Genes Encoding Transcription Factors Research Articles

CRISPR-Cas9-mediated enhancement of Beauveria bassiana virulence with overproduction of oosporein

Biocontrol agents play a pivotal role in managing pests and contribute to sustainable agriculture. Recent advancements in genetic engineering can facilitate the development of entomopathogenic fungi with desired traits to enhance biocontrol efficacy. In this study, a CRISPR-Cas9 ribonucleoprotein system was utilized to genetically improve the virulence of Beauveria bassiana, a broad-spectrum insect pathogen used in biocontrol of arthropod pests worldwide. CRISPR-Cas9-based disruption of the transcription factor-encoding gene Bbsmr1 led to derepression of the oosporein biosynthetic gene cluster resulting in overproduction of the red-pigmented dibenzoquinone oosporein involved in host immune evasion, thus increasing fungal virulence. Mutants defective for Bbsmr1 displayed a remarkable enhanced insecticidal activity by reducing lethal times and concentrations, while concomitantly presenting negligible or minor pleiotropic effects. In addition, these mutants displayed faster germination on the insect cuticle which correlated with higher density of free-floating blastospores in the hemolymph and accelerated mortality of the host. These findings emphasize the utility of genetic engineering in developing enhanced fungal biocontrol agents with customized phenotypic traits, and provide an efficient and versatile genetic transformation tool for application in other beneficial entomopathogenic fungi.

Characterization and expression analysis of the B3 gene family during seed development in Akebia trifoliata

BackgroundB3 genes encode transcription factors that play key roles in plant growth and development. However, the specific B3 genes involved in the seed development of Akebia trifoliata remain unexplored.ResultsA total of 72 AktB3 genes were identified and classified into five subfamilies (ARF, LAV, RAV, HSI, and REM) based on phylogenetic analysis. These 72 AktB3 genes were unevenly distributed across 16 chromosomes. Collinear analysis indicated that segmental duplication has played a significant role in the evolution of AktB3 genes, and underwent purification selection. Expression profiling across seed development stages revealed that seven AktB3 genes, particularly from the LAV subfamily (AktABI3, AktFUS3, AktLEC2), were up-regulated at 70 days after flowering (DAF). Notably, the expression of oleosin exhibited a strong positive correlation with LAV subfamily genes, highlighting their potential roles as hub genes in lipid metabolism and seed development. Yeast two-hybrid (Y2H) and yeast one-hybrid (Y1H) experiments confirmed that AktFUS3-1, AktFUS3-2, and AktLEC2 form protein complexes and individually bind to the AktOLE1 promoter, thereby regulating downstream gene expression. These results provide direct evidence of the cooperative role these transcription factors play in controlling lipid metabolism, particularly related to oleosin proteins. Additionally, miRNA sequencing across three seed developmental stages identified 591 miRNAs and 1,673 target gene pairs. A total of 23 AktB3 genes were predicted to be targets of 20 miRNAs, with 11 miRNAs specifically targeting the ARF subfamily genes. Particularly, miR160-x, miR160-z, and miR167-z were predicted to target ARF subfamily genes, potentially influencing seed development. Moreover, the miRNA-B3 regulatory modules, especially involving ARF genes and miR160/167, require further study to clarify their roles in seed development.ConclusionsThese findings contribute valuable resources for future functional studies of the molecular regulatory networks governing seed development in A. trifoliata.

MYB168 and WRKY20 transcription factors synergistically regulate lignin monomer synthesis during potato tuber wound healing.

Lignin is a critical component of the closing layer of the potato (Solanum tuberosum L.) tuber during healing; however, the molecular mechanism of its formation remains poorly understood. To elucidate the molecular mechanism of tuber healing, we screened the genes encoding transcription factors that regulate lignin synthesis(StMYB24/49/105/144/168, StWRKY19/20/22/23/34) and the key genes involved in lignin monomer synthesis (PHENYLALANINE AMMONIA LYASE 5 (StPAL5) and CINNAMYL ALCOHOL DEHYDROGENASE 14 (StCAD14)) for induced expression after wounding using transcriptome data. DLR, Y1H, EMSA, and ChIP-qPCR assays revealed that StMYB168 could bind directly to the StPAL5 and StCAD14 promoters to activate their expression and that StWRKY20 enhanced this regulation with a synergistic effect. Y2H, BiFC, and Co-IP assays showed that StMYB168 interacted with StWRKY20 to form a MYB-WRKY complex. Furthermore, transient overexpression of StMYB168 and StWRKY20 in Nicotiana benthamiana leaves upregulated the expression of NbPAL and NbCAD10 and promoted lignin accumulation in the leaves. In addition, overexpression of StWRKY20 and StMYB168 together resulted in higher expression levels of NbPAL and NbCAD10 and higher levels of lignin monomer and total lignin. In contrast, silencing of StMYB168 and StWRKY20 in potato significantly reduced the lignin content of wounded tubers. In conclusion, StMYB168 and StWRKY20 are important regulators of lignin biosynthesis in potato tubers during healing and can positively regulate lignin biosynthesis by forming a complex. The elucidation of this regulatory module provides information on the regulatory mechanism of lignin monomer synthesis in wounded tubers at the transcriptional level.

Almost 40 years of studying homeobox genes in C. elegans.

Homeobox genes are among the most deeply conserved families of transcription factor-encoding genes. Following their discovery in Drosophila, homeobox genes arrived on the Caenorhabditis elegans stage with a vengeance. Between 1988 and 1990, just a few years after their initial discovery in flies and vertebrates, positional cloning and sequence-based searches showed that C. elegans contains HOX cluster genes, an apparent surprise given the simplicity and non-segmented body plan of the nematode, as well as many other non-clustered homeobox genes of all major subfamilies (e.g. LIM, POU, etc.). Not quite 40 years later, we have an exceptionally deep understanding of homeodomain protein expression and function in C. elegans, revealing their prevalent role in nervous system development. In this Spotlight, we provide a historical perspective and a non-comprehensive journey through the C. elegans homeobox field and discuss open questions and future directions.

Identification of genes associated with sex expression and sex determination in hemp (Cannabis sativa L.).

Dioecy in flowering plants has evolved independently many different times, and thus the genetic mechanisms underlying sex determination are diverse. In hemp (Cannabis sativa), sex is controlled by a pair of sex chromosomes (XX for females and XY for males). In an attempt to understand the molecular mechanism responsible for sex expression in hemp plants, we carried out RNA-Seq of male and female plants at different developmental stages. Using a pipeline involving differential gene expression analysis and weighted gene co-expression network analysis, we identified genes important for male and female flower development. We also demonstrate that sex-biased expression is already established at very early vegetative stages, before the onset of reproductive development, and several genes encoding transcription factors of the REM, bZIP and MADS family as candidate sex determination genes in hemp. Our findings demonstrate that the gene regulatory networks governing male and female development in hemp diverge already at a very early stage, leading to profound morphological differences in male and female hemp plants.

Choline chloride and N-allylglycine promote plant growth by increasing the efficiency of photosynthesis.

We previously reported that choline chloride and N-allylglycine stimulate photosynthesis in wheat protoplasts. Treatment of Arabidopsis thaliana and Brassica rapa plants with both compounds promoted growth and photosynthesis. To clarify the relationship between the enhancement of photosynthesis and increased growth, A. thaliana T87 cells, which show photosynthesis-dependent growth, and YG1 cells, which use sugar in the medium for growth, were treated with choline chloride or N-allylglycine. Only the T87 cells showed increased growth, suggesting that choline chloride and N-allylglycine promote growth by increasing photosynthetic activity. Transcriptome analysis using choline chloride and N-allylglycine-treated plants showed that the most abundant transcripts corresponded to photosynthetic electron transfer-related genes among the genes upregulated by both compounds. Furthermore, the compounds also upregulated genes encoding transcription factors that may control the expression of these photosynthetic genes. These results suggest that choline chloride and N-allylglycine promote photosynthesis through increased expression of photosynthetic electron transfer-related genes.

Gene identification for ocular congenital cranial motor neuron disorders using human sequencing, zebrafish screening, and protein binding microarrays.

To functionally evaluate novel human sequence-derived candidate genes and variants for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). Through exome and genome sequencing of a genetically unsolved human oCCDD cohort, we previously identified variants in 80 strong candidate genes. Here, we further prioritized a subset of these (43 human genes, 57 zebrafish genes) using a G0 CRISPR/Cas9-based knockout assay in zebrafish and generated F2 germline mutants for seventeen. We tested the functionality of variants of uncertain significance in known and novel candidate transcription factor-encoding genes through protein binding microarrays. We first demonstrated the feasibility of the G0 screen by targeting known oCCDD genes phox2a and mafba. 70-90% of gene-targeted G0 zebrafish embryos recapitulated germline homozygous null-equivalent phenotypes. Using this approach, we then identified three novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) with putative contributions to human and zebrafish cranial motor development. In addition, protein binding microarrays demonstrated reduced or abolished DNA binding of human variants of uncertain significance in known and novel sequence-derived transcription factors PHOX2A (p.(Trp137Cys)), MAFB (p.(Glu223Lys)), and OLIG2 (p.(Arg156Leu)). This study nominates three strong novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) and supports the functionality and putative pathogenicity of transcription factor candidate variants PHOX2A p.(Trp137Cys), MAFB p.(Glu223Lys), and OLIG2 p.(Arg156Leu). Our findings support that G0 loss-of-function screening in zebrafish can be coupled with human sequence analysis and protein binding microarrays to aid in prioritizing oCCDD candidate genes/variants.

Multi-Omics Analysis Revealed the rSNPs Potentially Involved in T2DM Pathogenic Mechanism and Metformin Response.

The goal of our study was to identify and assess the functionally significant SNPs with potentially important roles in the development of type 2 diabetes mellitus (T2DM) and/or their effect on individual response to antihyperglycemic medication with metformin. We applied a bioinformatics approach to identify the regulatory SNPs (rSNPs) associated with allele-asymmetric binding and expression events in our paired ChIP-seq and RNA-seq data for peripheral blood mononuclear cells (PBMCs) of nine healthy individuals. The rSNP outcomes were analyzed using public data from the GWAS (Genome-Wide Association Studies) and Genotype-Tissue Expression (GTEx). The differentially expressed genes (DEGs) between healthy and T2DM individuals (GSE221521), including metformin responders and non-responders (GSE153315), were searched for in GEO RNA-seq data. The DEGs harboring rSNPs were analyzed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). We identified 14,796 rSNPs in the promoters of 5132 genes of human PBMCs. We found 4280 rSNPs to associate with both phenotypic traits (GWAS) and expression quantitative trait loci (eQTLs) from GTEx. Between T2DM patients and controls, 3810 rSNPs were detected in the promoters of 1284 DEGs. Based on the protein-protein interaction (PPI) network, we identified 31 upregulated hub genes, including the genes involved in inflammation, obesity, and insulin resistance. The top-ranked 10 enriched KEGG pathways for these hubs included insulin, AMPK, and FoxO signaling pathways. Between metformin responders and non-responders, 367 rSNPs were found in the promoters of 131 DEGs. Genes encoding transcription factors and transcription regulators were the most widely represented group and many were shown to be involved in the T2DM pathogenesis. We have formed a list of human rSNPs that add functional interpretation to the T2DM-association signals identified in GWAS. The results suggest candidate causal regulatory variants for T2DM, with strong enrichment in the pathways related to glucose metabolism, inflammation, and the effects of metformin.

Transcriptional cascades during fasting amplify gluconeogenesis and instigate a secondary wave of ketogenic gene transcription.

During fasting, bodily homeostasis is maintained due to hepatic production of glucose (gluconeogenesis) and ketone bodies (ketogenesis). The main hormones governing hepatic fuel production are glucagon and glucocorticoids that initiate transcriptional programs aimed at supporting gluconeogenesis and ketogenesis. Using primary mouse hepatocytes as an exvivo model, we employed transcriptomic analysis (RNA-seq), genome-wide profiling of enhancer dynamics (ChIP-seq), perturbation experiments (inhibitors, shRNA), hepatic glucose production measurements and computational analyses. We found that in addition to the known metabolic genes transcriptionally induced by glucagon and glucocorticoids, these hormones induce a set of genes encoding transcription factors (TFs) thereby initiating transcriptional cascades. Upon activation by glucocorticoids, the glucocorticoid receptor (GR) induced the genes encoding two TFs: CCAAT/enhancer-binding protein beta (C/EBPβ) and peroxisome proliferator-activated receptor alpha (PPARα). We found that the GR-C/EBPβ cascade mainly serves as a secondary amplifier of primary hormone-induced gene programs. C/EBPβ augmented gluconeogenic gene expression and hepatic glucose production. Conversely, the GR-PPARα cascade initiated a secondary transcriptional wave of genes supporting ketogenesis. The cascade led to synergistic induction of ketogenic genes which is dependent on protein synthesis. Genome-wide analysis of enhancer dynamics revealed numerous enhancers activated by the GR-PPARα cascade. These enhancers were proximal to ketogenic genes, enriched for the PPARα response element and showed increased PPARα binding. This study reveals abundant transcriptional cascades occurring during fasting. These cascades serve two separated purposes: the amplification of the gluconeogenic transcriptional program and the induction of a gene program aimed at enhancing ketogenesis.

Loss-of-function of the Zinc Finger Homeobox 4 (ZFHX4) gene underlies a neurodevelopmental disorder.

8q21.11 microdeletions encompassing the gene encoding transcription factor ZFHX4, have previously been associated by us with a syndromic form of intellectual disability, hypotonia, decreased balance and hearing loss. Here, we report on 57 individuals, 52 probands and 5 affected family members, with protein truncating variants (n=36), (micro)deletions (n=20) or an inversion (n=1) affecting ZFHX4 with variable developmental delay and intellectual disability, distinctive facial characteristics, morphological abnormalities of the central nervous system, behavioral alterations, short stature, hypotonia, and occasionally cleft palate and anterior segment dysgenesis. The phenotypes associated with 8q21.11 microdeletions and ZFHX4 intragenic loss-of-function variants largely overlap, identifying ZFHX4 as the main driver for the microdeletion syndrome, although leukocyte-derived DNA shows a mild common methylation profile for (micro)deletions only. We identify ZFHX4 as a transcription factor that is increasingly expressed during human brain development and neuronal differentiation. Furthermore, ZFHX4 interacting factors identified via IP-MS in neural progenitor cells, suggest an important role for ZFHX4 in cellular and developmental pathways, especially during histone modifications, cytosolic transport and development. Additionally, using CUT&RUN, we observed that ZFHX4 binds with the promoter regions of genes with crucial roles in embryonic, neuron and axon development. Since loss-of-function variants in ZFHX4 are found with consistent dysmorphic facial features, we investigated whether the disruption of zfhx4 causes craniofacial abnormalities in zebrafish. First-generation (F0) zfhx4 crispant zebrafish, (mosaic) mutant for zfhx4 loss-of-function variants, have significantly shorter Meckel's cartilages and smaller ethmoid plates compared to control zebrafish. Furthermore, behavioral assays show a decreased movement frequency in the zfhx4 crispant zebrafish in comparison with control zebrafish larvae. Although further research is needed, our in vivo work suggests a role for zfhx4 in facial skeleton patterning, palatal development and behavior.

Analysis of the Spatholobus suberectus full-length transcriptome identified an R2R3-MYB transcription factor-encoding gene SsMYB158 that regulates flavonoid biosynthesis

Spatholobus suberectus Dunn (Leguminosae) has been used for medicinal purposes for a long period. Flavonoids are the major bioactive components of S. suberectus. However, there is still limited knowledge of the exact method via which transcription factors (TFs) regulate flavonoid biosynthesis. The full-length transcriptome of S. suberectus was analyzed using SMRT sequencing; 61,548 transcripts were identified, including 12,311 new gene loci, 53,336 novel transcripts, 44,636 simple sequence repeats, 36,414 complete coding sequences, 871 long non-coding RNAs and 6781 TFs. The SsMYB158 TF, which is associated with flavonoid biosynthesis, belongs to the R2R3-MYB class and is localized subcellularly to the nucleus. The overexpression of SsMYB158 in Nicotiana benthamiana and the transient overexpression of SsMYB158 in S. suberectus resulted in a substantial enhancement in both flavonoids and catechin levels. In addition, there was a remarkable upregulation in the expression of essential enzyme-coding genes associated with the flavonoid biosynthesis pathways. Our study revealed SsMYB158 as a critical regulator of flavonoid biosynthesis in S. suberectus and laying the foundation for its molecular breeding.

Transcriptomic profiling of dynamic alternative splicing during the early response to Ralstonia solanacearum infection in tobacco roots

The pathogenic bacterium, Ralstonia solanacearum, causes bacterial wilt disease in many crops, which leads to significant yield losses worldwide. Although genes associated with resistance to this pathogen have been isolated and characterized in crops, the molecular mechanisms underlying the plant–pathogen interactions remain to be elucidated. Here, we performed a comparative transcriptional profiling analysis of tobacco (Nicotiana tabacum) cultivars C048 (susceptible) and C244 (resistant) in response to R. solanacearum infection. We found that the number of down- and up-regulated genes increased dramatically 3 h post inoculation (hpi), peaked 24 hpi, and then decreased 48 and 72 hpi, representing a “transcriptomic shock”. Of these genes, those associated with biotic and abiotic stresses and secondary metabolism were up-regulated, whereas those associated with primary metabolism were down-regulated. Alternative splicing (AS) modulates root defense against R. solanacearum by fine-tuning gene expression during the transcriptomic responses to pathogen invasion. The numbers of skipped exon (SE) and mutually exclusive exon (MXE) type AS events were reduced by approximately 60–80% in roots 9–72 hpi compared to those occurring 0–3 hpi. On the contrary, the number of differential alternative splicing (DAS) events showing a change in isoform ratio between samples increased, and most of them were associated with the down-regulation of corresponding gene expression. In addition, genes encoding transcription factors and leucine-rich repeat domain proteins that showed changes in both expression level and AS profile during pathogen infection were identified. Our study offers novel insights into the mechanisms underlying the transcriptional and post-transcriptional regulation of the tobacco response to R. solanacearum infection and will benefit the molecular breeding of pathogen-resistant tobacco in the future.

Single-cell landscape of alternative polyadenylation in human lymphoid hematopoiesis.

Alternative polyadenylation (APA) is an essential post-transcriptional process that produces mature mRNA isoforms by regulating the usage of polyadenylation sites (PASs). APA is involved in lymphocyte activation; however, its role throughout the entire differentiation trajectory remains elusive. Here, we analyzed single-cell 3'-end transcriptome data from healthy subjects to construct a dynamic-APA landscape from hematopoietic stem and progenitor cells (HSPCs) to terminally differentiated lymphocytes. This analysis covered 19973 cells of 12 clusters from five lineages (B cells, CD4+ T cells, CD8+ T cells, natural killer cells, and plasmacytoid dendritic cells). A total of 2364 genes exhibited differential 3'UTR PAS usage, and 3021 genes displayed differential intronic cleavage during lymphoid differentiation. We observed a global trend of 3'UTR shortening during lymphoid differentiation. Nevertheless, specific events of both 3'UTR shortening and lengthening were also identified within each cluster. The APA patterns delineated three differentiation stages: HSPCs, precursor cells, and mature cells. Moreover, we demonstrated that the conversion of naïve T cells to memory T cells was accompanied by dynamic APA in transcription factor-encoding genes (TCF7 and NFATC2IP), immune function-related genes (BCL2, CD5, CD28, GOLT1B, and TMEM59), and protein ubiquitination-related genes (UBE2G1, YPEL5, and SUMO3). These findings expand our understanding of the underlying molecular mechanisms of APA and facilitate studies on the regulatory role of APA in lymphoid hematopoiesis.

A single gene mutation underpins metabolic adaptation and acquisition of filamentous competence in the emerging fungal pathogen Candida auris.

Filamentous cell growth is a vital property of fungal pathogens. The mechanisms of filamentation in the emerging multidrug-resistant fungal pathogen Candida auris are poorly understood. Here, we show that exposure of C. auris to glycerol triggers a rod-like filamentation-competent (RL-FC) phenotype, which forms elongated filamentous cells after a prolonged culture period. Whole-genome sequencing analysis reveals that all RL-FC isolates harbor a mutation in the C2H2 zinc finger transcription factor-encoding gene GFC1 (Gfc1 variants). Deletion of GFC1 leads to an RL-FC phenotype similar to that observed in Gfc1 variants. We further demonstrate that GFC1 mutation causes enhanced fatty acid β-oxidation metabolism and thereby promotes RL-FC/filamentous growth. This regulation is achieved through a Multiple Carbon source Utilizer (Mcu1)-dependent mechanism. Interestingly, both the evolved RL-FC isolates and the gfc1Δ mutant exhibit an enhanced ability to colonize the skin. Our results reveal that glycerol-mediated GFC1 mutations are beneficial during C. auris skin colonization and infection.

ZmWRKY30 modulates drought tolerance in maize by influencing myo-inositol and reactive oxygen species homeostasis.

Maize (Zea mays L.) is an important food crop with a wide range of uses in both industry and agriculture. Drought stress during its growth cycle can greatly reduce maize crop yield and quality. However, the molecular mechanisms underlying maize responses to drought stress remain unclear. In this work, a WRKY transcription factor-encoding gene, ZmWRKY30, from drought-treated maize leaves was screened out and characterized. ZmWRKY30 gene expression was induced by dehydration treatments. The ZmWRKY30 protein localized to the nucleus and displayed transactivation activity in yeast. Compared with wild-type (WT) plants, Arabidopsis lines overexpressing ZmWRKY30 exhibited a significantly enhanced drought stress tolerance, as evidenced by the improved survival rate, increased antioxidant enzyme activity by superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), elevated proline content, and reduced lipid peroxidation recorded after drought stress treatment. In contrast, the mutator (Mu)-interrupted ZmWRKY30 homozygous mutant (zmwrky30) was more sensitive to drought stress than its null segregant (NS), characterized by the decreased survival rate, reduced antioxidant enzyme activity (SOD, POD, and CAT) and proline content, as well as increased malondialdehyde accumulation. RNA-Seq analysis further revealed that, under drought conditions, the knockout of the ZmWRKY30 gene in maize affected the expression of genes involved in reactive oxygen species (ROS), proline, and myo-inositol metabolism. Meanwhile, the zmwrky30 mutant exhibited significant downregulation of myo-inositol content in leaves under drought stress. Combined, our results suggest that ZmWRKY30 positively regulates maize responses to water scarcity. This work provides potential target genes for the breeding of drought-tolerant maize.

Metabolomic and transcriptomic analyses of monoterpene biosynthesis in Muscat and Neutral grape hybrids

Monoterpenes are typical aroma components of muscat grape (Vitis vinifera L.) cultivars. However, differences in the molecular mechanism of monoterpene biosynthesis between Muscat and non-Muscat grapes remain unclear. Here, comprehensive transcriptomic and metabolomic analyses of two F1 hybrids with different aroma profiles obtained from crosses between 'Muscat Hamburg' and 'Red Globe' grapes were performed to examine the unique monoterpene accumulation pattern and related candidate genes. The concentrations of most monoterpenes differed at various times, in the hybrid progeny, increasing from the pre-veraison stage to the veraison stage, followed by a decreasing trend from veraison to maturity. Within the F1 generation, the concentrations of accumulated monoterpenes were significantly higher in Muscat grapes than in neutral grapes. Furthermore, linalool, α-terpineol, and l-α-terpineol may be important odorants affecting the floral characteristics of Muscat grapes due to their high odor activity values. Transcriptional analysis identified three VvDXS (LOC100249323, LOC100268079, and LOC100247834), 1 VvHDS (LOC100257071), one VvMVK (LOC100251148), one VvHMGR (LOC100265082), and one VvGGPPS (LOC100257234) genes encoding proteins involved in the MEP and MVA pathways. Eleven TPS genes potentially involved in monoterpene regulation were also identified. Among them, VvTPS (LOC100853562) showed consistency with the accumulation of monoterpenes and had a high expression level; MYB (LOC100255488), GATA (LOC100262474), and ERF (LOC100255943, LOC100260401) were selected as candidate genes encoding transcription factors. The findings in this study provide new insights for further functional validation and investigation of genes related to monoterpene synthesis in Muscat aroma grapes.

Deletion of Trps1 regulatory elements recapitulates postnatal hip joint abnormalities and growth retardation of Trichorhinophalangeal syndrome in mice.

Trichorhinophalangeal syndrome (TRPS) is a genetic disorder caused by point mutations or deletions in the gene-encoding transcription factor TRPS1. TRPS patients display a range of skeletal dysplasias, including reduced jaw size, short stature, and a cone-shaped digit epiphysis. Certain TRPS patients experience early onset coxarthrosis that leads to a devastating drop in their daily activities. The etiologies of congenital skeletal abnormalities of TRPS were revealed through the analysis of Trps1 mutant mouse strains. However, early postnatal lethality in Trps1 knockout mice has hampered the study of postnatal TRPS pathology. Here, through epigenomic analysis we identified two previously uncharacterized candidate gene regulatory regions in the first intron of Trps1. We deleted these regions, either individually or simultaneously, and examined their effects on skeletal morphogenesis. Animals that were deleted individually for either region displayed only modest phenotypes. In contrast, the Trps1Δint/Δint mouse strain with simultaneous deletion of both genomic regions exhibit postnatal growth retardation. This strain displayed delayed secondary ossification center formation in the long bones and misshaped hip joint development that resulted in acetabular dysplasia. Reducing one allele of the Trps1 gene in Trps1Δint mice resulted in medial patellar dislocation that has been observed in some patients with TRPS. Our novel Trps1 hypomorphic strain recapitulates many postnatal pathologies observed in human TRPS patients, thus positioning this strain as a useful animal model to study postnatal TRPS pathogenesis. Our observations also suggest that Trps1 gene expression is regulated through several regulatory elements, thus guaranteeing robust expression maintenance in skeletal cells.

Ectopic Pitx2 expression and sinoatrial node dysfunction in mice recapitulating a novel cardiac syndrome identified in unrelated families with deletions in a gene desert on chromosome 4q25

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Research Councin/NWO Introduction Several unrelated families presenting with a complex, heterogeneous cardiac syndrome including sinoatrial node dysfunction and atrial fibrillation were found to share overlapping deletions in a 1.5 Megabase pair gene desert on chromosome 4q25. These overlapping regions contain evolutionarily conserved CTCF binding sites that modulate the separation of topologically associating domains containing the transcription factor-encoding gene PITX2 and noncoding RNA genes. Aim To understand mechanistically how the deletion in the gene desert causes sinoatrial node dysfunction and atrial arrhythmias. Methods Mice lacking the orthologue of the minimally overlapping genomic region deleted in affected patients were generated. Electrophysiology, morphology, tissue composition, and transcriptomes were analysed in this mouse model. Results These mice recapitulated major morpho-functional features of the syndrome, including sinoatrial node dysfunction and atrial arrhythmogenesis . Out of the 15 genes flanking the gene desert, only Pitx2 expression was deregulated in the sinoatrial node. During development, cardiac Pitx2c expression is normally restricted to the left side where it drives left sinus venosus and left atrial morphogenesis and prevents the formation of a left-sided sinoatrial node. The PITX2 locus has also been associated with atrial fibrillation in GWAS. Immunohistochemistry together with 3D volume analysis showed that Pitx2c was ectopically expressed in half of the pacemaker cardiomyocytes of the developing and postnatal sinoatrial node while its expression in the left atrium was maintained. Key transcription factors that drive pacemaker cell differentiation, Tbx3, Isl1 and Shox2, were lost where Pitx2c was ectopically expressed. Moreover, a conserved long non-coding RNA (Playrr) previously linked to Pitx2 regulation in the developing dorsal mesentery, was downregulated in the sinoatrial node of homozygous mutants. The downregulation of the pacemaker gene program additionally coincided with the activation of the atrial myocardial gene program in half of the pacemaker cardiomyocytes. Conclusion We generated a mouse model of a novel human cardiac syndrome including sinoatrial node dysfunction associated with the deletion of critical CTCF binding sites in a gene desert on chromosome 4q25. This deletion induced ectopic Pitx2c expression in the sinoatrial node alongside a substantial loss of pacemaker cardiomyocyte identity and a gain in a working atrial myocardium-like phenotype in the sinoatrial node of affected mice.Loss of pacemaker cardiomyocyte identitySinoatrial node dysfunction in mutants

Comparative Transcriptome Analysis Revealed Candidate Gene Modules Involved in Salt Stress Response in Sweet Basil and Overexpression of ObWRKY16 and ObPAL2 Enhanced Salt Tolerance of Transgenic Arabidopsis.

Sweet basil (Ocimum basilicum L.) is an important aromatic plant with high edibility and economic value, widely distributed in many regions of the tropics including the south of China. In recent years, environmental problems, especially soil salinization, have seriously restricted the planting and spread of sweet basil. However, the molecular mechanism of the salt stress response in sweet basil is still largely unknown. In this study, seed germination, seedling growth, and chlorophyll synthesis in sweet basil were inhibited under salt stress conditions. Through comparative transcriptome analysis, the gene modules involved in the metabolic processes, oxidative response, phytohormone signaling, cytoskeleton, and photosynthesis were screened out. In addition, the landscape of transcription factors during salt treatment in sweet basil was displayed as well. Moreover, the overexpression of the WRKY transcription factor-encoding gene, ObWRKY16, and the phenylalanine ammonia-lyase-encoding gene, ObPAL2, enhanced the seed germination, seedling growth, and survival rate, respectively, of transgenic Arabidopsis, suggesting that they might be important candidates for the creation of salt-tolerant sweet basil cultivars. Our data enrich the study on salt responses in sweet basil and provide essential gene resources for genetic improvements in sweet basil in the future.

Genome-wide analysis of long noncoding RNAs as cis-acting regulators of transcription factor-encoding genes in IgA nephropathy.

IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis in the world, but the disease pathogenesis noncoding is yet to be elucidated. Previous studies have revealed regulatory functions for long noncoding RNA (lncRNA) in various diseases; however, the roles of lncRNA in IgAN and regulation of transcription factors (TFs) have been scarcely investigated. Renal tissue samples (n = 5) from patients with IgAN and control samples (n = 4) were collected and RNA sequencing (RNA-seq) was performed. Four software programs were employed for lncRNA prediction. GO (Gene Ontology)/KEGG (Kyoto Encyclopedia of Genes and Genomes) were employed for analysis of the identified differentially expressed genes (DEGs). A regulatory network model of DE lncRNA-TF-DEG was developed, and the levels of expression of key lncRNAs, TFs, and corresponding target genes were assessed using qRT-PCR and immunofluorescence. The current study identified 674 upregulated and 1,011 downregulated DE mRNAs and 260 upregulated and 232 downregulated DE lncRNAs in IgAN samples compared with control samples. The upregulated DE mRNAs showed enrichment in cell adhesion and collagen glial fiber organization pathways. The DE lncRNAs-DE mRNAs showing co-expression are associated with transmembrane transport. A novel regulatory network model of lncRNA-TF-DEG has been developed. This study identified seven TFs that are cis-regulated by 6 DE lncRNAs, and show co-expression with 132 DEGs (correlation coefficient ≥ 0.8, P ≤ 0.01), generating 158 pairs that showed co-expression. The lncRNAs NQO1-DT and RP5-1057120.6 were found to be highly expressed in IgAN samples. The TFs vitamin D Receptor (VDR) and NFAT5, along with their target genes were also aberrantly expressed. Key lncRNAs and TFs centrally associated with IgAN have been identified in this study. A regulatory network model of lncRNA-TF-mRNA was constructed. Further studies on the genes identified herewith could provide insight into the pathogenesis of IgAN.