuate nucleus of the Sim1-Cre:Rosa-mEGFP male mice, there was decreased alpha-melanocyte-stimulating hormone in proopiomelanocortin neurons projecting to the PVN, with increased cell apoptosis, neuronal loss, and microgliosis. These defects were undetectable in the arcuate nucleus of female mice exposed to the HF diet. Thus, injury to Sim1 neurons of the PVN is a shared feature of exposure to HF diet in mice of both sexes, while injury to proopiomelanocortin neurons in arcuate nucleus is specific to male mice. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

The barren stalk2 Gene Is Required for Axillary Meristem Development in Maize

The diversity of plant architecture is determined by axillary meristems (AMs). AMs are produced from small groups of stem cells in the axils of leaf primordia and generate vegetative branches and reproductive inflorescences. Previous studies identified genes critical for AM development that function in auxin biosynthesis, transport, and signaling. barren stalk1 (ba1), a basic helix–loop–helix transcription factor, acts downstream of auxin to control AM formation. Here, we report the cloning and characterization of barren stalk2 (ba2), a mutant that fails to produce ears and has fewer branches and spikelets in the tassel, indicating that ba2 functions in reproductive AM development. Furthermore, the ba2 mutation suppresses tiller growth in the teosinte branched1 mutant, indicating that ba2 also plays an essential role in vegetative AM development. The ba2 gene encodes a protein that co-localizes and heterodimerizes with BA1 in the nucleus. Characterization of the genetic interaction between ba2 and ba1 demonstrates that ba1 shows a gene dosage effect in ba2 mutants, providing further evidence that BA1 and BA2 act together in the same pathway. Characterization of the molecular and genetic interaction between ba2 and additional genes required for the regulation of ba1 further supports this finding. The ba1 and ba2 genes are orthologs of rice genes, LAX PANICLE1 (LAX1) and LAX2, respectively, hence providing insights into pathways controlling AMs development in grasses.

The FZD7-TWIST1 axis is responsible for anoikis resistance and tumorigenesis in ovarian carcinoma.

Frizzled family receptor 7 (FZD7), a Wnt signaling receptor, is associated with the maintenance of stem cell properties and cancer progression. FZD7 has emerged as a potential therapeutic target because it is capable of transducing both canonical and noncanonical Wnt signals. In this study, we investigated the regulatory pathway downstream of FZD7 and its functional roles. We found that FZD7 expression was crucial to the maintenance of the mesenchymal phenotype, anoikis resistance, and spheroid and tumor formation in ovarian cancer (OC). We identified TWIST1 as the crucial downstream effector of the FZD7 pathway. TWIST1, a basic helix loop helix transcription factor, is known to associate with mesenchymal and cancer stem cell phenotypes. Manipulating TWIST1 expression mimicked the functional consequences observed in the FZD7 model, and overexpression of TWIST1 partially rescued the functional phenotypes abolished by FZD7 knockdown. We further proved that FZD7 regulated TWIST1 expression through epigenetic modifications of H3K4me3 and H3K27ac at the TWIST1 proximal promoter. We also identified that the FZD7‐TWIST1 axis regulates the expression of BCL2, a gene that controls apoptosis. Identification of this FZD7‐TWIST1‐BCL2 pathway reaffirms the mechanism of anoikis resistance in OC. We subsequently showed that the FZD7‐TWIST1 axis can be targeted by using a small molecule inhibitor of porcupine, an enzyme essential for secretion and functional activation of Wnts. In conclusion, our results identified that the FZD7‐TWIST1 axis is important for tumorigenesis and anoikis resistance, and therapeutic inhibition results in cell death in OCs.

A Robust Auxin Response Network Controls Embryo and Suspensor Development through a Basic Helix Loop Helix Transcriptional Module.

Land plants reproduce sexually by developing an embryo from a fertilized egg cell. However, embryos can also be formed from other cell types in many plant species. Thus, a key question is how embryo identity in plants is controlled, and how this process is modified during nonzygotic embryogenesis. The Arabidopsis (Arabidopsis thaliana) zygote divides to produce an embryonic lineage and an extra-embryonic suspensor. Yet, normally quiescent suspensor cells can develop a second embryo when the initial embryo is damaged, or when response to the signaling molecule auxin is locally blocked. Here we used auxin-dependent suspensor embryogenesis as a model to determine transcriptome changes during embryonic reprogramming. We found that reprogramming is complex and accompanied by large transcriptomic changes before anatomical changes. This analysis revealed a strong enrichment for genes encoding components of auxin homeostasis and response among misregulated genes. Strikingly, deregulation among multiple auxin-related gene families converged upon the re-establishment of cellular auxin levels or response. This finding points to a remarkable degree of feedback regulation to create resilience in the auxin response during embryo development. Starting from the transcriptome of auxin-deregulated embryos, we identified an auxin-dependent basic Helix Loop Helix transcription factor network that mediates the activity of this hormone in suppressing embryo development from the suspensor.

The Degradation and Subcellular Localization of an Oncogenic protein complex in T-cell Leukemia

Background and Hypothesis: 
 LIM domain Only-2 (LMO2) is a key oncogenic driver of human T-cell acute lymphoblastic leukemia. LMO2 functions as part of a large multisubunit complex that regulates gene expression. LMO2 itself does not bind DNA but it does bind class II basic helix loop helix transcription factors, TAL1 or LYL1, which are also part of the LMO2-associated complex. We recently identified LIM domain binding protein 1 (LDB1) as an obligate LMO2 partner that is required for LMO2 protein stability in T-ALL. We believe the interaction between LDB1 and LMO2 is crucial to understanding the pathogenesis of T-ALL and is a potential therapeutic target for this aggressive leukemia. We hypothesize that in the absence of LDB1, LMO2 is rapidly degraded. Nonetheless, the mechanisms for the localization and degradation of LMO2 and its partners have not been fully explored. 
 Experimental Design or Project Methods: 
 In this study, we analyzed the first order decay of the LMO2-associated complex including LMO2, SSBP2, SSBP3, TAL1, LYL1, and LDB1. We analyzed the subcellular localization of these same proteins distinguishing between the nucleus and the cytoplasm. Our studies were enabled by cloning the HALO tag to the NH2-terminus of these proteins. Fluorescent small molecules such as HALO ligand, R110, were used for intracellular labeling followed by pulse chase analysis via flow cytometry for half-life and imaging by confocal microscopy. 
 Results: 
 We observed a hierarchy of protein stability: LDB1 had the longest half-life, followed by SSBP3, SSBP2, Tal1, LMO2 and Lyl1had the shortest half-lives. Co-expression of LDB1 conferred enhanced staility upon all protein components. All of the protein components studied showed a predominantly nuclear distribution with some in the cytoplasm. 
 Conclusion and Potential Impact: 
 The prolonged protein stability of LDB1 allows it to confer enhanced stability to LMO2 and bHLH proteins TAL1 and LYL1. Thus, targeting the assembly of the multisubunit LMO2 complex.

A basic helix loop helix transcription factor, AaMYC2-Like positively regulates artemisinin biosynthesis in Artemisia annua L.

Artemisinin based combination therapies are still an extensively used remedy for the cure of Malaria. Although artemisinin biosynthetic pathway has been fully worked out, only few information is available on its regulatory mechanism in plants. In the present study, a new member of MYC type basic helix loop helix transcription factor was isolated from Artemisia annua and named as, AaMYC2-Like. The deduced protein sequence of AaMYC2-Like confirmed the presence of conserved DNA binding, dimerization, nuclear localization and helix loop helix domains. The expression analysis of AaMYC2-Like in various tissues established the highest transcript levels in leaves and lowest in roots which were in accordance with the trichome density, the biofactories for artemisinin biosynthesis. Moreover AaMYC2-Like was induced in presence of methyl jasmonate (MeJA), abscisic acid 2,4-dichlorophenoxy acetic acid, methyl viologen, heat, cold etc. The maximum elicitation of AaMYC2-Like transcript levels in presence of MeJA as compared to other phytohormones confirmed AaMYC2-Like as jasmonic acid responsive MYC type transcription factor. Further AaMYC2-Like was found to be transcriptionally active protein and localized to the nucleus. The transient overexpression of AaMYC2-Like in Artemisia annua leaves increased transcript levels of three key enzymes involved in artemisinin biosynthetic pathway such as cytochrome P-450 dependent hydroxylase, double bond reductase and aldehyde dehydrogenase. Consequently artemisinin content was also dramatically elevated in transiently over expressing AaMYC2-Like transgenic plants, confirming prominent role of AaMYC2-Like in the regulation of key biosynthetic genes. Taken together these results strongly suggest that AaMYC2-Like is an important MYC homolog which plays prominent role in the regulation of artemisinin biosynthetic pathway.

Clusterin, a Novel DEC1 Target, Modulates DNA Damage-Mediated Cell Death.

Differentiated embryonic chondrocyte expressed gene 1 (DEC1, also known as Sharp2/Stra13/BHLHE40) is a basic helix-loop-helix transcription factor that plays an important role in circadian rhythms, cell proliferation, apoptosis, cellular senescence, hypoxia response, and epithelial-to-mesenchymal transition of tumor cells. Secretory clusterin (sCLU) is a cytoprotective protein that guards against genotoxic stresses. Here, clusterin (CLU) was identified as a novel target gene of DEC1 and suppresses DNA damage-induced cell death in tumor cells. Mechanistically, based on chromatin immunoprecipitation and luciferase assays, DEC1 binds to and activates the promoter of the CLU gene. DEC1 and DNA-damaging agents induce sCLU expression, whereas DEC1 knockdown decreases the expression of sCLU upon DNA damage. Moreover, the data demonstrate that DEC1 inhibits, whereas sCLU knockdown enhances, DNA damage-induced cell death in MCF7 breast cancer cells. Given that DEC1 and sCLU are frequently overexpressed in breast cancers, these data provide mechanistic insight into DEC1 as a prosurvival factor by upregulating sCLU to reduce the DNA damage-induced apoptotic response. Together, this study reveals sCLU as a novel target of DEC1 which modulates the sensitivity of the DNA damage response.Implications: DEC1 and sCLU are frequently overexpressed in breast cancer, and targeting the sCLU-mediated cytoprotective signaling pathway may be a novel therapeutic approach. Mol Cancer Res; 16(11); 1641-51. ©2018 AACR.

The regulatory module MdPUB29‐MdbHLH3 connects ethylene biosynthesis with fruit quality in apple

The plant hormone ethylene is critical for climacteric fruit ripening, while glucose and anthocyanins determine the fruit quality of climacteric fruits such as apple. Understanding the exact molecular mechanism for this process is important for elucidating the interconnection of ethylene and fruit quality. Overexpression of apple MdbHLH3 gene, an anthocyanin-related basic helix-loop-helix transcription factor (bHLH TF) gene, promotes ethylene production, and transgenic apple plantlets and trees exhibit ethylene-related root developmental abnormalities, premature leaf senescence, and fruit ripening. Biochemical analyses demonstrate that MdbHLH3 binds to the promoters of three genes that are involved in ethylene biosynthesis, including MdACO1, MdACS1, and MdACS5A, activating their transcriptional expression, thereby promoting ethylene biosynthesis. High glucose-inhibited U-box-type E3 ubiquitin ligase MdPUB29, the ortholog of Arabidopsis AtPUB29 in apple, influences the expression of ethylene biosynthetic genes and ethylene production by direct ubiquitination of the MdbHLH3 protein. Our findings provide new insights into the ubiquitination of MdbHLH3 by glucose-inhibited ubiquitin E3 ligase MdPUB29 in the regulation of ethylene biosynthesis as well as indicate that the regulatory module MdPUB29-MdbHLH3 connects ethylene biosynthesis with fruit quality in apple.

Identification of key genes in prostate cancer gene expression profile by bioinformatics.

The aim of this study was to identify key candidate genes in prostate cancer. The gene expression profiles of GSE32448, GSE45016, GSE46602 and GSE104749 were downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) between prostate cancer and normal samples were identified by R language. The gene ontology functional and pathway enrichment analyses of DEGs were performed by the Database for Annotation, Visualization and Integrated Discovery software followed by the construction of protein-protein interaction network. Hub gene identification was performed by the plug-in cytoHubba in Cytoscape software. The 217 DEGs were significantly enriched in biological processes including epithelial cell differentiation, response to estradiol and several pathways, mainly associated with protein digestion and absorption pathway in prostate cancer. Epithelial cell adhesion molecule, twist family basic helix-loop-helix transcription factor 1, CD38 molecule and vascular endothelial growth factor A were identified as hub genes. The expression levels of hub genes were consistent with data obtained in The Cancer Genome Atlas for prostate adenocarcinoma. These hub genes may be used as potential targets for prostate cancer diagnosis and treatment.