Mechanisms of Regulation of the CHRDL1 Gene by the TWIST2 and ADD1/SREBP1c Transcription Factors.

Setleis syndrome (SS) is a rare focal facial dermal dysplasia caused by recessive mutations in the basic helix-loop-helix (bHLH) transcription factor, TWIST2. Expression microarray analysis showed that the chordin-like 1 (CHRDL1) gene is up-regulated in dermal fibroblasts from three SS patients with the Q119X TWIST2 mutation. METHODS: Putative TWIST binding sites were found in the upstream region of the CHRDL1 gene and examined by electrophoretic mobility shift (EMSA) and reporter gene assays. RESULTS: EMSAs showed specific binding of TWIST1 and TWIST2 homodimers, as well as heterodimers with E12, to the more distal E-boxes. An adjoining E-box was bound by ADD1/SREBP1c. EMSA analysis suggested that TWIST2 and ADD1/SREBP1c could compete for binding. Luciferase (luc) reporter assays revealed that the CHRDL1 gene upstream region drives its expression and ADD1/SREBP1c increased it 2.6 times over basal levels. TWIST2, but not the TWIST2-Q119X mutant, blocked activation by ADD1/SREBP1c, but overexpression of TWIST2-Q119X increased luc gene expression. In addition, EMSA competition assays showed that TWIST2, but not TWIST1, competes with ADD1/SREBP1c for DNA binding to the same site. CONCLUSIONS: Formation of an inactive complex between the TWIST2 Q119X and Q65X mutant proteins and ADD1/SREBP1c may prevent repressor binding and allow the binding of other regulators to activate CHRDL1 gene expression.

Homodimeric and Heterodimeric Interactions among Vertebrate Basic Helix-Loop-Helix Transcription Factors.

The basic helix-loop-helix transcription factor (bHLH TF) family is involved in tissue development, cell differentiation, and disease. These factors have transcriptionally positive, negative, and inactive functions by combining dimeric interactions among family members. The best known bHLH TFs are the E-protein homodimers and heterodimers with the tissue-specific TFs or ID proteins. These cooperative and dynamic interactions result in a complex transcriptional network that helps define the cell's fate. Here, the reported dimeric interactions of 67 vertebrate bHLH TFs with other family members are summarized in tables, including specifications of the experimental techniques that defined the dimers. The compilation of these extensive data underscores homodimers of tissue-specific bHLH TFs as a central part of the bHLH regulatory network, with relevant positive and negative transcriptional regulatory roles. Furthermore, some sequence-specific TFs can also form transcriptionally inactive heterodimers with each other. The function, classification, and developmental role for all vertebrate bHLH TFs in four major classes are detailed.

Comparative anatomy and genetic bases of fruit development in selected Rubiaceae (Gentianales).

PREMISE: The Rubiaceae are ideal for studying the diversity of fruits that develop from flowers with inferior ovary. We aimed to identify morpho-anatomical changes during fruit development that distinguish those derived from the carpel versus the extra-carpellary tissues. In addition, we present the fruit genetic core regulatory network in selected Rubiaceae species and compare it in terms of copy number and expression patterns to model core eudicots in the Brassicaceae and the Solanaceae. METHODS: We used light microscopy to follow morphoanatomical changes in four selected species with different fruit types. We generated reference transcriptomes for seven selected Rubiaceae species and isolated homologs of major transcription factors involved in fruit development histogenesis, assessed their homology, identified conserved and new protein motifs, and evaluated their expression in three species with different fruit types. RESULTS: Our studies revealed ovary-derived pericarp tissues versus floral-cup-derived epicarp tissues. Gene evolution analyses of FRUITFULL, SHATTERPROOF, ALCATRAZ, INDEHISCENT and REPLUMLESS homologs suggest that the gene complement in Rubiaceae is simpler compared to that in Brassicaceae or Solanaceae. Expression patterns of targeted genes vary in response to the fruit type and the developmental stage evaluated. CONCLUSIONS: Morphologically similar fruits can have different anatomies as a result of convergent tissues developed from the epicarps covering the anatomical changes from the pericarps. Expression analyses suggest that the fruit patterning regulatory network established in model core eudicots cannot be extrapolated to asterids with inferior ovaries.

Expression Profiling Identifies TWIST2 Target Genes in Setleis Syndrome Patient Fibroblast and Lymphoblast Cells.

Background: Setleis syndrome (SS) is a focal facial dermal dysplasia presenting with bilateral temporal skin lesions, eyelash abnormalities and absent meibomian glands. SS is a rare autosomal recessive disorder caused by mutations in the TWIST2 gene, which codes for a transcription factor of the bHLH family known to be involved in skin and facial development. Methods: We obtained gene expression profiles by microarray analyses from control and SS patient primary skin fibroblast and lymphoblastoid cell lines. Results: Out of 983 differentially regulated genes in fibroblasts (fold change ≥ 2.0), 479 were down-regulated and 509 were up-regulated, while in lymphoblasts, 1248 genes were down-regulated and 73 up-regulated. RT-PCR reactions confirmed altered expression of selected genes. Conclusions: TWIST2 is described as a repressor, but expression profiling suggests an important role in gene activation as well, as evidenced by the number of genes that are down-regulated, with a much higher proportion of down-regulated genes found in lymphoblastoid cells from an SS patient. As expected, both types of cell types showed dysregulation of cytokine genes. These results identify potential TWIST2 target genes in two important cell types relevant to rare disorders caused by mutations in this bHLH gene.

The Transcription Factor SCX is a Potential Serum Biomarker of Fibrotic Diseases.

Fibrosing diseases are causes of morbidity and mortality around the world, and they are characterized by excessive extracellular matrix (ECM) accumulation. The bHLH transcription factor scleraxis (SCX) regulates the synthesis of ECM proteins in heart fibrosis. SCX expression was evaluated in lung fibroblasts and tissue derived from fibrotic disease patients and healthy controls. We also measured SCX in sera from 57 healthy controls, and 56 Idiopathic Pulmonary Fibrosis (IPF), 40 Hypersensitivity Pneumonitis (HP), and 100 Systemic Sclerosis (SSc) patients. We report high SCX expression in fibroblasts and tissue from IPF patients versus controls. High SCX-serum levels were observed in IPF (0.663 ± 0.559 ng/mL, p < 0.01) and SSc (0.611 ± 0.296 ng/mL, p < 0.001), versus controls (0.351 ± 0.207 ng/mL) and HP (0.323 ± 0.323 ng/mL). Serum levels of the SCX heterodimerization partner, TCF3, did not associate with fibrotic illness. IPF patients with severely affected respiratory capacities and late-stage SSc patients presenting anti-topoisomerase I antibodies and interstitial lung disease showed the highest SCX-serum levels. SCX gain-of-function induced the expression of alpha-smooth muscle actin (α-SMA/ACTA2) in fibroblasts when co-overexpressed with TCF3. As late and severe stages of the fibrotic processes correlated with high circulating SCX, we postulate it as a candidate biomarker of fibrosis and a potential therapeutic target.

Overexpression of bHLH domain of HIF-1 failed to inhibit the HIF-1 transcriptional activity in hypoxia.

BACKGROUND: Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition. METHODS: We inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl2) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT. RESULTS: Hypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and ß-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl2. The gene expression of VEGF, vimentin, and ß-catenin and protein level of ß-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of ß-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant. CONCLUSION: bHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.

A key for hypoxia genetic adaptation in alpaca could be a HIF1A truncated bHLH protein domain / Una clave para la adaptacion genética de la hypoxia en las alpacas podria ser un dominio de proteina bHLH truncado con HIF1A

Animals exposed to hypobaric hypoxia triggers a physiological hypoxia response via Hypoxia Inducible Factor (HIF) proteins that functions as transcriptional complexes. As the South American camelids inhabit at high Andean altitudes we have asked if they have developed genetic adaptations to live at high altitudes. In the present study we investigate genetic structures of the HIF1A proteins carried by members of the superorder Cetartiodactyla. During our investigation we discovered the existence of a genetic event that caused the loss of most of the bHLH domain in the proteins borne by the Alpaca and other members of the Cetartiodactyla superorder; we designate them as bHLH short sequences. Further analysis at the nucleotide level revealed in the 12 short sequences included in the study the presence at the 5´end of the bHLH domains stop codons. Seven out of the 12 short HIF1A proteins, have an identical or almost identical nucleotide sequence at their 5´end with a same TAA stop codon and at the same position. As the mutations affects to both the Artiodactyls and Cetaceans, we postulate that the mutation(s) occurred before their divergence about 55 million years ago. The relevance of these findings for genetic adaptation of Alpacas to hypobaric hypoxia of high altitude conditions is discussed.

Los animales expuestos a hypoxia hipobárica generan una respuesta hipóxica fisiológica debido a unas proteinas de Factor-Hipoxia Inducible (HIF) que funcionan como complejos transcripcionales. Debido a que los camelidos Americanos habitan en las grandes alturas andinas, nos hemos preguntado si han desarrollado una adaptación genética para vivir a grandes alturas. Eneste estudio hemos investigado la estructura genética de las proteinas HIF1A que llevan consigo los miembros de la superorden de los cetartiodáctilos. Durante nuestra investigación, descubrimos la existencia de un evento genético que causó la perdida de la mayoría del dominio bHLH en las proteinas transmitidas por la alpaca y otros miembros de la superorden de los cetartiodáctilos; las hemos designado como secuencias cortas de bHLH. Análisis posteriores a nivel nucleótido revelaron que en la doceava secuencia corta incluida en el studio, hubo presencia de codones de terminación en el extreme 5' del dominio de bHLH. Siete de las doce proteinas cortas HIF1A, tiene una secuencia idéntica o casi idéntica de nucleotidos en su extremo 5', con el mismo codón de terminación TAA y en la misma posición. Debido a que la mutación afecta tanto a Artiodáctilos como Cetáceos, proponemos que la mutación(es) ocurrió antes de su divergencia hace unos 55 millones de años. Analizamos la relevancia de estos descubrimientos sobre la adaptación genética de las alpacas a la hipoxia hipobárica en condiciones de grandes alturas.

Regulatory Potential of bHLH-Type Transcription Factors on the Road to Rubber Biosynthesis in Hevea brasiliensis.

Natural rubber is the main component of latex obtained from laticifer cells of Hevea brasiliensis. For improving rubber yield, it is essential to understand the genetic molecular mechanisms responsible for laticifer differentiation and rubber biosynthesis. Jasmonate enhances both secondary laticifer differentiation and rubber biosynthesis. Here, we carried out time-course RNA-seq analysis in suspension-cultured cells treated with methyljasmonic acid (MeJA) to characterize the gene expression profile. Gene Ontology (GO) analysis showed that the term "cell differentiation" was enriched in upregulated genes at 24 hours after treatment, but inversely, the term was enriched in downregulated genes at 5 days, indicating that MeJA could induce cell differentiation at an early stage of the response. Jasmonate signaling is activated by MYC2, a basic helix-loop-helix (bHLH)-type transcription factor (TF). The aim of this work was to find any links between transcriptomic changes after MeJA application and regulation by TFs. Using an in vitro binding assay, we traced candidate genes throughout the whole genome that were targeted by four bHLH TFs: Hb_MYC2-1, Hb_MYC2-2, Hb_bHLH1, and Hb_bHLH2. The latter two are highly expressed in laticifer cells. Their physical binding sites were found in the promoter regions of a variety of other TF genes, which are differentially expressed upon MeJA exposure, and rubber biogenesis-related genes including SRPP1 and REF3. These studies suggest the possibilities that Hb_MYC2-1 and Hb_MYC2-2 regulate cell differentiation and that Hb_bHLH1 and Hb_bHLH2 promote rubber biosynthesis. We expect that our findings will help to increase natural rubber yield through genetic control in the future.

Overexpression of bHLH domain of HIF-1 failed to inhibit the HIF-1 transcriptional activity in hypoxia

BACKGROUND: Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition. METHODS: We inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl2) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT. RESULTS: Hypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and ß-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl2. The gene expression of VEGF, vimentin, and ß-catenin and protein level of ß-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of ß-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant. CONCLUSION: bHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.

ID Proteins May Reduce Aggressiveness of Thyroid Tumors.

ID genes have an important function in the cell cycle, and ID proteins may help identify aggressive tumors, besides being considered promising therapeutic targets. However, their role in thyroid tumors is still poorly understood. We examined ID expression and their correlation with diagnostic and prognostic features aiming to find a clinical application in differentiated thyroid carcinoma (DTC) cases. mRNA levels of ID1, ID2, ID3, and ID4 genes were quantified and their expression was observed by immunohistochemistry in 194 thyroid samples including 68 goiters, 16 follicular adenomas, 75 classic papillary thyroid carcinomas, 18 follicular variants of papillary thyroid carcinoma, 5 follicular thyroid carcinomas, and 1 anaplastic thyroid cancer, besides 11 normal thyroid tissues. DTC patients were managed according to standard protocols and followed up for M = 28 ± 16 months. ID2, ID3, and ID4 mRNA levels were higher in benign (2.0 ± 1.9; 0.6 ± 0.6; and 0.7 ± 1.0 AU, respectively) than those in malignant nodules (0.30 ± 0.62; 0.3 ± 0.3; and 0.2 ± 0.3 AU, respectively, p < 0.0001 for all three genes) and were associated with no extra thyroid invasion or metastasis at diagnosis. ID3 nuclear protein expression was higher in benign than that in malignant cells (5.2 ± 0.9 vs 3.0 ± 1.8 AU; p < 0.0001). On the contrary, the cytoplasmic expression of ID3 was higher in malignant than that in benign lesions (5.7 ± 1.5 vs 4.0 ± 1.4 AU; p < 0.0001). Our data indicate that ID genes are involved in thyroid tumorigenesis and suggest these genes act impeding the evolution of more aggressive phenotypes. The different patterns of their tissue expression may help identify malignancy and characterize thyroid lesion aggressiveness.

Genetic Switches between Cancer and Emphysema Resolution of Cigarette-Smoke Induced Inflammation.

Cigarette smoke initiates an inflammatory response that has aftermath long after quitting. We segregated former smokers, according to their lung function and their co-founding diseases, in 3 groups: Cancer, Emphysema and COPD. Then we searched for outlier genes in intersections of Venn diagrams where we identified 6 subsets and 23 genes that may be responsible for disease outcome. Genes expressed in the cancer patients with or without emphysema (PPA subset) were BHLH, FPRL2, CD49D, DEADH, NRs4A3, MBLL, GNS, BE675435, ISGF-3, and FLJ23462. Patients with emphysema as co-founding disease, with or without cancer (APP), had only ANXA2 in common. Genes expressed only in non-cancer patients (AAP subset) of COPD group were IL-1A, SOX13, RPP38; TBXA2R, NPEPL1, CFLAR, TFEB, PRKCBP1, IGF1R, DDX11, and KCNAB1. HIV-1Rev was the gene expressed in cancer patients with emphysema (APA subset). Then, we also looked at out-layers genes significantly expressed in all patients (PPP subset with 5066 genes), the down-regulated in Emphysema were MMP9, PLUNC, CEACAM5, and NR4A1 while the up-regulated were F2R, COL15A1, PDE4C, and BGN. We chose genes and checked them at the protein level on immune cells, this showed that neutrophils from Cancer group had increased expression of CD49d, and their total number was also increased in bronchial-alveolar lavage (154%). Macrophages in the lung of patients with emphysema were associated with a significant increase of adhesion molecule CD58 and to significant CD95 decrease, indicating they do not die. Besides, macrophages downregulated MMP9 in the lung compared to blood macrophages. Overall, we find that cancer progression requires a stickier and greater number of neutrophils in the lung while emphysema requires stickier and longevous macrophages to lead matrix destruction, and together with higher expression of SOX13 and RPP38, may promote autoimmunity. We also identified two genes, ANXA2 and HIV1-rev, that may be a pivot between cancer and emphysema outcome of inflammation.

The antagonistic basic helix-loop-helix partners BEE and IBH1 contribute to control plant tolerance to abiotic stress.

The bHLH family is composed by canonical and non-canonical transcription factors (TFs) that differ in the presence or absence of their DNA-binding domain, respectively. Since both types of bHLH proteins are able to dimerize, their relative abundance impacts their biological activity. Among this TF family BEE and IBH are canonical and non-canonical bHLHs, respectively and previous reports indicated that BEE2 and IBH1 dimerize. Wondering whether BEE TFs participate in the abiotic stress response and how the dimerization with IBH1 could regulate their role in Arabidopsis, double bee1/bee2 and triple bee1/bee2/bee3 mutants were tested under salinity and drought stresses. The bee1/bee2/bee3 mutant showed an enhanced tolerance whereas the double mutant behaved similar to wild type plants. These results indicated that BEE genes play a role in the stress response and also put in evidence the redundancy within the BEE family. Moreover, ectopic expression of IBH1 on different mutant backgrounds improved plant tolerance to abiotic stress, independently of the background. However, the yield of these transgenic plants was penalized with abortive seeds. Our results suggest that BEE genes are negative regulators of physiological responses to abiotic stress whereas IBH1 is a positive modulator via different pathways, one of them involving BEE TFs.

Overexpression of Hevea brasiliensis HbICE1 Enhances Cold Tolerance in Arabidopsis.

Rubber trees (Hevea brasiliensis) were successfully introduced to south China in the 1950s on a large-scale; however, due to the climate, are prone to cold injury during the winter season. Increased cold tolerance is therefore an important goal, yet the mechanism underlying rubber tree responses to cold stress remains unclear. This study carried out functional characterization of HbICE1 (Inducer of CBF Expression 1) from H. brasiliensis. A nucleic protein with typical features of ICEs, HbICE1 was able to bind to MYC recognition sites and had strong transactivation activity. HbICE1 was constitutively expressed in all tested tissues, with highest levels in the bark, and was up-regulated when subjected to various stresses including cold, dehydration, salinity and wounding. When overexpressed in Arabidopsis, 35S::HbICE1 plants showed enhanced cold resistance with increased proline content, reduced malondialdehyde (MDA) metabolism and electrolyte leakage, and decreased reactive oxygen species (ROS) accumulation. Expression of the cold responsive genes (COR15A, COR47, RD29A, and KIN1) was also significantly promoted in 35S::HbICE1 compared to wild-type plants under cold stress. Differentially expressed genes (DEGs) analysis showed that cold treatment changed genes expression profiles involved in many biological processes and phytohormones perception and transduction. Ethylene, JA, ABA, as well as ICE-CBF signaling pathways might work synergistically to cope with cold tolerance in rubber tree. Taken together, these findings suggest that HbICE1 is a member of the ICE gene family and a positive regulator of cold tolerance in H. brasiliensis.

Apotransferrin-induced recovery after hypoxic/ischaemic injury on myelination.

We have previously demonstrated that aTf (apotransferrin) accelerates maturation of OLs (oligodendrocytes) in vitro as well as in vivo. The purpose of this study is to determine whether aTf plays a functional role in a model of H/I (hypoxia/ischaemia) in the neonatal brain. Twenty-four hours after H/I insult, neonatal rats were intracranially injected with aTf and the effects of this treatment were evaluated in the CC (corpus callosum) as well as the SVZ (subventricular zone) at different time points. Similar to previous studies, the H/I event produced severe demyelination in the CC. Demyelination was accompanied by microglial activation, astrogliosis and iron deposition. Ferritin levels increased together with lipid peroxidation and apoptotic cell death. Histological examination after the H/I event in brain tissue of aTf-treated animals (H/I aTF) revealed a great number of mature OLs repopulating the CC compared with saline-treated animals (H/I S). ApoTf treatment induced a gradual increase in MBP (myelin basic protein) and myelin lipid staining in the CC reaching normal levels after 15 days. Furthermore, significant increase in the number of OPCs (oligodendroglial progenitor cells) was found in the SVZ of aTf-treated brains compared with H/I S. Specifically, there was a rise in cells positive for OPC markers, i.e. PDGFRα and SHH(+) cells, with a decrease in cleaved-caspase-3(+) cells compared with H/I S. Additionally, neurospheres from aTf-treated rats were bigger in size and produced more O4/MBP(+) cells. Our findings indicate a role for aTf as a potential inducer of OLs in neonatal rat brain in acute demyelination caused by H/I and a contribution to the differentiation/maturation of OLs and survival/migration of SVZ progenitors after demyelination in vivo.