miR156-SPL and miR169-NF-YA Modules Regulate the Induction of Somatic Embryogenesis in Arabidopsis via LEC- and Auxin-Related Pathways.

The embryogenic transition of plant somatic cells to produce somatic embryos requires extensive reprogramming of the cell transcriptome. The prominent role of transcription factors (TFs) and miRNAs in controlling somatic embryogenesis (SE) induction in plants was documented. The profiling of MIRNA expression in the embryogenic culture of Arabidopsis implied the contribution of the miR156 and miR169 to the embryogenic induction. In the present study, the function of miR156 and miR169 and the candidate targets, SPL and NF-YA genes, were investigated in Arabidopsis SE. The results showed that misexpression of MIRNA156 and candidate SPL target genes (SPL2, 3, 4, 5, 9, 10, 11, 13, 15) negatively affected the embryogenic potential of transgenic explants, suggesting that specific fine-tuning of the miR156 and target genes expression levels seems essential for efficient SE induction. The results revealed that SPL11 under the control of miR156 might contribute to SE induction by regulating the master regulators of SE, the LEC (LEAFY COTYLEDON) genes (LEC1, LEC2, FUS3). Moreover, the role of miR169 and its candidate NF-YA targets in SE induction was demonstrated. The results showed that several miR169 targets, including NF-YA1, 3, 5, 8, and 10, positively regulated SE. We found, that miR169 via NF-YA5 seems to modulate the expression of a master SE regulator LEC1/NF-YA and other auxin-related genes: YUCCA (YUC4, 10) and PIN1 in SE induction. The study provided new insights into miR156-SPL and miR169-NF-YA functions in the auxin-related and LEC-controlled regulatory network of SE.

Overexpression of the PtrNF-YA6 gene inhibits secondary cell wall thickening in poplar.

The NF-Y gene family in plants plays a crucial role in numerous biological processes, encompassing hormone response, stress response, as well as growth and development. In this study, we first used bioinformatics techniques to identify members of the NF-YA family that may function in wood formation. We then used molecular biology techniques to investigate the role and molecular mechanism of PtrNF-YA6 in secondary cell wall (SCW) formation in Populus trichocarpa. We found that PtrNF-YA6 protein was localized in the nucleus and had no transcriptional activating activity. Overexpression of PtrNF-YA6 had an inhibitory effect on plant growth and development and significantly suppressed hemicellulose synthesis and SCW thickening in transgenic plants. Yeast one-hybrid and ChIP-PCR assays revealed that PtrNF-YA6 directly regulated the expression of hemicellulose synthesis genes (PtrGT47A-1, PtrGT8C, PtrGT8F, PtrGT43B, PtrGT47C, PtrGT8A and PtrGT8B). In conclusion, PtrNF-YA6 can inhibit plant hemicellulose synthesis and SCW thickening by regulating the expression of downstream SCW formation-related target genes.

ZmmiR169q/ZmNF-YA8 is a module that homeostatically regulates primary root growth and salt tolerance in maize.

In response to salt stress, plants alter the expression of manifold gene networks, enabling them to survive and thrive in the face of adversity. As a result, the growth and development of plant roots could be drastically altered, with significant inhibition of the growth of root meristematic zones. Although it is known that root growth is primarily regulated by auxins and cytokinins, the molecular regulatory mechanism by which salt stress stunts root meristems remains obscure. In this study, we found that the ZmmiR169q/ZmNF-YA8 module regulates the growth of maize taproots in response to salt stress. Salt stress downregulates ZmmiR169q expression, allowing for significant upregulation of ZmNF-YA8, which, in turn, activates ZmERF1B, triggering the upregulation of ASA1 and ASA2, two rate-limiting enzymes in the biosynthesis of tryptophan (Trp), leading to the accumulation of auxin in the root tip, thereby inhibiting root growth. The development of the maize root is stymied as meristem cell division and meristematic zone expansion are both stifled. This study reveals the ZmmiR169q/ZmNF-YA8 module's involvement in maintaining an equilibrium in bestowing plant salt tolerance and root growth and development under salt stress, providing new insights into the molecular mechanism underlying the homeostatic regulation of plant development in response to salt stress.

CDCA8 induced by NF-YA promotes hepatocellular carcinoma progression by regulating the MEK/ERK pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most lethal malignant tumors. Cell division cycle associated 8 (CDCA8) is an important multifactorial regulator in cancers. However, its up and downstream targets and effects in HCC are still unclear. METHODS: A comprehensive bioinformatics analysis was performed using The Cancer Genome Atlas dataset (TCGA) to explore novel core oncogenes. We quantified CDCA8 levels in HCC tumors using qRT-PCR. HCC cell's proliferative, migratory, and invasive abilities were detected using a Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, clone formation, and a Transwell assay. An orthotopic tumor model and tail vein model were constructed to determine the effects of CDCA8 inhibition in vivo. The mechanism underlying CDCA8 was investigated using RNA sequencing. The prognostic value of CDCA8 was assessed with immunohistochemical staining of the tissue microarrays. RESULTS: CDCA8 was identified as a novel oncogene during HCC development. The high expression of CDCA8 was an independent predictor for worse HCC outcomes both in publicly available datasets and in our cohort. We found that CDCA8 knockdown inhibited HCC cell proliferation, colony formation, and migration by suppressing the MEK/ERK pathway in vitro. Moreover, CDCA8 deficiency significantly inhibited tumorigenesis and metastasis. Next-generation sequencing and laboratory validation showed that CDCA8 silencing inhibited the expression of TPM3, NECAP2, and USP13. Furthermore, NA-YA overexpression upregulated the expression of CDCA8. CDCA8 knockdown could attenuate NF-YA-mediated cell invasion in vitro. The expression of NF-YA alone or in combined with CDCA8 were validated as significant independent risk factors for patient survival. CONCLUSION: Our findings revealed that the expression of CDCA8 alone or in combined with NF-YA contributed to cancer progression, and could serve as novel potential therapeutic targets for HCC patients.

Identification and expression analysis of maize NF-YA subunit genes.

NF-YAs encode subunits of the nuclear factor-Y (NF-Y) gene family. NF-YAs represent a kind of conservative transcription factor in plants and are involved in plant growth and development, as well as resistance to biotic and abiotic stress. In this study, 16 maize (Zea mays) NF-YA subunit genes were identified using bioinformatics methods, and they were divided into three categories by a phylogenetic analysis. A conserved domain analysis showed that most contained a CCAAT-binding transcription factor (CBFB) _NF-YA domain. Maize NF-YA subunit genes showed very obvious tissue expression characteristics. The expression level of the NF-YA subunit genes significantly changed under different abiotic stresses, including Fusarium graminearum infection and salicylic acid (SA) or jasmonic acid (JA) treatments. After inoculation with Setosphaeria turcica and Cochliobolus heterostrophus, the lesion areas of nfya01 and nfya06 were significantly larger than that of B73, indicating that ZmNFYA01 and ZmNFYA06 positively regulated maize disease resistance. ZmNFYA01 and ZmNFYA06 may regulated maize disease resistance by affecting the transcription levels of ZmPRs. Thus, NF-YA subunit genes played important roles in promoting maize growth and development and resistance to stress. The results laid a foundation for clarifying the functions and regulatory mechanisms of NF-YA subunit genes in maize.

Genome-Wide Identification and Characterization of the NF-YA Gene Family and Its Expression in Response to Different Nitrogen Forms in Populus × canescens.

The NF-YA gene family is a class of conserved transcription factors that play important roles in plant growth and development and the response to abiotic stress. Poplar is a model organism for studying the rapid growth of woody plants that need to consume many nutrients. However, studies on the response of the NF-YA gene family to nitrogen in woody plants are limited. In this study, we conducted a systematic and comprehensive bioinformatic analysis of the NF-YA gene family based on Populus × canescens genomic data. A total of 13 PcNF-YA genes were identified and mapped to 6 chromosomes. According to the amino acid sequence characteristics and genetic structure of the NF-YA domains, the PcNF-YAs were divided into five clades. Gene duplication analysis revealed five pairs of replicated fragments and one pair of tandem duplicates in 13 PcNF-YA genes. The PcNF-YA gene promoter region is rich in different cis-acting regulatory elements, among which MYB and MYC elements are the most abundant. Among the 13 PcNF-YA genes, 9 contained binding sites for P. × canescens miR169s. In addition, RT-qPCR data from the roots, wood, leaves and bark of P. × canescens showed different spatial expression profiles of PcNF-YA genes. Transcriptome data and RT-qPCR analysis showed that the expression of PcNF-YA genes was altered by treatment with different nitrogen forms. Furthermore, the functions of PcNF-YA genes in transgenic poplar were analyzed, and the potential roles of PcNF-YA genes in the response of poplar roots to different nitrogen forms were revealed, indicating that these genes regulate root growth and development.

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.

Ectopic expression of AtNF-YA6-VP16 in petals results in a novel petal phenotype in Torenia fournieri.

MAIN CONCLUSION: A novel Torenia phenotype having separate petals was obtained by the combination of NF-YA6-VP16 with a floral organ-specific promoter. Genetic engineering techniques helped in obtaining novel flower colors and shapes, in particular, by introducing functionally modified transcription factors (TFs) to ornamental flower species. Herein, we used functionally modified Arabidopsis TFs fused with the repression domain SRDX and the activation domain VP16 to screen for novel floral traits in Torenia fournieri Lind (torenia). We avoided undesired phenotypes unrelated to flowers by expressing these TFs through a floral organ-specific promoter belonging to the class-B genes, GLOBOSA (TfGLO). Fourteen constructs were produced to express functionally modified Arabidopsis TFs in which each of SRDX and VP16 was fused into 7 TFs that were used for the collective transformation of Torenia plants. Among the obtained transgenic plants, phenotypes with novel floral traits reflected in separate petals within normally gamopetalous flower lines. Sequencing analysis revealed that the transgenic plants contained nuclear factor-YA6 (NF-YA6) fused with the VP16. In the margin between the lips of the petals and tube in the TfGLOp:NF-YA6-VP16 plants, staminoid organs have been developed to separate petals. In the petals of the TfGLOp:NF-YA6-VP16 plants, the expression of a Torenia class C gene, PLENA (TfPLE), was found to be ectopically increased. Moreover, expression of TfPLE-VP16 under the control of the TfGLO promoter brought a similar staminoid phenotype observed in the TfGLOp:NF-YA6-VP16 plants. These results suggest that the introduction of the TfGLOp:NF-YA6-VP16 induced TfPLE expression, resulting in the formation of staminoid petals and separation of them.

The miR169n-NF-YA8 regulation module involved in drought resistance in Brassica napus L.

As an ancient and conserved plant microRNA (miRNA) family, miR169 targets nuclear factor Y subunit alpha (NF-YA) family members. The miR169-NF-YA module is associated with plant development and various stress responses. However, the function of miR169 in response to drought stress in rapeseed (Brassica napus L.) is unclear. In the present study, we showed that miR169n acted as a negative regulator of drought resistance in rapeseed by targeting a nuclear factor Y-A gene, NF-YA8. miR169n was strongly down-regulated by drought stress. Expression of a miR169n target mimicry construct (MIM169n) which functioned as a sponge to trap miR169n resulted in enhanced resistance of transgenic plants to both osmotic stress at the post-germination stage and drought stress at the seedling stage. MIM169n plants had a higher relative water content (RWC) and proline content, lower relative electrolyte leakage (REL), and showed higher antioxidative capability compared with those of control (CK) plants under drought stress. Moreover, NF-YA8 was verified as a target of miR169n, and overexpression of NF-YA8 led to improved tolerance of rapeseed to osmotic stress at the post-germination stage. Overall, our findings implied that the miR169n-NF-YA8 regulatory module could serve as a potential target for genetic improvement of drought resistance in B. napus.

Rapid excavating a FLOWERING LOCUS T-regulator NF-YA using genotyping-by-sequencing.

Soybean (Glycine max (L.) Merrill) is one of the most important crop plants in the world as an important source of protein for both human consumption and livestock fodder. As flowering time contributes to yield, finding new QTLs and further identifying candidate genes associated with various flowering time are fundamental to enhancing soybean yield. In this study, a set of 120 recombinant inbred lines (RILs) which was developed from a cross of two soybean cultivars, Suinong4 (SN4) and ZK168, were genotyped by genotyping-by-sequencing (GBS) approach and phenotyped to expand the cognitive of flowering time by quantitative trait loci (QTL) analysis. Eventually, three stable QTLs related to flowering time which were detected separately located on chromosome 14, 18, and 19 under long-day (LD) conditions. We predicted candidate genes for each QTL and carried out association analyses between the putative causal alleles and flowering time. Moreover, a transient transfection assay was performed and showed that NUCLEAR FACTOR YA 1b (GmNF-YA1b) as a strong candidate for the QTL on chromosome 19 might affect flowering time by suppressing the expression of FLOWERING LOCUS T (GmFT) genes in soybean. QTLs detected in this study would provide fundamental resources for finding candidate genes and clarify the mechanisms of flowering which would be helpful for breeding novel high-yielding soybean cultivars. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01237-w.

Lotus japonicus Nuclear Factor YA1, a nodule emergence stage-specific regulator of auxin signalling.

Organogenesis of legume root nodules begins with the nodulation factor-dependent stimulation of compatible root cells to initiate divisions, signifying an early nodule primordium formation event. This is followed by cellular differentiation, including cell expansion and vascular bundle formation, and we previously showed that Lotus japonicus NF-YA1 is essential for this process, presumably by regulating three members of the SHORT INTERNODES/STYLISH (STY) transcription factor gene family. In this study, we used combined genetics, genomics and cell biology approaches to characterize the role of STY genes during root nodule formation and to test a hypothesis that they mediate nodule development by stimulating auxin signalling. We show here that L. japonicus STYs are required for nodule emergence. This is attributed to the NF-YA1-dependent regulatory cascade, comprising STY genes and their downstream targets, YUCCA1 and YUCCA11, involved in a local auxin biosynthesis at the post-initial cell division stage. An analogous NF-YA1/STY regulatory module seems to operate in Medicago truncatula in association with the indeterminate nodule patterning. Our data define L. japonicus and M. truncatula NF-YA1 genes as important nodule emergence stage-specific regulators of auxin signalling while indicating that the inductive stage and subsequent formation of early nodule primordia are mediated through an independent mechanism(s).

Genome-wide identification of NF-YA gene family in cotton and the positive role of GhNF-YA10 and GhNF-YA23 in salt tolerance.

Nuclear factor YA (NF-YA) genes play important roles in many biological processes, such as leaf growth, nitrogen nutrition, drought resistance, and salt stress. The functions of NF-YA genes in cotton have not been elucidated. The current study identified a total of 16, 16, 31, and 29 genes from Gossypium raimondii, G. arboretum, G. barbadense, and G. hirsutum, respectively. The NF-YA genes in cotton were phylogenetically classified into 4 groups. Analysis of gene structure, conserved motifs and multiple sequence alignments supported the evolutionary conservation of NF-YA family genes in cotton. Analysis of the expression patterns of GhNF-YAs in cotton suggested that GhNF-YAs play important roles in plant growth, development, and stress responses. The quantitative real-time PCR (qRT-PCR) validation of selected genes suggested that GhNF-YA genes are induced in response to salt, drought, ABA, and MeJA treatments. GhNF-YA genes may regulate salt and drought stress via the ABA or MeJA pathway. Silencing of GhNF-YA10 and GhNF-YA23 significantly reduced the salt tolerance of cotton seedlings, indicating that these genes participate in the regulation of the response of cotton to salt stress. These results establish a foundation for subsequent functional studies of the NF-YA gene family in cotton.

NF-YA promotes the cell proliferation and tumorigenic properties by transcriptional activation of SOX2 in cervical cancer.

NF-YA is considered as a crucial regulator for the maintenance of cancer stem cell (CSC) and involved in various types of malignant tumours. However, the exact function and molecular mechanisms of NF-YA in the progression of cervical cancer remains poorly understood. Here, the expression of NF-YA detected by immunohistochemistry was gradually increased from normal cervical tissues, to the high-grade squamous intraepithelial lesions, and then to cervical cancer tissues. NF-YA promoted the cell proliferation and tumorigenic properties of cervical cancer cells as well as tumorsphere formation and chemoresistance in vitro. The luciferase reporter assay combined with mutagenesis analyses and Western blotting showed that NF-YA trans-activated the expression of SOX2 in cervical cancer. Furthermore, quantitative chromatin immunoprecipitation (qChIP) and electrophoretic mobility shift assay (EMSA) confirmed that NF-YA protein directly bound to the CCAAT box region located upstream of the SOX2 promoter. Together, our data demonstrated that NF-YA was highly expressed in cervical cancer and promoted the cell proliferation, tumorigenicity and CSC characteristic by trans-activating the expression of SOX2.

Arabidopsis NUCLEAR FACTOR Y A8 inhibits the juvenile-to-adult transition by activating transcription of MIR156s.

Vegetative (juvenile-to-adult) and flowering (vegetative-to-reproductive) phase changes are crucial in the life cycle of higher plants. MicroRNA156 (miR156) and its target SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes are master regulators that determine vegetative phase changes. The miR156 level gradually declines as a plant ages and its expression is rapidly repressed by sugar. However, the underlying regulatory mechanism of transcriptional regulation of the MIR156 gene remains largely unknown. In this study, we demonstrated that Arabidopsis NUCLEAR FACTOR Y A8 (NF-YA8) binds directly to CCAAT cis-elements in the promoters of multiple MIR156 genes, thus activating their transcription and inhibiting the juvenile-to-adult transition. NF-YA8 was highly expressed in juvenile-stage leaves, and significantly repressed with developmental age and by sugar signals. Our results suggest that NF-YA8 acts as a signaling hub, integrating internal developmental age and sugar signals to regulate the transcription of MIR156s, thus affecting the juvenile-to-adult and flowering transitions.

NF-YA Overexpression in Lung Cancer: LUAD.

The trimeric transcription factor (TF) NF-Y regulates the CCAAT box, a DNA element enriched in promoters of genes overexpressed in many types of cancer. The regulatory NF-YA is present in two major isoforms, NF-YAl ("long") and NF-YAs ("short"). There is growing indication that NF-YA levels are increased in tumors. Here, we report interrogation of RNA-Seq TCGA (The Cancer Genome Atlas)-all 576 samples-and GEO (Gene Expression Ominibus) datasets of lung adenocarcinoma (LUAD). NF-YAs is overexpressed in the three subtypes, proliferative, inflammatory, and TRU (terminal respiratory unit). CCAAT is enriched in promoters of tumor differently expressed genes (DEG) and in the proliferative/inflammatory intersection, matching with KEGG (Kyoto Encyclopedia of Genes and Genomes) terms cell-cycle and signaling. Increasing levels of NF-YAs are observed from low to high CpG island methylator phenotypes (CIMP). We identified 166 genes overexpressed in LUAD cell lines with low NF-YAs/NF-YAl ratios: applying this centroid to TCGA samples faithfully predicted tumors' isoform ratio. This signature lacks CCAAT in promoters. Finally, progression-free intervals and hazard ratios concurred with the worst prognosis of patients with either a low or high NF-YAs/NF-YAl ratio. In conclusion, global overexpression of NF-YAs is documented in LUAD and is associated with aggressive tumor behavior; however, a similar prognosis is recorded in tumors with high levels of NF-YAl and overexpressed CCAAT-less genes.

Mutant analysis in the nonlegume Parasponia andersonii identifies NIN and NF-YA1 transcription factors as a core genetic network in nitrogen-fixing nodule symbioses.

●Nitrogen-fixing nodulation occurs in 10 taxonomic lineages, with either rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. In the legume-rhizobium endosymbiosis, both processes are guarded by the transcription factor NODULE INCEPTION (NIN) and its downstream target genes of the NUCLEAR FACTOR Y (NF-Y) complex. ●It is hypothesized that nodulation has a single evolutionary origin c. 110 Ma, followed by many independent losses. Despite a significant body of knowledge of the legume-rhizobium symbiosis, it remains elusive which signalling modules are shared between nodulating species in different taxonomic clades. We used Parasponia andersonii to investigate the role of NIN and NF-YA genes in rhizobium nodulation in a nonlegume system. ●Consistent with legumes, P. andersonii PanNIN and PanNF-YA1 are coexpressed in nodules. By analyzing single, double and higher-order CRISPR-Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF-YA1 are PanNIN-dependent and that PanNF-YA1 is specifically required for intracellular rhizobium infection. ●This demonstrates that NIN and NF-YA1 have conserved symbiotic functions. As Parasponia and legumes diverged soon after the birth of the nodulation trait, we argue that NIN and NF-YA1 represent core transcriptional regulators in this symbiosis.

Discovery, characterization and potential roles of a novel NF-YAx splice variant in human neuroblastoma.

BACKGROUND: Identification of novel cancer-associated splice variants is of potential diagnostic, prognostic and therapeutic importance. NF-Y transcription factor is comprised of NF-YA, NF-YB and NF-YC subunits, binds inverted CCAAT-boxes in ≈70% of gene promoters, regulates > 1000 cancer-associated genes and proteins involved in proliferation, staminality, differentiation, apoptosis, metabolism and is subject to component alternative splicing. RT-PCR evaluation of alternative NF-YA splicing in primary human neuroblastomas (NBs), led to discovery of a novel NF-YAx splice variant, also expressed during mouse embryo development and induced by doxorubicin in NB cells. Here, we report the discovery and characterisation of NF-YAx and discus its potential roles in NB. METHODS: NF-YAx cDNA was RT-PCR-cloned from a stage 3 NB (provided by the Italian Association of Haematology and Paediatric Oncology, Genova, IT), sequenced and expressed as a protein using standard methods and compared to known fully-spliced NF-YAl and exon B-skipped NF-YAs isoforms in: EMSAs for capacity to form NF-Y complexes; by co-transfection, co-immunoprecipitation and Western blotting for capacity to bind Sp1; by IF for localisation; in AO/EtBr cell-death and colony formation assays for relative cytotoxicity, and by siRNA knockdown, use of inhibitors and Western blotting for potential mechanisms of action. Stable SH-SY5Y transfectants of all three NF-YA isoforms were also propagated and compared by RT-PCR and Western blotting for differences in cell-death and stem cell (SC)-associated gene expression, in cell-death assays for sensitivity to doxorubicin and in in vitro proliferation, substrate-independent growth and in vivo tumour xenograft assays for differences in growth and tumourigenic capacity. RESULTS: NF-YAx was characterized as a novel variant with NF-YA exons B, D and partial F skipping, detected in 20% of NF-YA positive NBs, was the exclusive isoform in a stage 3 NB, expressed in mouse stage E11.5-14 embryos and induced by doxorubicin in SH-SY5Y NB cells. The NF-YAx protein exhibited nuclear localisation, competed with other isoforms in CCAAT box-binding NF-Y complexes but, in contrast to other isoforms, did not bind Sp1. NF-YAx expression in neural-related progenitor and NB cells repressed Bmi1 expression, induced KIF1Bß expression and promoted KIF1Bß-dependent necroptosis but in NB cells also selected tumourigenic, doxorubicin-resistant, CSC-like sub-populations, resistant to NF-YAx cytotoxicity. CONCLUSIONS: The discovery of NF-YAx in NBs, its expression in mouse embryos and induction by doxorubicin in NB cells, unveils a novel NF-YA splice mechanism and variant, regulated by and involved in development, genotoxic-stress and NB. NF-YAx substitution of other isoforms in NF-Y complexes and loss of capacity to bind Sp1, characterises this novel isoform as a functional modifier of NF-Y and its promotion of KIF1Bß-dependent neural-lineage progenitor and NB cell necroptosis, association with doxorubicin-induced necroptosis and expression in mouse embryos coinciding with KIF1Bß-dependent sympathetic neuroblast-culling, confirm a cytotoxic function and potential role in suppressing NB initiation. On the other hand, the in vitro selection of CSC-like NB subpopulations resistant to NF-YAx cytotoxicity not only helps to explain high-level exclusive NF-YAx expression in a stage 3 NB but also supports a role for NF-YAx in disease progression and identifies a potential doxorubicin-inducible mechanism for post-therapeutic relapse.

NF-YA Overexpression in Lung Cancer: LUSC.

The CCAAT box is recognized by the trimeric transcription factor NF-Y, whose NF-YA subunit is present in two major splicing isoforms, NF-YAl ("long") and NF-YAs ("short"). Little is known about the expression levels of NF-Y subunits in tumors, and nothing in lung cancer. By interrogating RNA-seq TCGA and GEO datasets, we found that, unlike NF-YB/NF-YC, NF-YAs is overexpressed in lung squamous cell carcinomas (LUSC). The ratio of the two isoforms changes from normal to cancer cells, with NF-YAs becoming predominant in the latter. NF-YA increased expression correlates with common proliferation markers. We partitioned all 501 TCGA LUSC tumors in the four molecular cohorts and verified that NF-YAs is similarly overexpressed. We analyzed global and subtype-specific RNA-seq data and found that CCAAT is the most abundant DNA matrix in promoters of genes overexpressed in all subtypes. Enriched Gene Ontology terms are cell-cycle and signaling. Survival curves indicate a worse clinical outcome for patients with increasing global amounts of NF-YA; same with hazard ratios with very high and, surprisingly, very low NF-YAs/NF-YAl ratios. We then analyzed gene expression in this latter cohort and identified a different, pro-migration signature devoid of CCAAT. We conclude that overexpression of the NF-Y regulatory subunit in LUSC has the scope of increasing CCAAT-dependent, proliferative (NF-YAshigh) or CCAAT-less, pro-migration (NF-YAlhigh) genes. The data further reinstate the importance of analysis of single isoforms of TFs involved in tumor development.

Overexpression of soybean miR169c confers increased drought stress sensitivity in transgenic Arabidopsis thaliana.

The miR169 family, a large-scale microRNA gene family conserved in plants, is involved in stress responses, although how soybean miR169 functions in response to drought stress remains unclear. We show that gma-miR169c exerts a negative regulatory role in the response to drought stress by inhibiting the expression of its target gene, nuclear factor Y-A (NF-YA). A real-time RT-PCR analysis indicated that gma-miR169c is widely expressed in soybean tissues and induced by polyethylene glycol (PEG), high salt, cold stress and abscisic acid (ABA). Histochemical ß-glucuronidase (GUS) staining showed that the gma-miR169c promoter drives GUS reporter gene expression in various transgenic Arabidopsis tissues, and the stress-induced pattern was confirmed in transgenic Arabidopsis and transgenic soybean hairy roots. Arabidopsis overexpressing gma-miR169c is more sensitive to drought stress, with reduced survival, accelerated leaf water loss, and shorter root length than wild-type plants. We identified a precise cleavage site for 10 gma-miR169c targets and found reduced transcript levels of the AtNFYA1 and AtNFYA5 transcription factors in gma-miR169c-overexpressing Arabidopsis and reduced expression of the stress response genes AtRD29A, AtRD22, AtGSTU25 and AtCOR15A. These results indicate that gma-miR169c plays a negative regulatory role in drought stress and is a candidate miRNA for improving plant drought adaptation.

p53-targeted lincRNA-p21 acts as a tumor suppressor by inhibiting JAK2/STAT3 signaling pathways in head and neck squamous cell carcinoma.

BACKGROUND: Long intergenic noncoding RNA p21 (lincRNA-p21) is considered a target of wild-type p53, but little is known about its regulation by mutant p53 and its functions during the progression of head and neck squamous cell carcinoma (HNSCC). METHODS: RNAscope was used to detect the expression and distribution of lincRNA-p21. Chromatin immunoprecipitation and electrophoretic mobility shift assays were performed to analyze the transcriptional regulation of lincRNA-p21 in HNSCC cells. The biological functions of lincRNA-p21 were investigated in vitro and in vivo. RNA immunoprecipitation and pull-down assays were used to detect the direct binding of lincRNA-p21. RESULTS: Lower lincRNA-p21 expression was observed in HNSCC tissues and indicated worse prognosis. Both wild and mutant type p53 transcriptionally regulated lincRNA-p21, but nuclear transcription factor Y subunit alpha (NF-YA) was essential for mutant p53 in the regulation of lincRNA-p21. Ectopic expression of lincRNA-p21 significantly inhibited cell proliferation capacity in vitro and in vivo and vice versa. Moreover, the overexpression of lincRNA-p21 induced G1 arrest and apoptosis. Knockdown NF-YA expression reversed tumor suppressor activation of lincRNA-p21 in mutant p53 cells, not wild-type p53 cells. A negative correlation was observed between lincRNA-p21 and the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) in HNSCC tissues. High lincRNA-p21 expression inhibited Janus kinase 2 (JAK2)/STAT3 signal activation and vice versa. Further, we observed direct binding to STAT3 by lincRNA-p21 in HNSCC cells, which suppressed STAT3-induced oncogenic potential. CONCLUSIONS: Our results revealed the transcriptional regulation of lincRNA-p21 by the mutant p53/NF-YA complex in HNSCC. LincRNA-p21 acted as a tumor suppressor in HNSCC progression, which was attributed to direct binding to STAT3 and blocking of JAK2/STAT3 signaling.