Genome-wide screening and identification of nuclear Factor-Y family genes and exploration their function on regulating abiotic and biotic stress in potato (Solanum tuberosum L.).

The Nuclear Factor-Y (NF-Y) transcription factor (TF), which includes three distinct subunits (NF-YA, NF-YB and NF-YC), is known to manipulate various aspects of plant growth, development, and stress responses. Although the NF-Y gene family was well studied in many species, little is known about their functions in potato. In this study, a total of 37 potato NF-Y genes were identified, including 11 StNF-YAs, 20 StNF-YBs, and 6 StNF-YCs. The genetic features of these StNF-Y genes were investigated by comparing their evolutionary relationship, intron/exon organization and motif distribution pattern. Multiple alignments showed that all StNF-Y proteins possessed clearly conserved core regions that were flanked by non-conserved sequences. Gene duplication analysis indicated that nine StNF-Y genes were subjected to tandem duplication and eight StNF-Ys arose from segmental duplication events. Synteny analysis suggested that most StNF-Y genes (33 of 37) were orthologous to potato's close relative tomato (Solanum lycopersicum L.). Tissue-specific expression of the StNF-Y genes suggested their potential roles in controlling potato growth and development. The role of StNF-Ys in regulating potato responses to abiotic stress (ABA, drought and salinity) was also confirmed: twelve StNF-Y genes were up-regulated and another two were down-regulated under different abiotic treatments. In addition, genes responded differently to pathogen challenges, suggesting that StNF-Y genes may play distinct roles under certain biotic stress. In summary, insights into the evolution of NF-Y family members and their functions in potato development and stress responses are provided.

Genome-wide analysis of the NF-Y gene family and their roles in relation to fruit development in Tartary buckwheat (Fagopyrum tataricum).

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor playing crucial roles in various biological process in plant. However, thorough research on NF-Y gene family of Tartary buckwheat (Fagopyrum tataricum) is little. In this study, 38 FtNF-Y genes (12 FtNF-YAs, 17 FtNF-YBs, and 9 FtNF-YCs) were identified and renamed on the basis of their subfamily and chromosomal location. Their gene structure, genomic mapping, motif composition, conserved domain, phylogenetic relationships, cis-acting elements and gene expression were investigated. Illustration of gene structures and conserved domains of FtNF-Ys revealed their functional conservation and specificity. Construction of phylogenetic trees of NF-Ys in Tartary buckwheat, Arabidopsis, tomato, rice and banana, allowed us to predict functional similarities among NF-Ys from different species. Gene expression analysis displayed that twenty-four FtNF-Ys were expressed in all the tissues and the transcript levels of them were different, suggesting their function varieties. Moreover, expression profiles of twenty FtNF-Ys along five different fruit development stages acquired by real-time quantitative PCR (RT-qPCR) demonstrated distinct abundance diversity at different stages, providing some clues of potential fruit development regulators. Our study could provide helpful reference information for further function characterization of FtNF-Ys and for the fruit quality enhancement of Tartary buckwheat.

Identification, expression, and putative target gene analysis of nuclear factor-Y (NF-Y) transcription factors in tea plant (Camellia sinensis).

MAIN CONCLUSION: Genome-wide identification and characterization of nuclear factor-Y family in tea plants, and their expression profiles and putative targets provide the basis for further elucidation of their biological functions. The nuclear factor-Y (NF-Y) transcription factors (TFs) are crucial regulators of plant growth and physiology. However, the NF-Y TFs in tea plant (Camellia sinensis) have not yet been elucidated, and its biological functions, especially the putative target genes within the genome range, are still unclear. In this study, we identified 35 CsNF-Y encoding genes in the tea plant genome, including 10 CsNF-YAs, 15 CsNF-YBs and 10 CsNF-YCs. Their conserved domains and motifs, phylogeny, duplication event, gene structure, and promoter were subsequently analyzed. Tissue expression analysis revealed that CsNF-Ys exhibited three distinct expression patterns in eight tea tree tissues, among which CsNF-YAs were moderately expressed. Drought and abscisic acid (ABA) treatment indicated that CsNF-YAs may have a greater impact than other subunit members. Furthermore, through the genome-wide investigation of the presence of the CCAAT box, we found that CsNF-Ys may participate in the development of tea plants by regulating target genes of multiple physiological pathways, including photosynthesis, chlorophyll metabolism, fatty acid biosynthesis, and amino acid metabolism pathways. Our findings will contribute to the functional analysis of NF-Y genes in woody plants and the cultivation of high-quality tea plant cultivars.

CCAAT-displacement protein/cut homeobox transcription factor (CUX1) represses estrogen receptor-alpha (ER-α) in triple-negative breast cancer cells and can be antagonized by muscadine grape skin extract (MSKE).

Triple-Negative Breast Cancers (TNBCs) are the most difficult to treat subtype of breast cancer and are often associated with high nuclear expression of Snail and Cathepsin L (Cat L) protease. We have previously shown that Snail can increase Cat L expression/activity in prostate and breast cancer cells. This study investigated the role of CUX1 (a downstream substrate of Cat L) in TNBC. We showed that Cat L and CUX1 were highly expressed in TNBC patient tissue/cell lines, as compared to ER-positive samples, using cBioportal data and western blot/zymography analyses. Additionally, luciferase reporter and chromatin immunoprecipitation assays showed that CUX1 directly bound to estrogen receptor-alpha (ER-α) promoter in MDA-MB-468, a representative TNBC cell line, and that CUX1 siRNA could restore ER-α transcription and protein expression. Furthermore, Snail and CUX1 expression in various TNBC cell lines was inhibited by muscadine grape skin extract (MSKE, a natural grape product rich in anthocyanins) or Cat L inhibitor (Z-FY-CHO) leading to decreased cell invasion and migration. MSKE decreased cell viability and increased expression of apoptotic markers in MDA-MB-468 cells, with no effect on non-tumorigenic MCF10A cells. MSKE also decreased CUX1 binding to ER-α promoter and restored ER-α expression in TNBC cells, while both MSKE and CUX1 siRNA restored sensitivity to estradiol and 4-hydoxytamoxifen as shown by increased cell viability. Therefore, CUX1 activated by Snail-Cat L signaling may contribute to TNBC via ER-α repression, and may be a viable target for TNBC using natural products such as MSKE that targets cancer and not normal cells.

Shaoyao Gancao Tang (SG-Tang), a formulated Chinese medicine, reduces aggregation and exerts neuroprotection in spinocerebellar ataxia type 17 (SCA17) cell and mouse models.

Spinocerebellar ataxia (SCA) type 17 is an autosomal dominant ataxia caused by expanded polyglutamine (polyQ) tract in the TATA-box binding protein (TBP). Substantial studies have shown involvement of compromised mitochondria biogenesis regulator peroxisome proliferator-activated receptor gamma-coactivator 1 alpha (PGC-1α), nuclear factor erythroid 2-related factor 2 (NRF2), nuclear factor-Y subunit A (NFYA), and their downstream target genes in the pathogenesis of polyQ-expansion diseases. The extracts of Paeonia lactiflora (P. lactiflora) and Glycyrrhiza uralensis (G. uralensis) have long been used as a Chinese herbal medicine (CHM). Shaoyao Gancao Tang (SG-Tang) is a formulated CHM made of P. lactiflora and G. uralensis at a 1:1 ratio. In the present study, we demonstrated the aggregate-inhibitory and anti-oxidative effect of SG-Tang in 293 TBP/Q79 cells. We then showed that SG-Tang reduced the aggregates and ameliorated the neurite outgrowth deficits in TBP/Q79 SH-SY5Y cells. SG-Tang upregulated expression levels of NFYA, PGC-1α, NRF2, and their downstream target genes in TBP/Q79 SH-SY5Y cells. Knock down of NFYA, PGC-1α, and NRF2 attenuated the neurite outgrowth promoting effect of SG-Tang on TBP/Q79 SH-SY5Y cells. Furthermore, SG-Tang inhibited aggregation and rescued motor-deficits in SCA17 mouse model. The study results suggest the potential of SG-Tang in treating SCA17 and probable other polyQ diseases.

A phenotypic Caenorhabditis elegans screen identifies a selective suppressor of antipsychotic-induced hyperphagia.

Antipsychotic (AP) drugs are used to treat psychiatric disorders but are associated with significant weight gain and metabolic disease. Increased food intake (hyperphagia) appears to be a driving force by which APs induce weight gain but the mechanisms are poorly understood. Here we report that administration of APs to C. elegans induces hyperphagia by a mechanism that is genetically distinct from basal food intake. We exploit this finding to screen for adjuvant drugs that suppress AP-induced hyperphagia in C. elegans and mice. In mice AP-induced hyperphagia is associated with a unique hypothalamic gene expression signature that is abrogated by adjuvant drug treatment. Genetic analysis of this signature using C. elegans identifies two transcription factors, nhr-25/Nr5a2 and nfyb-1/NFYB to be required for AP-induced hyperphagia. Our study reveals that AP-induced hyperphagia can be selectively suppressed without affecting basal food intake allowing for novel drug discovery strategies to combat AP-induced metabolic side effects.

Radical scavenging-linked anti-adipogenic activity of Alnus firma extracts.

The purpose of the present study was to investigate the antioxidant activity and anti-adipogenic effect of extracts from Alnus firma (A. firma), which is an edible plant that grows in mountainous areas. The total phenolic, flavonoid and anthocyanin content as well as the antioxidant activity of a 70% ethanolic extract of A. firma (AFE) was assessed. Furthermore, the effects of AFE on lipid accumulation and reactive oxygen species (ROS) production during adipogenesis of 3T3-L1 cells were investigated. The results revealed that the total phenolic, flavonoid and pro-anthocyanidin content of AFE as 436.26±3.30 mg gallic acid equivalents/g, 73.82±0.54 mg quercetin equivalents/g and 149.25±6.06 mg catechin equivalents/g, respectively. In addition, AFE exerted significant antioxidant effects in terms of 1,1-diphenyl-2-picryl hydrazyl radical scavenging activity, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity, reducing power, oxygen radical absorbance capacity and nitric oxide radical scavenging activity. As for its anti-adipogenic activity, AFE significantly inhibited ROS production and lipid accumulation during adipogenesis of 3T3-L1 cells compared with those in control cells. In addition, AFE regulated adipogenic transcription factors including peroxisome proliferator­activated receptor-γ, CCAAT/enhance-binding protein α and adipocyte protein 2. These results indicated that A. firma is a potential candidate for a functional food supplement.

Discovery of Novel Human Gene Regulatory Modules from Gene Co-expression and Promoter Motif Analysis.

Deciphering gene regulatory networks requires identification of gene expression modules. We describe a novel bottom-up approach to identify gene modules regulated by cis-regulatory motifs from a human gene co-expression network. Target genes of a cis-regulatory motif were identified from the network via the motif's enrichment or biased distribution towards transcription start sites in the promoters of co-expressed genes. A gene sub-network containing the target genes was extracted and used to derive gene modules. The analysis revealed known and novel gene modules regulated by the NF-Y motif. The binding of NF-Y proteins to these modules' gene promoters were verified using ENCODE ChIP-Seq data. The analyses also identified 8,048 Sp1 motif target genes, interestingly many of which were not detected by ENCODE ChIP-Seq. These target genes assemble into house-keeping, tissues-specific developmental, and immune response modules. Integration of Sp1 modules with genomic and epigenomic data indicates epigenetic control of Sp1 targets' expression in a cell/tissue specific manner. Finally, known and novel target genes and modules regulated by the YY1, RFX1, IRF1, and 34 other motifs were also identified. The study described here provides a valuable resource to understand transcriptional regulation of various human developmental, disease, or immunity pathways.

The garlic NF-YC gene, AsNF-YC8, positively regulates non-ionic hyperosmotic stress tolerance in tobacco.

To investigate the relationship between nuclear factor Y (NF-Y) and stress tolerance in garlic, we cloned a NF-Y family gene AsNF-YC8 from garlic, which was largely upregulated at dehydrate stage. Expression pattern analyses in garlic revealed that AsNF-YC8 is induced through abscisic acid (ABA) and abiotic stresses, such as NaCl and PEG. Compared with wild-type plants, the overexpressing-AsNF-YC8 transgenic tobacco plants showed higher seed germination rates, longer root length and better plant growth under salt and drought stresses. Under drought stress, the transgenic plants maintained higher relative water content (RWC), net photosynthesis, lower levels of malondialdehyde (MDA), and less ion leakage (IL) than wild-type control plants. These results indicate the high tolerance of the transgenic plants to drought stress compared to the WT. The transgenic tobacco lines accumulated less reactive oxygen species (ROS) and exhibited higher antioxidative enzyme activities compared with wild-type (WT) plants under drought stress, which suggested that the overexpression of AsNF-YC8 improves the antioxidant defense system by regulating the activities of these antioxidant enzymes, which in turn protect transgenic lines against drought stress. These results suggest that AsNF-YC8 plays an important role in tolerance to drought and salt stresses.

Transcriptional suppression of CTP:phosphoethanolamine cytidylyltransferase by 25-hydroxycholesterol is mediated by nuclear factor-Y and Yin Yang 1.

Pcyt2 (CTP:phosphoethanolamine cytidylyltransferase) is the rate-limiting enzyme in mammalian PE (phosphatidylethanolamine) biosynthesis. Previously, we reported that Pcyt2 mRNA levels increased in several types of cells after serum starvation, an effect that could be suppressed by supplementation with low-density lipoprotein or 25-HC (25-hydroxycholesterol). Transcription of Hmgcr, which encodes 3-hydroxy-3-methylglutaryl-CoA reductase, is also suppressed by 25-HC in the same dose-dependent manner. Nevertheless, a sterol-regulatory element was not detected in the Pcyt2 promoter region. The important element for transcriptional control of Pcyt2 by 25-HC (1.25 µM) was determined to reside between -56 and -36 on the basis of analysis with several Pcyt2 promoter deletion-luciferase reporters in NIH 3T3 cells. Using the yeast one-hybrid system, we found that NF-Y (nuclear factor-Y) binds at C(-37)CAAT(-41) and YY1 (Yin Yang1) binds at C(-42)AT(-40) in the Pcyt2 promoter. Endogenous NF-Y and YY1 bind clearly and competitively to these sites and are important for basal Pcyt2 transcription. Moreover, NF-Y binds to the Hmgcr promoter at C(-14)CA(-12) in gel-shift analysis, and suppression of the basal luciferase activity of the Hmgcr promoter-reporter construct (-30/+61) by 25-HC was abolished when C(-14)CA(-12) was mutated. Furthermore, transcriptional suppression of Pcyt2 by 25-HC was reduced following knockdown targeting of NF-YA or YY1. ChIP analysis revealed that 25-HC inhibited the interaction between NF-Y and RNA polymerase II on the Pcyt2 and Hmgcr promoters. On the basis of these results, we conclude that NF-Y and YY1 are important for the basal transcription of Pcyt2 and that NF-Y is involved in the inhibitory effects of 25-HC on Pcyt2 transcription.

ZHX2 enhances the cytotoxicity of chemotherapeutic drugs in liver tumor cells by repressing MDR1 via interfering with NF-YA.

We previously reported the tumor suppressor function of Zinc-fingers and homeoboxes 2 (ZHX2) in hepatocellular carcinoma (HCC). Other studies indicate the association of increased ZHX2 expression with improved response to high dose chemotherapy in multiple myeloma. Here, we aim to test whether increased ZHX2 levels in HCC cells repress multidrug resistance 1(MDR1) expression resulting in increased sensitivity to chemotherapeutic drugs. We showed evidence that increased ZHX2 levels correlated with reduced MDR1 expression and enhanced the cytotoxicity of CDDP and ADM in different HCC cell lines. Consistently, elevated ZHX2 significantly reduced ADM efflux in HepG2 cells and greatly increased the CDDP-mediated suppression of liver tumor growth in vivo. Furthermore, immunohistochemical staining demonstrated the inverse correlation of ZHX2 and MDR1 expression in HCC tissues. Luciferase report assay showed that ZHX2 repressed the MDR1 promoter activity, while knockdown of NF-YA or mutating the NF-Y binding site eliminated this ZHX2-mediated repression of MDR1 transcription. Co-IP and ChIP assay further suggested that ZHX2 interacted with NF-YA and reduced NF-Y binding to the MDR1 promoter. Taken together, we clarify that ZHX2 represses NF-Y-mediated activation of MDR1 transcription and, in doing so, enhances the effects of chemotherapeutics in HCC cells both in vitro and in vivo.

Overexpression of wheat NF-YA10 gene regulates the salinity stress response in Arabidopsis thaliana.

The nuclear factor Y (NF-Y) transcription factor is formed by the interaction of three distinct subunits (NF-YA, -YB and -YC). It targets the CCAAT box, a common cis-element in eukaryotic promoters. Here, the bread wheat gene TaNF-YA10-1 has been isolated from the salinity tolerant cultivar SR3. Recombinant TaNF-YA10-1 was heterologously produced in Escherichia coli, and the purified protein successfully bound to the CCAAT motif in vitro. TaNF-YA10-1 was down-regulated by the imposition of salinity and abscisic acid (ABA). The constitutive expression of TaNF-YA10-1 in Arabidopsis thaliana significantly increased the plant's sensitivity to salinity and repressed its sensitivity to ABA as judged from the seed germination, cotyledon greening and the relative root growth. The transcription of stress-related genes AtRAB18, AtRD29B, AtABI5, AtCBF1 and AtCBF3 was downregulated in TaNF-YA10-1 overexpression transgenic plants. The data provide supportive evidence that TaNFYA10-1 is involved in the regulation of growth under salinity stress conditions.

[Analysis of codon use features of CBF gene in Camellia sinensis].

CBF (C-repeat-binding factor) transcription factor exists widely in all kinds of plants. It is an important regulative factor in the process of plant resistance adversity. In this paper, Camellia sinensis CBF1 gene sequence was analyzed by Codon W, CHIPS, and CUSP programs online, and then compared with C. sinensis genes, genomes in other species, and CBF genes from 39 plant species. It is important to identify the codon usage of CsCBF1 gene and select appropriate expression systems. The results showed that CsCBF1 gene and selected 70 C. sinensis genes had distinct usage differences. CsCBF1 gene was bias toward the synonymous codons with G and C at the third codon position, but 70 C. sinensis genes were bias toward the synonymous codons with A and T. The differences in codon usage frequency between CsCBF1 gene and dicotyledons such as Arabidopsis thaliana and Nicotiana tobacum were less than monocotyledons such as wheat (Triticum aestivum) and corn (Zea mays). Therefore, A. thaliana and N. tobacum expression systems may be more suitable for the expression of CsCBF1 gene. The analysis results of CBF genes from 40 plant species also showed that most of the CBF genes were bias toward the synonymous codons with G and C at the third codon position. The reason of this phenomenon is possible due to special functions of these genes.

Expression of CCAAT-binding factor antisense transcripts in reproductive tissues affects plant fertility.

We have previously isolated a CCAAT-binding factor B subunit gene ( BnCBF-B) from Brassica napus that is widely expressed in different plant tissues and whose role is still unknown. To investigate the importance of this transcription factor subunit in plant reproductive tissues, we targeted antisense BnCBF-B transcripts to the tapetum of transgenic B. napus plants. Of the 24 independent transformants, 13 yielded reduced quantities of viable pollen, of which five were unable to produce the elongated siliques indicative of normal seed set. The decrease in pollen viability probably resulted from the precocious degeneration of the tapetal cell layer observed in these plants. Surprisingly, the male-sterile phenotype was also accompanied by a decrease in female fertility, which could be due to the expression of the antisense BnCBF-B transcripts in the female reproductive structures of the transgenic plants. These results suggest that the BnCBF-B gene plays a critical non-redundant role in plant reproductive tissues.

Functional identification of an Arabidopsis snf4 ortholog by screening for heterologous multicopy suppressors of snf4 deficiency in yeast.

Yeast Snf4 is a prototype of activating gamma-subunits of conserved Snf1/AMPK-related protein kinases (SnRKs) controlling glucose and stress signaling in eukaryotes. The catalytic subunits of Arabidopsis SnRKs, AKIN10 and AKIN11, interact with Snf4 and suppress the snf1 and snf4 mutations in yeast. By expression of an Arabidopsis cDNA library in yeast, heterologous multicopy snf4 suppressors were isolated. In addition to AKIN10 and AKIN11, the deficiency of yeast snf4 mutant to grown on non-fermentable carbon source was suppressed by Arabidopsis Myb30, CAAT-binding factor Hap3b, casein kinase I, zinc-finger factors AZF2 and ZAT10, as well as orthologs of hexose/UDP-hexose transporters, calmodulin, SMC1-cohesin and Snf4. Here we describe the characterization of AtSNF4, a functional Arabidopsis Snf4 ortholog, that interacts with yeast Snf1 and specifically binds to the C-terminal regulatory domain of Arabidopsis SnRKs AKIN10 and AKIN11.