Screening and Identification of Transcription Factors Potentially Regulating Foxl2 Expression in Chlamys farreri Ovary

Foxl2 is an evolutionarily conserved female sex gene, which is specifically expressed in the ovary and mainly involved in oogenesis and ovarian function maintenance. However, little is known about the mechanism that regulates Foxl2 specific expression during the ovary development. In the present study, we constructed the gonadal yeast one-hybrid (Y1H) library of Chlamysfarreri with ovaries and testes at different developmental stages using the Gateway technology. The library capacity was more than 1.36 × 107 CFU, and the length of the inserted fragment was 0.75 Kb~2 Kb, which fully met the demand of yeast library screening. The highly transcriptional activity promoter sequence of C. farreri Foxl2 (Cf-Foxl2) was determined at −1000~−616 bp by dual-luciferase reporter (DLR) assay and was used as bait to screen possible transcription factors from the Y1H library. Eleven candidate factors, including five unannotated factors, were selected based on Y1H as well as their expressional differences between ovaries and testes and were verified for the first time to be involved in the transcriptional regulation of Cf-Foxl2 by RT-qPCR and DLR. Our findings provided valuable data for further studying the specific regulation mechanism of Foxl2 in the ovary.

Differentially Expressed Genes Related to Flowering Transition between Once- and Continuous-Flowering Roses

Roses are the most important cut flower crops and widely used woody ornamental plants in gardens throughout the world, and they are model plants for studying the continuous-flowering trait of woody plants. To analyze the molecular regulation mechanism of continuous flowering, comparative transcriptome data of once- and continuous-flowering roses in our previous study were used to conduct weighted gene co-expression network analysis (WGCNA) to obtain the candidate genes related to flowering transitions. The expression patterns of candidate genes at different developmental stages between Rosa chinensis “Old Blush” (continuous-flowering cultivar) and R. “Huan Die” (once-flowering cultivar) were investigated, and the relationship of the key gene with the endogenous hormone was analyzed. The results showed that the expression trends of VIN3-LIKE 1 (VIL1), FRIGIDA- LIKE 3 (FRI3), APETALA 2- LIKE (AP2-like) and CONSTANS-LIKE 2 (CO-like 2) genes were significantly different between “Old Blush” and “Huan Die”, and the expression trends of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and CO-like 2 were consistent in the flowering transition of “Old Blush” under different environments. The changes in cytokinin and gibberellic acid (GA3) content were different in the two rose cultivars. The overall change trend of the abscisic acid and GA3 in the flowering transition of “Old Blush” under different environments was consistent. The promoter sequence of CO-like 2 contained a P-box element associated with gibberellin response, as well as binding sites for transcription factors. In a word, we found CO-like 2 associated with continuous flowering and some factors that may synergistically regulate continuous flowering. The results provided a reference for elucidating the molecular regulatory mechanisms of continuous-flowering traits in roses.

Prediction and Experimental Verification of a Hierarchical Transcription Factor Regulatory Network of Porcine Myoglobin (Mb)

Myoglobin is a key chemical component that determines meat’s color and affects consumers’ purchase intentions. In this work, we firstly identified the promoter sequence of the Mb gene from the primary assembly of high-throughput genome sequencing in pigs, and predicted its potential transcription factors by LASAGNA. Through the data mining of the mRNA expression profile of longissimus dorsi muscle of different pig breeds, we constructed a hierarchical interplay network of Mb-TFs (Myoglobin-Transcription Factors), consisting of 16 adaptive transcription factors and 23 secondary transcription factors. The verification of gene expression in longissimus dorsi muscle showed that the Mb mRNA and encoded protein were significantly (p < 0.05) more abundant in Bama pigs than Yorkshire pigs. The qRT-PCR (Real-Time Quantitative Reverse Transcription PCR) validation on genes of the Mb-TFs network showed that FOS, STAT3, STAT1, NEFL21, NFE2L2 and MAFB were significant positive regulatory core transcription factors of Mb-TFs network in Bama pigs, whereas ATF3 was the secondary transcription factor most responsible for the activation of the above transcription factors. Our study provides a new strategy to unravel the mechanism of pork color formation, based on public transcriptome and genome data analysis.

Transcriptional and Epigenetic Bioinformatic Analysis of Claudin-9 Regulation in Gastric Cancer

Gastric cancer is a heterogeneous disease that represents 5% to 10% of all new cancer cases worldwide. Advances in histological diagnosis and the discovery of new genes have admitted new genomic classifications. Nevertheless, the bioinformatic analysis of gastric cancer databases has favored the detection of specific differentially expressed genes with biological significance. Claudins, a family of proteins involved in tight junction physiology, have emerged as the key regulators of cellular processes, such as growth, proliferation, and migration, associated with cancer progression. The expression of Claudin-9 in the gastric cancer tissue has been linked to poor prognosis, however, its transcriptional and epigenetic regulations demand a more comprehensive analysis. Using the neural network promoter prediction, TransFact, Uniprot-KB, Expasy-SOPMA, protein data bank, proteomics DB, Interpro, BioGRID, String, and the FASTA protein sequence databases and software, we found the following: (1) the promoter sequence has an unconventional structure, including different transcriptional regulation elements distributed throughout it, (2) GATA 4, GATA 6, and KLF5 are the key regulators of Claudin-9 expression, (3) Oct1, NF-κB, AP-1, c-Ets-1, and HNF-3β have the higher binding affinity to the CLDN9 promoter, (4) Claudin-9 interacts with cell differentiation and development proteins, (5) CLDN9 is highly methylated, and (6) Claudin-9 expression is associated with poor survival. In conclusion, Claudin-9 is a protein that should be considered a diagnostic marker as its gene promoter region binds to the transcription factors associated with the deregulation of cell control, enhanced cell proliferation, and metastasis.

Using Machine Learning Approaches to Predict Target Gene Expression in Rice T-DNA Insertional Mutants

To change the expression of the flanking genes by inserting T-DNA into the genome is commonly used in rice functional gene research. However, whether the expression of a gene of interest is enhanced must be validated experimentally. Consequently, to improve the efficiency of screening activated genes, we established a model to predict gene expression in T-DNA mutants through machine learning methods. We gathered experimental datasets consisting of gene expression data in T-DNA mutants and captured the PROMOTER and MIDDLE sequences for encoding. In first-layer models, support vector machine (SVM) models were constructed with nine features consisting of information about biological function and local and global sequences. Feature encoding based on the PROMOTER sequence was weighted by logistic regression. The second-layer models integrated 16 first-layer models with minimum redundancy maximum relevance (mRMR) feature selection and the LADTree algorithm, which were selected from nine feature selection methods and 65 classified methods, respectively. The accuracy of the final two-layer machine learning model, referred to as TIMgo, was 99.3% based on fivefold cross-validation, and 85.6% based on independent testing. We discovered that the information within the local sequence had a greater contribution than the global sequence with respect to classification. TIMgo had a good predictive ability for target genes within 20 kb from the 35S enhancer. Based on the analysis of significant sequences, the G-box regulatory sequence may also play an important role in the activation mechanism of the 35S enhancer.

Identification and Expression Characterization of the Smad3 Gene and SNPs Associated with Growth Traits in the Hard Clam (Meretrix meretrix)

It has been demonstrated that the sekelsky mothers against decapentaplegic homolog 3 (Smad3) plays an important role in the growth and development of vertebrates. However, little is known about the association between the Smad3 gene and the growth traits of mollusks. In this study, Smad3 from the hard clam Meretrix meretrix (Mm-Smad3) was cloned, characterized, and screened for growth-related single nucleotide polymorphisms (SNPs) in its exons. The full-length cDNA of Mm-Smad3 was 1938 bp, encoding a protein with 428 amino acid residues. The protein sequence included an MH1 (27–135 aa) and MH2 domain (233–404 aa). Promoter analysis showed that the promoter sequence of Mm-Smad3 was 2548 bp, and the binding sites of Pit-1a, Antp, Hb, and other transcription factors are related to the growth and development of hard clams. The phylogenetic tree was divided into two major clusters, including mollusks and vertebrate. The expression level of Mm-Smad3 was predominantly detected in the mantle and foot, while extremely less expression was observed in the digestive gland. The low expression level of Mm-Smad3 was detected at the stages of unfertilized mature eggs, fertilized eggs, four-cell embryos, blastula, gastrulae, trochophore, and D-shaped larvae, whereas an opposite trend was observed regarding the highest expression at the umbo larvae stage (p < 0.05). In the mantle repair experiment, the time-course expression profiles showed that compared to the expression level at 0 h, Mm-Smad3 significantly decreased at 6 h (p < 0.05) but increased at 12 and 48 h. Further, the association analysis identified 11 SNPs in the exons of Mm-Smad3, of which three loci (c.597 C > T, c.660 C > T, c.792 A > T) were significantly related to the growth traits of clam (p < 0.05). Overall, our findings indicated that Mm-Smad3 is a growth-related gene and the detected SNP sites provide growth-related markers for molecular marker-assisted breeding of this species.

Comparison of RNA synthesis initiation properties of non-segmented negative strand RNA virus polymerases

It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.

Communication and Cross-Regulation Between Multiple Concatenated Enzymatic Reaction Networks

Nature connects multiple fuel-driven chemical/enzymatic reaction networks (CRNs/ERNs) via cross-regulation to hierarchically control biofunctions for a tailored adaption in complex sensory landscapes. In contrast, emerging artificial fuel-driven systems most-ly focus on a single CRN and their implementation to direct self-assembly or material responses. In this work, we introduce a facile example of communication and cross-regulation among multiple DNA-based ERNs regulated by a concatenated RNA transcription regulator. For this purpose, we run two fuel-driven DNA-based ERNs by concurrent NAD+-fueled ligation and restriction via endo-nucleases (REases) in parallel. ERN one allows for the dynamic steady-state formation of the promoter sequence for T7 RNA poly-merase, which activates RNA transcription. The produced RNA regulator can repress or promote the second ERN via RNA-mediated strand displacement. Furthermore, adding RNase H to degrade the produced RNA can restart the reaction or tune the lag time of two ERNs, giving rise to a repression-recovery and promotion-stop processes. We believe that concatenation of multiple CRNs provides a basis for the design of more elaborate autonomous regulatory mechanisms in systems chemistry and synthetic biology.

Identification of genome-wide distribution of cyanobacterial group 2 sigma factor SigE accountable for its regulon

Sigma factors are the subunits of bacterial RNA polymerase that govern the expression of genes by recognizing the promoter sequence. Cyanobacteria, which are oxygenic phototrophic eubacteria, have multiple alternative sigma factors that respond to various environmental stresses. The subgroup highly homologous to the primary sigma factor (SigA) is called the group 2 sigma factor. The model cyanobacterium, Synechocystis sp. PCC 6803, has four group 2 sigma factors (SigB-E) conserved within the phylum Cyanobacteria. Among the group 2 sigma factors in Synechocystis sp. PCC 6803, SigE is unique because it alters metabolism by inducing the expression of genes related to sugar catabolism and nitrogen metabolism. However, the features of promoter sequence of the SigE regulon remains elusive. Here, we identified the direct targets of SigA and SigE by chromatin immunoprecipitation sequencing (ChIP-seq). We then showed that the binding sites of SigE and SigA overlapped substantially, but SigE exclusively localized to SigE-dependent promoters. We also found consensus sequences from SigE-dependent promoters and confirmed their importance. ChIP-seq analysis showed both the redundancy and specificity of SigE compared with SigA, integrating information obtained from a previously adopted genetic approach and in vitro assays. The features of SigE elucidated in our study indicate its similarity with group 2 sigma factors of other bacteria, even though they are evolutionally irrelevant. Our approach is also applicable to other organisms and organelles, such as plant plastids, which have multiple group 2 sigma factors.