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

Atoh8 in Development and Disease

Atoh8 belongs to a large superfamily of transcriptional regulators called basic helix-loop-helix (bHLH) proteins. bHLH proteins have been identified in a wide range of organisms from yeast to humans. The members of this special group of transcription factors were found to be involved not only in embryonic development but also in disease initiation and its progression. Given their importance in several fundamental processes, the translation, subcellular location and turnover of bHLH proteins is tightly regulated. Alterations in the expression of bHLH proteins have been associated with multiple diseases also in context with Atoh8 which seems to unfold its functions as both transcriptional activator and repressor. Like many other bHLH transcription factors, so far, Atoh8 has also been observed to be involved in both embryonic development and carcinogenesis where it mainly acts as tumor suppressor. This review summarizes our current understanding of Atoh8 structure, function and regulation and its complex and partially controversial involvement in development and disease.

Inactivating NHLH2 Variants Cause Idiopathic Hypogonadotropic Hypogonadism and Obesity in Humans

Metabolism has a role in determining the time of pubertal development and fertility. Nonetheless, molecular/cellular pathways linking metabolism/body weight to puberty/reproduction are unknown. The KNDy (Kisspeptin/Neurokinin B/Dynorphin) neurons in the arcuate (ARC) nucleus of the hypothalamus constitute the GnRH (Gonadotrophin-releasing hormone) pulse generator. We previously created a mouse model with a whole-body targeted deletion of nescient helix-loop-helix 2 (Nhlh2; N2KO), a class II member of the basic helix-loop-helix (bHLH) family of transcription factors. As this mouse model features pubertal failure and late-onset obesity, we wanted to study whether NHLH2 represents a candidate molecule to link metabolism and puberty in the hypothalamus. Exome sequencing of a large Idiopathic Hypogonadotropic Hypogonadism (IHH) cohort revealed obese patients with rare sequence variants in NHLH2, which were characterized by in silico protein analysis, chromatin immunoprecipitation, and luciferase reporter assays. In vitro heterologous expression studies demonstrated that the variant p.R79C impairs Nhlh2 binding to the Mc4r promoter. Furthermore, p.R79C and other variants show impaired transactivation of the human KISS1 promoter. These are the first inactivating human variants that support NHLH2’s critical role in human puberty and body weight control. Failure to carry out this function results in the absence of pubertal development and late-onset obesity in humans.

Phylogenetic analysis of phytochrome A gene from Lablab purpureus (L.) Sweet

Background Phytochromes are the best characterized photoreceptors that perceive Red (R)/Far-Red (FR) signals and mediate key developmental responses in plants. It is well established that photoperiodic control of flowering is regulated by PHY A (phytochrome A) gene. So far, the members of PHY A gene family remains unexplored in Lablab purpureus, and therefore, their functions are still not deciphered. PHYA3 is the homologue of phytochrome A and known to be involved in dominant suppression of flowering under long day conditions by downregulating florigens in Glycine max. The present study is the first effort to identify and characterize any photoreceptor gene (PHYA3, in this study) in Lablab purpureus and decipher its phylogeny with related legumes. Results PHYA3 was amplified in Lablab purpureus cv GNIB-21 (photo-insensitive and determinate) by utilizing primers designed from GmPHYA3 locus of Glycine max. This study was successful in partially characterizing PHYA3 in Lablab purpureus (LprPHYA3) which is 2 kb longer and belongs to exon 1 region of PHYA3 gene. Phylogenetic analysis of the nucleotide and protein sequences of PHYA genes through MEGA X delineated the conservation and evolution of Lablab purpureus PHYA3 (LprPHYA3) probably from PHYA genes of Vigna unguiculata, Glycine max and Vigna angularis. A conserved basic helix-loop-helix motif bHLH69 was predicted having DNA binding property. Domain analysis of GmPHYA protein and predicted partial protein sequence corresponding to exon-1 of LprPHYA3 revealed the presence of conserved domains (GAF and PAS domains) in Lablab purpureus similar to Glycine max. Conclusion Partial characterization of LprPHYA3 would facilitate the identification of complete gene in Lablab purpureus utilizing sequence information from phylogenetically related species of Fabaceae. This would allow screening of allelic variants for LprPHYA3 locus and their role in photoperiod responsive flowering. The present study could aid in modulating photoperiod responsive flowering in Lablab purpureus and other related legumes in near future through genome editing.

Homologous BHLH transcription factors induce distinct deformations of torsionally-stressed DNA: a potential transcription regulation mechanism.

Changing torsional restraints on DNA is essential for the regulation of transcription. Torsional stress, introduced by RNA polymerase, can propagate along chromatin facilitating topological transitions and modulating the specific binding of transcription factors (TFs) to DNA. Despite the importance, the mechanistic details on how torsional stress impacts the TFs-DNA complexation remain scarce. Herein we address the impact of torsional stress on DNA complexation with homologous human basic-helix-loop-helix (BHLH) hetero- and homodimers: MycMax, MadMax, and MaxMax. The three TF dimers exhibit specificity towards the same DNA consensus sequences, the E-box response element, while regulating different transcriptional pathways. Using microseconds-long atomistic molecular dynamics simulations together with the torsional restraint that controls DNA total helical twist, we gradually over- and underwind naked and complexed DNA to a maximum of ±5°/b.p. step. We observe that the binding of the BHLH dimers results in a similar increase in DNA torsional rigidity. However, under torsional stress the BHLH dimers induce distinct DNA deformations, characterised by changes in DNA grooves geometry and a significant asymmetric DNA bending. Supported by bioinformatics analyses, our data suggest that torsional stress may contribute to the execution of differential transcriptional programs of the homologous TFs by modulating their collaborative interactions.

HPVE6-USP46 Mediated Cdt2 Stabilization Reduces Set8 Mediated H4K20-Methylation to Induce Gene Expression Changes

E6 from high-risk strains of HPV is well known to transform cells by deregulating p53. We reported that in HPV transformed cell-lines E6 from high-risk HPV can recruit the USP46 deubiquitinase to substrates such as Cdt2 and stabilize the latter, and that USP46 is important for growth of HPV induced tumors in xenografts. Here we show that in cervical cancer biopsies the stabilization of Cdt2 in the HPV-induced cancers leads to the decrease of a CRL4-Cdt2 substrate, the histone H4K20 mono-methyltransferase Set8, and decrease in H4K20me1 or H4K20me3 that can be detected by immunohistochemistry. In HPV-transformed cancer cell lines in vitro, knockdown of E6 decreases Cdt2 and increases Set8. Co-knockdown of Set8 shows that some of the gene expression changes produced by E6 knockdown is due to the increase of Set8. EGFR and EGFR regulated genes were identified in this set of genes. Turning to the mechanism by which E6 stabilizes Cdt2, we find that a purified E6:USP46 complex has significantly more de-ubiquitinase activity in vitro than USP46 alone, demonstrating that E6 can directly interact with USP46 in the absence of other proteins and that it can substitute for the known activators of USP46, UAF1 and WDR20. Deletion mapping of Cdt2 shows that there are three discrete, but redundant, parts of the substrate that are essential for stabilization by E6: USP46. The helix–loop–helix region or the WD40 repeat driven beta-propeller structure of Cdt2 are dispensable for the stabilization implying that interaction with DDB1 (and the rest of the CRL4 complex) or with the substrate of the CRL4-Cdt2 E3 ligase is not necessary for E6:USP46 to interact with and stabilize Cdt2. The identification of 50 amino acid stretches in the 731 amino acid Cdt2 protein as being important for the stabilization by E6 underlines the specificity of the process. In summary, E6 activates the deubiquitinase activity of USP46, stabilizes Cdt2 utilizing multiple sites on Cdt2, and leads to degradation of Set8 and changes in gene-expression in HPV-transformed cells.

Transcription factor-like 5 is a potential DNA/RNA-binding protein essential for maintaining male fertility in mice

Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, but the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion since the heterozygous males (Tcfl5+/-) were infertile. We did; however, observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/- mice. Mechanistically, we demonstrated that TCFL5 transcriptionally and post-transcriptionally regulated a set of genes participating in male germ cell development via TCFL5 ChIP-DNA and eCLIP-RNA high-throughput sequencing. We also identified a known RBP, FXR1 as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and acts as a dual-function protein that mediates DNA and RNA to regulate spermatogenesis.

Identification of Abscisic Acid-Dependent Phosphorylated Basic Helix-Loop-Helix Transcription Factors in Guard Cells of Vicia faba by Mass Spectrometry

An unknown 61 kDa protein is phosphorylated by abscisic acid (ABA)-activated protein kinase in response to ABA and binds to 14-3-3 protein in a phosphorylation-dependent manner in guard-cell protoplasts (GCPs) from Vicia faba. Subsequently, ABA-dependent phosphorylated proteins were identified as basic helix–loop–helix transcription factors, named ABA-responsive kinase substrates (AKSs) in GCPs from Arabidopsis thaliana. However, whether the 61 kDa protein in Vicia GCPs is an AKS is unclear. We performed immunoprecipitation of ABA-treated Vicia GCPs using anti-14-3-3 protein antibodies and identified several AKS isoforms in V. faba (VfAKSs) by mass spectrometry. The 61 kDa protein was identified as VfAKS1. Phosphoproteomic analysis revealed that VfAKSs are phosphorylated at Ser residues, which are important for 14-3-3 protein binding and monomerisation, in response to ABA in GCPs. Orthologs of AtABCG40, an ABA importer in guard cells, and CHC1, a clathrin heavy chain and a regulator of stomatal movement, also co-immunoprecipitated with 14-3-3 protein from guard cells.

A combined transcriptional and dynamic roadmap of single human pancreatic endocrine progenitors reveals proliferative capacity and differentiation continuum

Basic helix-loop-helix genes, particularly proneural genes, are well-described triggers of cell differentiation, yet limited information exists on their dynamics, notably in human development. Here, we focus on Neurogenin 3 (NEUROG3), which is crucial for pancreatic endocrine lineage initiation. Using a double reporter to monitor endogenous NEUROG3 transcription and protein expression in single cells in 2D and 3D models of human pancreas development, we show peaks of expression for the RNA and protein at 22 and 11 hours respectively, approximately two-fold slower than in mice, and remarkable heterogeneity in peak expression levels all triggering differentiation. We also reveal that some human endocrine progenitors proliferate once, mainly at the onset of differentiation, rather than forming a subpopulation with sustained proliferation. Using reporter index-sorted single-cell RNA-seq data, we statistically map transcriptome to dynamic behaviors of cells in live imaging and uncover transcriptional states associated with variations in motility as NEUROG3 levels change, a method applicable to other contexts.