Expression profiling of Nuclear Factor-Y (NF-Y) transcription factors during dehydration and salt stress in finger millet reveals potential candidate genes for multiple stress tolerance.

MAIN CONCLUSION: Expression profiling of NF-Y transcription factors during dehydration and salt stress in finger millet genotypes contrastingly differing in tolerance levels identifies candidate genes for further characterization and functional studies. The Nuclear Factor-Y (NF-Y) transcription factors are known for imparting abiotic stress tolerance in different plant species. However, there is no information on the role of this transcription factor family in naturally drought-tolerant crop finger millet (Eleusine coracana L.). Therefore, interpretation of expression profiles against drought and salinity stress may provide valuable insights into specific and/or overlapping expression patterns of Eleusine coracana Nuclear Factor-Y (EcNF-Y) genes. Given this, we identified 59 NF-Y (18 NF-YA, 23 NF-YB, and 18 NF-YC) encoding genes and designated them EcNF-Y genes. Expression profiling of these genes was performed in two finger millet genotypes, PES400 (dehydration and salt stress tolerant) and VR708 (dehydration and salt stress sensitive), subjected to PEG-induced dehydration and salt (NaCl) stresses at different time intervals (0, 6, and 12 h). The qRT-PCR expression analysis reveals that the six EcNF-Y genes namely EcNF-YA1, EcNF-YA5, EcNF-YA16, EcNF-YB6, EcNF-YB10, and EcNF-YC2 might be associated with tolerance to both dehydration and salinity stress in early stress condition (6 h), suggesting the involvement of these genes in multiple stress responses in tolerant genotype. In contrast, the transcript abundance of finger millet EcNF-YA5 genes was also observed in the sensitive genotype VR708 under late stress conditions (12 h) of both dehydration and salinity stress. Therefore, the EcNF-YA5 gene might be important for adaptation to salinity and dehydration stress in sensitive finger millet genotypes. Therefore, this gene could be considered as a susceptibility determinant, which can be edited to impart tolerance. The phylogenetic analyses revealed that finger millet NF-Y genes share strong evolutionary and functional relationship to NF-Ys governing response to abiotic stresses in rice, sorghum, maize, and wheat. This is the first report of expression profiling of EcNF-Ys genes identified from the finger millet genome and reveals potential candidate for enhancing dehydration and salt tolerance.

Genome-wide identification and expression analysis of NF-Y gene family in tobacco (Nicotiana tabacum L.).

Nuclear factor Y (NF-Y) gene family is an important transcription factor composed of three subfamilies of NF-YA, NF-YB and NF-YC, which is involved in plant growth, development and stress response. In this study, 63 tobacco NF-Y genes (NtNF-Ys) were identified in Nicotiana tabacum L., including 17 NtNF-YAs, 30 NtNF-YBs and 16 NtNF-YCs. Phylogenetic analysis revealed ten pairs of orthologues from tomato and tobacco and 25 pairs of paralogues from tobacco. The gene structure of NtNF-YAs exhibited similarities, whereas the gene structure of NtNF-YBs and NtNF-YCs displayed significant differences. The NtNF-Ys of the same subfamily exhibited a consistent distribution of motifs and protein 3D structure. The protein interaction network revealed that NtNF-YC12 and NtNF-YC5 exhibited the highest connectivity. Many cis-acting elements related to light, stress and hormone response were found in the promoter of NtNF-Ys. Transcriptome analysis showed that more than half of the NtNF-Y genes were expressed in all tissues, and NtNF-YB9/B14/B15/B16/B17/B29 were specifically expressed in roots. A total of 15, 12, 5, and 6 NtNF-Y genes were found to respond to cold, drought, salt, and alkali stresses, respectively. The results of this study will lay a foundation for further study of NF-Y genes in tobacco and other Solanaceae plants.

Genome-Wide Identification and Drought Stress Response Pattern of the NF-Y Gene Family in Cymbidium sinense.

Cymbidium sinense, a type of orchid plant, is more drought-resistant and ornamental than other terrestrial orchids. Research has shown that many members of the NUCLEAR FACTOR Y (NF-Y) transcription factor family are responsive to plant growth, development, and abiotic stress. However, the mechanism of the NF-Y gene family's response to abiotic stress in orchids has not yet been reported. In this study, phylogenetic analysis allowed for 27 CsNF-Y genes to be identified (5 CsNF-YAs, 9 CsNF-YBs, and 13 CsNF-YC subunits), and the CsNF-Ys were homologous to those in Arabidopsis and Oryza. Protein structure analysis revealed that different subfamilies contained different motifs, but all of them contained Motif 2. Secondary and tertiary protein structure analysis indicated that the CsNF-YB and CsNF-YC subfamilies had a high content of alpha helix structures. Cis-element analysis showed that elements related to drought stress were mainly concentrated in the CsNF-YB and CsNF-YC subfamilies, with CsNF-YB3 and CsNF-YC12 having the highest content. The results of a transcriptome analysis showed that there was a trend of downregulation of almost all CsNF-Ys in leaves under drought stress, while in roots, most members of the CsNF-YB subfamily showed a trend of upregulation. Additionally, seven genes were selected for real-time reverse transcription quantitative PCR (qRT-PCR) experiments. The results were generally consistent with those of the transcriptome analysis. The regulatory roles of CsNF-YB 1, 2, and 4 were particularly evident in the roots. The findings of our study may make a great contribution to the understanding of the role of CsNF-Ys in stress-related metabolic processes.

The pancancer overexpressed NFYC Antisense 1 controls cell cycle mitotic progression through in cis and in trans modes of action.

Antisense RNAs (asRNAs) represent an underappreciated yet crucial layer of gene expression regulation. Generally thought to modulate their sense genes in cis through sequence complementarity or their act of transcription, asRNAs can also regulate different molecular targets in trans, in the nucleus or in the cytoplasm. Here, we performed an in-depth molecular characterization of NFYC Antisense 1 (NFYC-AS1), the asRNA transcribed head-to-head to NFYC subunit of the proliferation-associated NF-Y transcription factor. Our results show that NFYC-AS1 is a prevalently nuclear asRNA peaking early in the cell cycle. Comparative genomics suggests a narrow phylogenetic distribution, with a probable origin in the common ancestor of mammalian lineages. NFYC-AS1 is overexpressed pancancer, preferentially in association with RB1 mutations. Knockdown of NFYC-AS1 by antisense oligonucleotides impairs cell growth in lung squamous cell carcinoma and small cell lung cancer cells, a phenotype recapitulated by CRISPR/Cas9-deletion of its transcription start site. Surprisingly, expression of the sense gene is affected only when endogenous transcription of NFYC-AS1 is manipulated. This suggests that regulation of cell proliferation is at least in part independent of the in cis transcription-mediated effect on NFYC and is possibly exerted by RNA-dependent in trans effects converging on the regulation of G2/M cell cycle phase genes. Accordingly, NFYC-AS1-depleted cells are stuck in mitosis, indicating defects in mitotic progression. Overall, NFYC-AS1 emerged as a cell cycle-regulating asRNA with dual action, holding therapeutic potential in different cancer types, including the very aggressive RB1-mutated tumors.

Expression and function of NF-Y subunits in cancer.

NF-Y is a Transcription Factor (TF) targeting the CCAAT box regulatory element. It consists of the NF-YB/NF-YC heterodimer, each containing an Histone Fold Domain (HFD), and the sequence-specific subunit NF-YA. NF-YA expression is associated with cell proliferation and absent in some post-mitotic cells. The review summarizes recent findings impacting on cancer development. The logic of the NF-Y regulome points to pro-growth, oncogenic genes in the cell-cycle, metabolism and transcriptional regulation routes. NF-YA is involved in growth/differentiation decisions upon cell-cycle re-entry after mitosis and it is widely overexpressed in tumors, the HFD subunits in some tumor types or subtypes. Overexpression of NF-Y -mostly NF-YA- is oncogenic and decreases sensitivity to anti-neoplastic drugs. The specific roles of NF-YA and NF-YC isoforms generated by alternative splicing -AS- are discussed, including the prognostic value of their levels, although the specific molecular mechanisms of activity are still to be deciphered.

Jasmonate signaling pathway confers salt tolerance through a NUCLEAR FACTOR-Y trimeric transcription factor complex in Arabidopsis.

Jasmonate (JA) is a well-known phytohormone essential for plant response to biotic stress. Recently, a crucial role of JA signaling in salt resistance has been highlighted; however, the specific regulatory mechanism remains largely unknown. In this study, we found that the NUCLEAR FACTOR-Y (NF-Y) subunits NF-YA1, NF-YB2, and NF-YC9 form a trimeric complex that positively regulates the expression of salinity-responsive genes, whereas JASMONATE-ZIM DOMAIN protein 8 (JAZ8) directly interacts with three subunits and acts as the key repressor to suppress both the assembly of the NF-YA1-YB2-YC9 trimeric complex and the transcriptional activation activity of the complex. When plants encounter high salinity, JA levels are elevated and perceived by the CORONATINE INSENSITIVE (COI) 1 receptor, leading to the degradation of JAZ8 via the 26S proteasome pathway, thereby releasing the activity of the NF-YA1-YB2-YC9 complex, initiating the activation of salinity-responsive genes, such as MYB75, and thus enhancing the salinity tolerance of plants.

Two different viral proteins suppress NUCLEAR FACTOR-YC-mediated antiviral immunity during infection in rice.

Plant viruses have multiple strategies to counter and evade the host's antiviral immune response. However, limited research has been conducted on the antiviral defense mechanisms commonly targeted by distinct types of plant viruses. In this study, we discovered that NUCLEAR FACTOR-YC (NF-YC) and NUCLEAR FACTOR-YA (NF-YA), 2 essential components of the NF-Y complex, were commonly targeted by viral proteins encoded by 2 different rice (Oryza sativa L.) viruses, rice stripe virus (RSV, Tenuivirus) and southern rice black streaked dwarf virus (SRBSDV, Fijivirus). In vitro and in vivo experiments showed that OsNF-YCs associate with OsNF-YAs and inhibit their transcriptional activation activity, resulting in the suppression of OsNF-YA-mediated plant susceptibility to rice viruses. Different viral proteins RSV P2 and SRBSDV SP8 directly disrupted the association of OsNF-YCs with OsNF-YAs, thereby suppressing the antiviral defense mediated by OsNF-YCs. These findings suggest an approach for conferring broad-spectrum disease resistance in rice and reveal a common mechanism employed by viral proteins to evade the host's antiviral defense by hindering the antiviral capabilities of OsNF-YCs.

The transcription factor NF-YA is crucial for neural progenitor maintenance during brain development.

In contrast to stage-specific transcription factors, the role of ubiquitous transcription factors in neuronal development remains a matter of scrutiny. Here, we demonstrated that a ubiquitous factor NF-Y is essential for neural progenitor maintenance during brain morphogenesis. Deletion of the NF-YA subunit in neural progenitors by using nestin-cre transgene in mice resulted in significant abnormalities in brain morphology, including a thinner cerebral cortex and loss of striatum during embryogenesis. Detailed analyses revealed a progressive decline in multiple neural progenitors in the cerebral cortex and ganglionic eminences, accompanied by induced apoptotic cell death and reduced cell proliferation. In neural progenitors, the NF-YA short isoform lacking exon 3 is dominant and co-expressed with cell cycle genes. ChIP-seq analysis from the cortex during early corticogenesis revealed preferential binding of NF-Y to the cell cycle genes, some of which were confirmed to be downregulated following NF-YA deletion. Notably, the NF-YA short isoform disappears and is replaced by its long isoform during neuronal differentiation. Forced expression of the NF-YA long isoform in neural progenitors resulted in a significant decline in neuronal count, possibly due to the suppression of cell proliferation. Collectively, we elucidated a critical role of the NF-YA short isoform in maintaining neural progenitors, possibly by regulating cell proliferation and apoptosis. Moreover, we identified an isoform switch in NF-YA within the neuronal lineage in vivo, which may explain the stage-specific role of NF-Y during neuronal development.

Nuclear factor Y is a pervasive regulator of neuronal gene expression.

Nervous system function relies on the establishment of complex gene expression programs that provide neuron-type-specific and core pan-neuronal features. These complementary regulatory paradigms are controlled by terminal selector and parallel-acting transcription factors (TFs), respectively. Here, we identify the nuclear factor Y (NF-Y) TF as a pervasive direct and indirect regulator of both neuron-type-specific and pan-neuronal gene expression. Mapping global NF-Y targets reveals direct binding to the cis-regulatory regions of pan-neuronal genes and terminal selector TFs. We show that NFYA-1 controls pan-neuronal gene expression directly through binding to CCAAT boxes in target gene promoters and indirectly by regulating the expression of terminal selector TFs. Further, we find that NFYA-1 regulation of neuronal gene expression is important for neuronal activity and motor function. Thus, our research sheds light on how global neuronal gene expression programs are buffered through direct and indirect regulatory mechanisms.

Characterization of NF-Y gene family and their expression and interaction analysis in Phalaenopsis orchid.

The complex of Nuclear Factor Ys (NF-Ys), a family of heterotrimeric transcription factors composed of three unique subunits (NF-YA, NF-YB, and NF-YC), binds to the CCAAT box of eukaryotic promoters to activate or repress transcription of the downstream genes involved into various biological processes in plants. However, the systematic characterization of NF-Y gene family has not been elucidated in Phalaenopsis. A total of 24 NF-Y subunits (4 NF-YA, 9 NF-YB, and 11 NF-YC subunits) were identified in Phalaenopsis genome, whose exon/intron structures were highly differentiated among the PhNF-Y subunits. The distribution of motifs between coding regions of PhNF-YA and PhNF-YB/C was distinct. Segmental and tandem duplication events among paralogous PhNF-Ys were occurred. Six pairs of orthologous NF-Ys from Phalaenopsis and Arabidopsis and five pairs of orthologous NF-Ys from Phalaenopsis and rice involved in the phylogenetic gene synteny were identified. The various cis-elements being responsive to low-temperature, drought and ABA were distributed in the promoters of PhNF-Ys. qRT-PCR analysis indicated all of PhNF-Ys displayed the spatial specificity of expression in different tissues. Moreover, the expression levels of multiple PhNF-Ys significantly changed responding to low-temperature and ABA treatment. Yeast two hybrid and bimolecular fluorescence complementation assays approved the interaction of PhNF-YA1/3 with PhNF-YB6/PhNF-YC7, respectively, as well as PhNF-YB6 with PhNF-YC7. PhNF-YA1/3, PhNF-YB6, and PhNF-YC7 proteins were all localized in the nucleus. Further, transient overexpression of PhNF-YB6 and PhNF-YC7 promoted PhFT3 and repressed PhSVP expression in Phalaenopsis. These findings will facilitate to explore the role of PhNF-Ys in floral transition in Phalaenopsis orchid.

Analysis of gene duplication within the Arabidopsis NUCLEAR FACTOR Y, subunit B (NF-YB) protein family reveals domains under both purifying and diversifying selection.

Gene duplication is an evolutionary mechanism that provides new genetic material. Since gene duplication is a major driver for molecular evolution, examining the fate of duplicated genes is an area of active research. The fate of duplicated genes can include loss, subfunctionalization, and neofunctionalization. In this manuscript, we chose to experimentally study the fate of duplicated genes using the Arabidopsis NUCLEAR FACTOR Y (NF-Y) transcription factor family. NF-Y transcription factors are heterotrimeric complexes, composed of NF-YA, NF-YB, and NF-YC. NF-YA subunits are responsible for nucleotide-specific binding to a CCAAT cis-regulatory element. NF-YB and NF-YC subunits make less specific, but essential complex-stabilizing contacts with the DNA flanking the core CCAAT pentamer. While ubiquitous in eukaryotes, each NF-Y family has expanded by duplication in the plant lineage. For example, the model plant Arabidopsis contains 10 each of the NF-Y subunits. Here we examine the fate of duplicated NF-YB proteins in Arabidopsis, which are composed of central histone fold domains (HFD) and less conserved flanking regions (N- and C-termini). Specifically, the principal question we wished to address in this manuscript was to what extent can the 10 Arabidopsis NF-YB paralogs functionally substitute the genes NF-YB2 and NF-YB3 in the promotion of photoperiodic flowering? Our results demonstrate that the conserved histone fold domains (HFD) may be under pressure for purifying (negative) selection, while the non-conserved N- and C-termini may be under pressure for diversifying (positive) selection, which explained each paralog's ability to substitute. In conclusion, our data demonstrate that the N- and C-termini may have allowed the duplicated genes to undergo functional diversification, allowing the retention of the duplicated genes.

Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3-FOXO1 in rhabdomyosarcoma.

Recurrent chromosomal rearrangements found in rhabdomyosarcoma (RMS) produce the PAX3-FOXO1 fusion protein, which is an oncogenic driver and a dependency in this disease. One important function of PAX3-FOXO1 is to arrest myogenic differentiation, which is linked to the ability of RMS cells to gain an unlimited proliferation potential. Here, we developed a phenotypic screening strategy for identifying factors that collaborate with PAX3-FOXO1 to block myo-differentiation in RMS. Unlike most genes evaluated in our screen, we found that loss of any of the three subunits of the Nuclear Factor Y (NF-Y) complex leads to a myo-differentiation phenotype that resembles the effect of inactivating PAX3-FOXO1. While the transcriptomes of NF-Y- and PAX3-FOXO1-deficient RMS cells bear remarkable similarity to one another, we found that these two transcription factors occupy nonoverlapping sites along the genome: NF-Y preferentially occupies promoters, whereas PAX3-FOXO1 primarily binds to distal enhancers. By integrating multiple functional approaches, we map the PAX3 promoter as the point of intersection between these two regulators. We show that NF-Y occupies CCAAT motifs present upstream of PAX3 to function as a transcriptional activator of PAX3-FOXO1 expression in RMS. These findings reveal a critical upstream role of NF-Y in the oncogenic PAX3-FOXO1 pathway, highlighting how a broadly essential transcription factor can perform tumor-specific roles in governing cellular state.

Genome-Wide Identification and Chilling Stress Analysis of the NF-Y Gene Family in Melon.

The nuclear factor Y (NF-Y) transcription factor contains three subfamilies: NF-YA, NF-YB, and NF-YC. The NF-Y family have been reported to be key regulators in plant growth and stress responses. However, little attention has been given to these genes in melon (Cucumis melo L.). In this study, twenty-five NF-Ys were identified in the melon genome, including six CmNF-YAs, eleven CmNF-YBs, and eight CmNF-YCs. Their basic information (gene location, protein characteristics, and subcellular localization), conserved domains and motifs, and phylogeny and gene structure were subsequently analyzed. Results showed highly conserved motifs exist in each subfamily, which are distinct between subfamilies. Most CmNF-Ys were expressed in five tissues and exhibited distinct expression patterns. However, CmNF-YA6, CmNF-YB1/B2/B3/B8, and CmNF-YC6 were not expressed and might be pseudogenes. Twelve CmNF-Ys were induced by cold stress, indicating the NF-Y family plays a key role in melon cold tolerance. Taken together, our findings provide a comprehensive understanding of CmNF-Y genes in the development and stress response of melon and provide genetic resources for solving the practical problems of melon production.

Crucial Abiotic Stress Regulatory Network of NF-Y Transcription Factor in Plants.

Nuclear Factor-Y (NF-Y), composed of three subunits NF-YA, NF-YB and NF-YC, exists in most of the eukaryotes and is relatively conservative in evolution. As compared to animals and fungi, the number of NF-Y subunits has significantly expanded in higher plants. The NF-Y complex regulates the expression of target genes by directly binding the promoter CCAAT box or by physical interaction and mediating the binding of a transcriptional activator or inhibitor. NF-Y plays an important role at various stages of plant growth and development, especially in response to stress, which attracted many researchers to explore. Herein, we have reviewed the structural characteristics and mechanism of function of NF-Y subunits, summarized the latest research on NF-Y involved in the response to abiotic stresses, including drought, salt, nutrient and temperature, and elaborated the critical role of NF-Y in these different abiotic stresses. Based on the summary above, we have prospected the potential research on NF-Y in response to plant abiotic stresses and discussed the difficulties that may be faced in order to provide a reference for the in-depth analysis of the function of NF-Y transcription factors and an in-depth study of plant responses to abiotic stress.

NFYAv1 is a Tumor-Promoting Transcript Associated with Poor Prognosis of Hepatocellular Carcinoma.

BACKGROUND Nuclear Transcription Factor Y Subunit Alpha (NFYA), together with NFYB and NFYC, form a sequence-specific heterotrimeric nuclear transcription factor (NFY), but their functional role in hepatocellular carcinoma (HCC) is still unclear. In this study, we explored the association between the NFY subunit genes and the survival of primary hepatocellular carcinoma (HCC) patients in The Cancer Genome Atlas (TCGA). The transcript-specific effect on HCC cell growth was studied. MATERIAL AND METHODS RNA-seq data from the Genotype-Tissue Expression Project (GTEx) and TCGA were analyzed in combination. In vitro cellular and molecular studies were conducted using SK-Hep-1 and Hep3B cells. Pearson's correlation coefficients were calculated to assess correlations. Welch's unpaired t test and one-way ANOVA with post hoc Tukey's multiple comparisons were performed. Kaplan-Meier (K-M) survival curves were assessed by conducting log-rank (Mantel-Cox) test. RESULTS NFYA was the only prognosis-related gene. Among the 2 splicing transcripts of NFYA, the long isoform (NFYAv1, NM_002505.5) but not the short-form (NFYAv2, NM_021705.4) was significantly associated with worse progression-free survival (PFS) (high [n=179] vs low [n=179], HR: 1.657, 95% CI: 1.228-2.235, P<0.001) and disease-specific survival (DSS) (high [n=175] vs low [n=175], HR: 1.986, 95% CI: 1.269-3.108, P<0.001) in HCC patients. GO/KEGG analysis in TCGA confirmed that NFYAv1 and NFYAv2 co-expressed (|Pearson's r|≥0.6) genes in primary HCC patients were enriched in quite different GO/KEGG terms. NFYAv1 knockdown significantly decreased cell viability and increased G0/G1 cell cycle arrest. The shRNA only targeting NFYAv1 had a significantly stronger growth-inhibiting effect than the shRNA targeting both NFYAv1 and NFYAv2. CONCLUSIONS This study showed that NFYAv1 is a tumor-promoting transcript associated with poor prognosis of HCC.

NF-YAl drives EMT in Claudinlow tumours.

NF-Y is a trimeric transcription factor whose binding site -the CCAAT box- is enriched in cancer-promoting genes. The regulatory subunit, the sequence-specificity conferring NF-YA, comes in two major isoforms, NF-YA long (NF-YAl) and short (NF-YAs). Extensive expression analysis in epithelial cancers determined two features: widespread overexpression and changes in NF-YAl/NF-YAs ratios (NF-YAr) in tumours with EMT features. We performed wet and in silico experiments to explore the role of the isoforms in breast -BRCA- and gastric -STAD- cancers. We generated clones of two Claudinlow BRCA lines SUM159PT and BT549 ablated of exon-3, thus shifting expression from NF-YAl to NF-YAs. Edited clones show normal growth but reduced migratory capacities in vitro and ability to metastatize in vivo. Using TCGA, including upon deconvolution of scRNA-seq data, we formalize the clinical importance of high NF-YAr, associated to EMT genes and cell populations. We derive a novel, prognostic 158 genes signature common to BRCA and STAD Claudinlow tumours. Finally, we identify splicing factors associated to high NF-YAr, validating RBFOX2 as promoting expression of NF-YAl. These data bring three relevant results: (i) the definition and clinical implications of NF-YAr and the 158 genes signature in Claudinlow tumours; (ii) genetic evidence of 28 amino acids in NF-YAl with EMT-promoting capacity; (iii) the definition of selected splicing factors associated to NF-YA isoforms.

miR-140-3P Induces Chemotherapy Resistance in Esophageal Carcinoma by Targeting the NFYA-MDR1 Axis.

Esophageal carcinoma (EC) is recognized as the 6th most frequent carcinoma in China, with esophageal squamous cell carcinoma (ESCC) being the predominant histologic type. Currently, chemotherapy is one among the most important therapy modalities for patients with ESCC. However, resistance to chemotherapeutic drugs leads to limited treatment options and poor prognosis. In our study, the analysis of small RNA sequencing and digital gene expression (DGE) profiling was done to recognize the microRNAs (miRNAs) and key genes related with drug resistance in ESCC. It was noticed that the hsa-miRNA-140-3p (miR-140-3p) expression was considerably higher in drug-resistant cells than in sensitive cells. In addition, DGE identified target genes of miR-140-3p might perform key roles in ESCC. Furthermore, this work exhibited that miR-140-3p represents the nuclear transcription factor Y subunit alpha (NFYA) gene by targeting its 3'-untranslated regions. Such an interaction might influence the formation of the transcription factor NFY trimer, which in turn may inhibit the transcription of the multidrug resistance 1 gene and, ultimately, to multidrug resistance in ESCC. The inhibition of miR-140-3p decreased resistance to oxaliplatin in EC. Therefore, miR-140-3p may serve as a molecular marker for treatment response, efficacy, and prognosis of chemotherapy in ESCC patients.

Five NUCLEAR FACTOR-Y subunit B genes in rapeseed (Brassica napus) promote flowering and root elongation in Arabidopsis.

MAIN CONCLUSION: Heterologous expression of BnNF-YB2, BnNF-YB3, BnNF-YB4, BnNF-YB5, or BnNF-YB6 from rapeseed promotes the floral process and also affects root development in Arabidopsis. The transcriptional regulator NUCLEAR FACTOR-Y (NF-Y) is a heterotrimeric complex composed of NF-YA, NF-YB, and NF-YC proteins and is ubiquitous in yeast, animal, and plant systems. In this study, we found that five NF-YB proteins from rapeseed (Brassica napus), including BnNF-YB2, BnNF-YB3, BnNF-YB4, BnNF-YB5, and BnNF-YB6 (BnNF-YB2/3/4/5/6), all function in photoperiodic flowering and root elongation. Sequence alignment and phylogenetic analysis showed that BnNF-YB2/3 and BnNF-YB4/5/6 were clustered with Arabidopsis AtNF-YB2 and AtNF-YB3, respectively, implying that these NF-YBs are evolutionarily and functionally conserved. In support of this hypothesis, the heterologous expression of individual BnNF-YB2, 3, 4, 5, or 6 in Arabidopsis promoted early flowering under a long-day photoperiod. Further analysis suggested that BnNF-YB 2/3/4/5/6 elevated the expression of key downstream flowering time genes including CO, FT, LFY and SOC1. Promoter-GUS fusion analysis showed that the five BnNF-YBs were expressed in a variety of tissues at various developmental stages and GFP fusion analysis revealed that all BnNF-YBs were localized to the nucleus. In addition, we demonstrated that the heterologous expression of individual BnNF-YB2/3/4/5/6 in Arabidopsis promoted root elongation and increased the number of root tips formed under both normal and treatment with simulators of abiotic stress conditions.

SP1 and NFY Regulate the Expression of PNPT1, a Gene Encoding a Mitochondrial Protein Involved in Cancer.

The Polyribonucleotide nucleotidyltransferase 1 gene (PNPT1) encodes polynucleotide phosphorylase (PNPase), a 3'-5' exoribonuclease involved in mitochondrial RNA degradation and surveillance and RNA import into the mitochondrion. Here, we have characterized the PNPT1 promoter by in silico analysis, luciferase reporter assays, electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP), siRNA-based mRNA silencing and RT-qPCR. We show that the Specificity protein 1 (SP1) transcription factor and Nuclear transcription factor Y (NFY) bind the PNPT1 promoter, and have a relevant role regulating the promoter activity, PNPT1 expression, and mitochondrial activity. We also found in Kaplan-Meier survival curves that a high expression of either PNPase, SP1 or NFY subunit A (NFYA) is associated with a poor prognosis in liver cancer. In summary, our results show the relevance of SP1 and NFY in PNPT1 expression, and point to SP1/NFY and PNPase as possible targets in anti-cancer therapy.

A conserved HSF:miR169:NF-YA loop involved in tomato and Arabidopsis heat stress tolerance.

Heat stress transcription factors (HSFs) and microRNAs (miRNAs) regulate different stress and developmental networks in plants. Regulatory feedback mechanisms are at the basis of these networks. Here, we report that plants improve their heat stress tolerance through HSF-mediated transcriptional regulation of MIR169 and post-transcriptional regulation of Nuclear Factor-YA (NF-YA) transcription factors. We show that HSFs recognize tomato (Solanum lycopersicum) and Arabidopsis MIR169 promoters using yeast one-hybrid/chromatin immunoprecipitation-quantitative PCR. Silencing tomato HSFs using virus-induced gene silencing (VIGS) reduced Sly-MIR169 levels and enhanced Sly-NF-YA9/A10 target expression. Further, Sly-NF-YA9/A10 VIGS knockdown tomato plants and Arabidopsis plants overexpressing At-MIR169d or At-nf-ya2 mutants showed a link with increased heat tolerance. In contrast, Arabidopsis plants overexpressing At-NF-YA2 and those expressing a non-cleavable At-NF-YA2 form (miR169d-resistant At-NF-YA2) as well as plants in which At-miR169d regulation is inhibited (miR169d mimic plants) were more sensitive to heat stress, highlighting NF-YA as a negative regulator of heat tolerance. Furthermore, post-transcriptional cleavage of NF-YA by elevated miR169 levels resulted in alleviation of the repression of the heat stress effector HSFA7 in tomato and Arabidopsis, revealing a retroactive control of HSFs by the miR169:NF-YA node. Hence, a regulatory feedback loop involving HSFs, miR169s and NF-YAs plays a critical role in the regulation of the heat stress response in tomato and Arabidopsis plants.