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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. [ABSTRACT FROM AUTHOR]

Heat stress is an essential factor affecting maize growth and yield formation. The exploration and mechanism analysis of related tolerance genes is an important field for breeding maize heat stress tolerant varieties. However, there is few study in this aspect. In this study, we identified a nuclear factor ZmNF-YC13 associated with heat stress response, and its encoding gene expression was rapidly induced by high temperature and osmotic stress. The promoter of Arabidopsis thaliana heat stress-inducible expression gene AtHSP70 was used to drive ZmNF-YC13 and the heat-inducible expression maize material of ZmNF-YC13 (HSP21Pro:ZmNF-YC13-myc) was successfully screened. Phenotypic analysis after high temperature treatment demonstrated that leaf length, leaf width, shoot thickness, fresh and dry weight of shoot and root were significantly higher than wild type. The relative expression level showed that ZmNF-YC13 could enhance the inducible level of downstream heat stress response genes in response to heat stress. Luciferase reporter assay and ChIP-qPCR assay also revealed that ZmNF-YC13 could regulate the expression of heat stress transcription factor ZmHsfA2c. These results confirmed preliminarily that ZmNF-YC13 could improve the heat tolerance of maize by regulating downstream heat stress response genes, which could provide a theoretical basis for marker-assisted selection and germplasm identification using the polymorphism of this locus. [ABSTRACT FROM AUTHOR]

Nuclear factor Y (NF-Y) is a class of transcription factors (TFs) with various biological functions that exist in almost all eukaryotes. In plants, heterotrimers composed of different NF-Y subunits are numerous and have different functions that can participate in the regulation of plant growth at various stages. However, NF-Y genes have not been systematically analyzed in chrysanthemum, only involving several NF-Y members. In this study, forty-six NF-Y members were identified from the diploid species Chrysanthemum seticuspe, including eight NF-YA members, twenty-one NF-YB members, and seventeen NF-YC members. These NF-Y genes were analyzed for their physicochemical characteristics, multiple alignments, conserved motifs, gene structure, promoter elements, and chromosomal location. Phylogenetic analysis revealed that only two gene pairs in C. seticuspe underwent gene duplication events. The Ka/Ks ratios were both less than one, indicating that the two pairs underwent purifying selection. Promoter element analysis showed that multiple abiotic stress and hormone response elements were present in the CsNF-Y genes, suggesting that these genes play an important role in the response to stress, growth, and development in plants. Further validation of candidate genes in response to drought regulation using RT-qPCR demonstrated that CsNF-Y genes in C. seticuspe play an important role in drought regulation. [ABSTRACT FROM AUTHOR]

Abstract Background Approaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patient outcomes and response to treatments. The transcription factor NF-Y participates to the progression from benign epithelium to both localized and metastatic PCa and is associated with aggressive transcriptional profile. The gene encoding for NF-YA, the DNA-binding subunit of NF-Y, produces two alternatively spliced transcripts, NF-YAs and NF-YAl. Bioinformatic analyses pointed at NF-YA splicing as a key transcriptional signature to discriminate between different tumor molecular subtypes. In this study, we aimed to determine the pathophysiological role of NF-YA splice variants in PCa and their association with aggressive subtypes. Methods Data on the expression of NF-YA isoforms were extracted from the TCGA (The Cancer Genome Atlas) database of tumor prostate tissues and validated in prostate cell lines. Lentiviral transduction and CRISPR-Cas9 technology allowed the modulation of the expression of NF-YA splice variants in PCa cells. We characterized 3D cell cultures through in vitro assays and RNA-seq profilings. We used the rank-rank hypergeometric overlap approach to identify concordant/discordant gene expression signatures of NF-YAs/NF-YAl-overexpressing cells and human PCa patients. We performed in vivo studies in SHO-SCID mice to determine pathological and molecular phenotypes of NF-YAs/NF-YAl xenograft tumors. Results NF-YA depletion affects the tumorigenic potential of PCa cells in vitro and in vivo. Elevated NF-YAs levels are associated to aggressive PCa specimens, defined by Gleason Score and TNM classification. NF-YAl overexpression increases cell motility, while NF-YAs enhances cell proliferation in PCa 3D spheroids and xenograft tumors. The transcriptome of NF-YAs-spheroids has an extensive overlap with localized and metastatic human PCa signatures. According to PCa PAM50 classification, NF-YAs transcript levels are higher in LumB, characterized by poor prognosis compared to LumA and basal subtypes. A significant decrease in NF-YAs/NF-YAl ratio distinguishes PCa circulating tumor cells from cancer cells in metastatic sites, consistently with pro-migratory function of NF-YAl. Stratification of patients based on NF-YAs expression is predictive of clinical outcome. Conclusions Altogether, our results indicate that the modulation of NF-YA isoforms affects prostate pathophysiological processes and contributes to cancer-relevant phenotype, in vitro and in vivo. Evaluation of NF-YA splicing may represent a new molecular strategy for risk assessment of PCa patients.

The nuclear factor Y (NF-Y) gene family is an important transcription factor family consisting of three subfamilies, NF-YA, NF-YB and NF-YC, which are widely involved in plant growth and development, stress responses and other processes. In this study, we identified 64 members of the NF-Y gene family in the M. sativa L. (Xinjiang Daye) genome, including 11 MsNF-YAs, 33 MsNF-YBs and 20 MsNF-YCs. Analysis of conserved motifs indicated that each unit included unique compounds of motifs, although certain members lost some motifs. Conserved functional domain analysis showed that each subunit contained a specific set of functional domains. Analysis of cis-acting elements in the promoter region of the MsNF-Y genes identified a series of cis-acting elements associated with stress responses. In addition, the transcriptome data and qRT-PCR analysis showed that MsNF-Y genes were significantly induced or downregulated by alkali treatment. The results of this study may help to establish a basis for further cloning and functional studies of NF-Y genes in Medicago sativa and other related legume species. [ABSTRACT FROM AUTHOR]

Nuclear factor Y (NF-Y) is a class of transcription factors (TFs) with various biological functions that exist in almost all eukaryotes. In plants, heterotrimers composed of different NF-Y subunits are numerous and have different functions that can participate in the regulation of plant growth at various stages. However, NF-Y genes have not been systematically analyzed in chrysanthemum, only involving several NF-Y members. In this study, forty-six NF-Y members were identified from the diploid species Chrysanthemum seticuspe, including eight NF-YA members, twenty-one NF-YB members, and seventeen NF-YC members. These NF-Y genes were analyzed for their physicochemical characteristics, multiple alignments, conserved motifs, gene structure, promoter elements, and chromosomal location. Phylogenetic analysis revealed that only two gene pairs in C. seticuspe underwent gene duplication events. The Ka/Ks ratios were both less than one, indicating that the two pairs underwent purifying selection. Promoter element analysis showed that multiple abiotic stress and hormone response elements were present in the CsNF-Y genes, suggesting that these genes play an important role in the response to stress, growth, and development in plants. Further validation of candidate genes in response to drought regulation using RT-qPCR demonstrated that CsNF-Y genes in C. seticuspe play an important role in drought regulation.

SUMMARY: The success of plant reproduction depends on the timely transition from the vegetative phase to reproductive growth, a process often referred to as flowering. Although several plant‐specific transcription factors belonging to the Teosinte Branched 1/Cycloidea/Proliferating Cell Factor (TCP) family are reportedly involved in the regulation of flowering in Arabidopsis, the molecular mechanisms, especially for Class I TCP members, are poorly understood. Here, we genetically identified Class I TCP7 as a positive regulator of flowering time. Protein interaction analysis indicated that TCP7 interacted with several Nuclear Factor‐Ys (NF‐Ys), known as the 'pioneer' transcription factors; CONSTANS (CO), a main photoperiod regulator of flowering. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was differentially expressed in the dominant‐negative mutant of TCP7 (lcu) and the loss‐of‐function mutant of Class I TCP members (septuple). Additionally, we obtained genetic and molecular evidence showing that TCP7 directly activates the flowering integrator gene, SOC1. Moreover, TCP7 synergistically activated SOC1 expression upon interacting with CO and NF‐Ys in vivo. Collectively, our results provide compelling evidence that TCP7 synergistically interacts with NF‐Ys to activate the transcriptional expression of the flowering integrator gene SOC1. Significance Statement: We demonstrated that TCP7 promoted flowering by interacting with NF‐Ys and CO and directly binding and regulating the expression of SOC1 in plants, which broaden our understanding of the regulatory mechanism of plant flowering transition, the function of NF‐Ys and plant‐specific transcription factor Class I TCP. [ABSTRACT FROM AUTHOR]

Background: Approaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patient outcomes and response to treatments. The transcription factor NF-Y participates to the progression from benign epithelium to both localized and metastatic PCa and is associated with aggressive transcriptional profile. The gene encoding for NF-YA, the DNA-binding subunit of NF-Y, produces two alternatively spliced transcripts, NF-YAs and NF-YAl. Bioinformatic analyses pointed at NF-YA splicing as a key transcriptional signature to discriminate between different tumor molecular subtypes. In this study, we aimed to determine the pathophysiological role of NF-YA splice variants in PCa and their association with aggressive subtypes. Methods: Data on the expression of NF-YA isoforms were extracted from the TCGA (The Cancer Genome Atlas) database of tumor prostate tissues and validated in prostate cell lines. Lentiviral transduction and CRISPR-Cas9 technology allowed the modulation of the expression of NF-YA splice variants in PCa cells. We characterized 3D cell cultures through in vitro assays and RNA-seq profilings. We used the rank-rank hypergeometric overlap approach to identify concordant/discordant gene expression signatures of NF-YAs/NF-YAl-overexpressing cells and human PCa patients. We performed in vivo studies in SHO-SCID mice to determine pathological and molecular phenotypes of NF-YAs/NF-YAl xenograft tumors. Results: NF-YA depletion affects the tumorigenic potential of PCa cells in vitro and in vivo. Elevated NF-YAs levels are associated to aggressive PCa specimens, defined by Gleason Score and TNM classification. NF-YAl overexpression increases cell motility, while NF-YAs enhances cell proliferation in PCa 3D spheroids and xenograft tumors. The transcriptome of NF-YAs-spheroids has an extensive overlap with localized and metastatic human PCa signatures. According to PCa PAM50 classification, NF-YAs transcript levels are higher in LumB, characterized by poor prognosis compared to LumA and basal subtypes. A significant decrease in NF-YAs/NF-YAl ratio distinguishes PCa circulating tumor cells from cancer cells in metastatic sites, consistently with pro-migratory function of NF-YAl. Stratification of patients based on NF-YAs expression is predictive of clinical outcome. Conclusions: Altogether, our results indicate that the modulation of NF-YA isoforms affects prostate pathophysiological processes and contributes to cancer-relevant phenotype, in vitro and in vivo. Evaluation of NF-YA splicing may represent a new molecular strategy for risk assessment of PCa patients. [ABSTRACT FROM AUTHOR]

Soybean is an important crop worldwide, but its production is severely affected by salt stress. Understanding the regulatory mechanism of salt response is crucial for improving the salt tolerance of soybean. Here, we reveal a role for nuclear factor Y subunit GmNFYA in salt tolerance of soybean likely through the regulation of histone acetylation. GmNFYA is induced by salt stress. Overexpression of GmNFYA significantly enhances salt tolerance in stable transgenic soybean plants by inducing salt‐responsive genes. Analysis in soybean plants with transgenic hairy roots also supports the conclusion. GmNFYA interacts with GmFVE, which functions with putative histone deacetylase GmHDA13 in a complex for transcriptional repression possibly by reducing H3K9 acetylation at target loci. Under salt stress, GmNFYA likely accumulates and competes with GmHDA13 for interaction with GmFVE, leading to the derepression and maintenance of histone acetylation for activation of salt‐responsive genes and finally conferring salt tolerance in soybean plants. In addition, a haplotype I GmNFYA promoter is identified with the highest self‐activated promoter activity and may be selected during future breeding for salt‐tolerant cultivars. Our study uncovers the epigenetic regulatory mechanism of GmNFYA in salt‐stress response, and all the factors/elements identified may be potential targets for genetic manipulation of salt tolerance in soybean and other crops. [ABSTRACT FROM AUTHOR]

Abstract 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 KIF1Bβ expression and promoted KIF1Bβ-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 KIF1Bβ-dependent neural-lineage progenitor and NB cell necroptosis, association with doxorubicin-induced necroptosis and expression in mouse embryos coinciding with KIF1Bβ-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.

Reactive oxygen species (ROS) are important signaling molecules in many physiological processes, yet excess ROS leads to cell damage and can lead to pathology. Accordingly, cells need to maintain tight regulation of ROS levels, and ROS-responsive transcriptional reprogramming is central to this process. Although it has long been recognized that oxidative stress leads to rapid, significant changes in gene expression, the impact of oxidative stress on the underlying chromatin accessibility landscape remained unclear. Here, we asked whether ROS-responsive transcriptional reprogramming is accompanied by reprogramming of the chromatin environment in MCF7 human breast cancer cells. Using a time-course exposure to multiple inducers of oxidative stress, we determined that the widespread ROS-responsive changes in gene expression induced by ROS occur with minimal changes to the chromatin environment. While we did observe changes in chromatin accessibility, these changes were: (1) far less numerous than gene expression changes after oxidative stress, and (2) occur within pre-existing regions of accessible chromatin. Transcription factor (TF) footprinting analysis of our ATAC-seq experiments identified 5 TFs or TF families with evidence for ROS-responsive changes in DNA binding: NRF2, AP-1, p53, NFY, and SP/KLF. Importantly, several of these (AP-1, NF–Y, and SP/KLF factors) have not been previously implicated as widespread regulators in the response to ROS. In summary, we have characterized genome-wide changes in gene expression and chromatin accessibility in response to ROS treatment of MCF7 cells, and we have found that regulation of the large-scale transcriptional response to excess ROS is primarily constrained by the cell's pre-existing chromatin landscape. [Display omitted] • Widespread ROS-responsive changes in gene expression occur with minimal changes to the chromatin accessibility landscape. • Quantitative changes in chromatin accessibility occur within pre-existing regions of accessible chromatin. • ROS-mediated gene induction is associated with increased NRF2, AP-1, and p53 footprints near target genes. • ROS-mediated gene repression is associated with decreased SP1 and NF–Y footprints near target genes. [ABSTRACT FROM AUTHOR]

Excessive accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER) can trigger ER stress. Although regulation of this stress response in plants is an important and effective means of mitigating the damage caused by stress, the associated genes and mechanisms in maize remain largely unknown. In this study, we identified the ER stress-responsive gene ZmNF-YC13, which encodes a protein localized in the nuclei and cytoplasm of maize cells. The overexpression of ZmNF-YC13 in maize and Arabidopsis was found to confer plant ER stress tolerance, and gene expression analyses revealed that the overexpression of this gene upregulates the expression of genes involved in protein folding and the ER-associated degradation (ERAD) system, including ERdj, BiP, and E3 ubiquitin ligase. Moreover, we established that ZmNF-YC13 activates the promoters of the chaperone genes ZmERdj and ZmBiP in protoplasts, which function in the elimination of unfolded and misfolded proteins in the ER. Based on our findings, we propose that ZmNF-YC13 positively regulates the ER stress response in maize by controlling chaperone genes, and we believe that these results could provide valuable information for further maize resistance breeding. [ABSTRACT FROM AUTHOR]

The three helical Histone Fold Motif (HFM) of core histone proteins provides an evolutionarily favored site for the protein–DNA interface. Despite significant variation in sequence, the HFM retains a distinctive structural fold that has diversified into several non-histone protein families. In this work, we explore the ancestry of non-histone HFM containing families in the plant kingdom. A sequence search algorithm was developed using iterative profile Hidden Markov Models to identify remote homologs of core-histone proteins. The resulting hits were functionally annotated, classified into families, and subjected to comprehensive phylogenetic analyses via Maximum likelihood and Bayesian methods. We have identified 4390 HFM containing proteins in the plant kingdom that are not histones, mostly existing as diverse transcription factor families, distributed widely within and across taxonomic groups. Patterns of homology suggest that core histone subunit H2A has evolved into newer families like NF-YC and DRAP1, whereas the H2B subunit of core histones shares a common ancestry with NF-YB and DR1 class of TFs. Core histone subunits H3 and H4 were found to have evolved into DPE and TAF proteins, respectively. Taken together these results provide insights into diversification events during the evolution of the HFM, including sub-functionalization and neo-functionalization of the HFM. [ABSTRACT FROM AUTHOR]

The expression of drought tolerance in wheat is based on a complex genetic mechanism and involves many genes. Gene expression is contingent upon various transcription factors which are proteins in nature, hence themselves being products of specific genes known as Transcription Factor genes. In the present studies, one such gene family; the NF-Y Gene Family has been explored for its role in conferring drought tolerance in wheat. One hundred genotypes of wheat were screened at seedling stage for morphological traits related with drought tolerance. Expression analysis of nine NF-Y genes was carried out to observe their expression patterns and drought responsiveness. The genotypes 29-SAWSN 11-12 (1014) and Chakwal-50 were the highest performers, while Nax-2 5924 was the lowest performer under drought. The reportedly drought responsive gene Traes_3AS_9151583B9.1 expressed differentially in case of drought tolerant and susceptible genotypes. Its ortholog, Traes_2BS_F23D9EA93.1 gave a similar expression pattern, thereby indicating its potential drought responsiveness. The results of expression analysis supported the results of phenotypic evaluation. The information generated might be useful for breeding drought tolerant wheat by harnessing the potential of drought responsive Ta-NFY genes. [ABSTRACT FROM AUTHOR]

The nuclear factor Y (NF-Y) gene family is an important transcription factor family consisting of three subfamilies, NF-YA, NF-YB and NF-YC, which are widely involved in plant growth and development, stress responses and other processes. In this study, we identified 64 members of the NF-Y gene family in the M. sativa L. (Xinjiang Daye) genome, including 11 MsNF-YAs, 33 MsNF-YBs and 20 MsNF-YCs. Analysis of conserved motifs indicated that each unit included unique compounds of motifs, although certain members lost some motifs. Conserved functional domain analysis showed that each subunit contained a specific set of functional domains. Analysis of cis-acting elements in the promoter region of the MsNF-Y genes identified a series of cis-acting elements associated with stress responses. In addition, the transcriptome data and qRT-PCR analysis showed that MsNF-Y genes were significantly induced or downregulated by alkali treatment. The results of this study may help to establish a basis for further cloning and functional studies of NF-Y genes in Medicago sativa and other related legume species.

Chronic and excessive alcohol consumption leads to alcoholic liver disease (ALD). However, the molecular mechanisms in the regulation of ALD have not been fully deciphered. Liver lipid accumulation is an important research direction in ALD. In this study, the physiological role of nuclear factor Y (NF–Y) in ALD and the related mechanisms were investigated using murine hepatocytes and an ethanol-induced liver injury mouse model. In this study, ethanol promoted hepatic NF–Y expression in a mouse model and Hepa1-6 mouse hepatocytes. Lentivirus-mediated NF–Y overexpression in Hepa1-6 cells markedly increased sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FASN) expression compared with empty vector control cells. Conversely, CRISPR/Cas9-mediated knockdown of NF–Y subunit A (NF-YA) attenuated FASN and SREBP1 expression. Mechanistically, luciferase reporter gene assays and chromatin immunoprecipitation (ChIP) analysis indicated that NF–Y activates the transcription of SREBP1 by directly binding to the CCAAT regulatory sequence motif in the promoter. Overall, our results reveal a previously unrecognized physiological function of NF–Y in ALD by activating sterol regulatory element-binding protein 1 (SREBP1). Modulation of hepatic NF–Y expression may therefore offer an attractive therapeutic approach to manage ALD. • Ethanol promotes NF–Y expression in vivo and in vitro. • NF–Y takes part in alcoholic liver disease by activating sterol regulatory element-binding protein 1(SREBP1). • NF–Y activates transcription of SREBP1 via directly binding to the CCAAT regulatory sequence motif in the promoter. [ABSTRACT FROM AUTHOR]

SUMMARY: NF‐Y transcription factor comprises three subunits: NF‐YA, NF‐YB and NF‐YC. NF‐YB and NF‐YC dimerize through their histone fold domain (HFD), which can bind DNA in a non‐sequence‐specific fashion while serving as a scaffold for NF‐YA trimerization. Upon trimerization, NF‐YA specifically recognizes the CCAAT box sequence on promoters and enhancers. In plants, each NF‐Y subunit is encoded by several genes giving rise to hundreds of potential heterotrimeric combinations. In addition, plant NF‐YBs and NF‐YCs interact with other protein partners to recognize a plethora of genomic motifs, as the CCT protein family that binds CORE sites. The NF‐Y subunit organization and its DNA‐binding properties, together with the NF‐Y HFD capacity to adapt different protein modules, represent plant‐specific features that play a key role in development, growth and reproduction. Despite their relevance, these features are still poorly understood at the molecular level. Here, we present the structures of Arabidopsis and rice NF‐YB/NF‐YC dimers, and of an Arabidopsis NF‐Y trimer in complex with the FT CCAAT box, together with biochemical data on NF‐Y mutants. The dimeric structures identify the key residues for NF‐Y HFD stabilization. The NF‐Y/DNA structure and the mutation experiments shed light on HFD trimerization interface properties and the NF‐YA sequence appetite for the bases flanking the CCAAT motif. These data explain the logic of plant NF‐Y gene expansion: the trimerization adaptability and the flexible DNA‐binding rules serve the scopes of accommodating the large number of NF‐YAs, CCTs and possibly other NF‐Y HFD binding partners and a diverse audience of genomic motifs. [ABSTRACT FROM AUTHOR]

As predominant pollinators, honeybees are important for crop production and terrestrial ecosystems. Recently, various environmental stresses have led to large declines in honeybee populations in many regions. The ability of honeybees to respond to these stresses is critical for their survival. However, the details of the stress defense mechanisms of honeybees have remained elusive. Here, we found that the Nuclear Factor Y (NF-Y) family (containing NF-YA, NF-YB, and NF-YC) is a novel stress mediator family that regulates honeybee environmental stress resistance. NF-YA localized in the nucleus, NF-YB accumulated in the cytoplasm, and NF-YC presented in both the nucleus and cytoplasm. NF-YC interacted with NF-YA and NF-YB in vitro and in vivo, and the nuclear import of NF-YB relied on its interaction with NF-YC. We further found that the expression of NF-Y was induced under multiple stress conditions. In addition, NF-Y regulated many stress responses and antioxidant genes at the transcriptome-wide level, and knockdown of NF-Y repressed the expression of stress-inducible genes, particularly LOC108003540 and LOC107994062, under adverse circumstances. Silencing NF-Y lowered honeybee stress resistance by reducing total antioxidant capacity and enhancing oxidative impairment. Collectively, these results indicate that NF-Y plays important roles in stress responses. Our study sheds light on the underlying defense mechanisms of honeybees under environmental stress. [ABSTRACT FROM AUTHOR]

The transition from vegetative to reproductive development in the temperate cereals is mainly regulated by seasonal cues including vernalization (determined mainly by VRN1 and VRN2 genes) and photoperiod (determined mainly by PPD1 and CO2 genes). The wheat VRN3 gene, which is similar to Arabidopsis FT, plays a central role in the integration of the competing signals from these two pathways. Under long days, VRN3 transcription is down-regulated by VRN2, a unique flowering repressor in cereals, and up-regulated by CO2. Overexpression of VRN3 overcomes VRN2 repression and promotes VRN1 transcription and flowering initiation. Using yeast two- and three-hybrid assays we show here that the CCT domains present in VRN2 and CO2 proteins interact with the same subset of eight NF-Y proteins, and that these CCT proteins compete with NF-YA for interactions with NF-YB proteins. We have confirmed all these interactions in vitro, and the interactions between VRN2 and two of the three NF-YB proteins were further confirmed in planta. In addition, we show that mutations in the CCT domain of VRN2 that eliminate the vernalization requirement in winter wheat also reduce the strength of the interactions between VRN2 and NF-Y proteins, and the ability of VRN2 to compete with CO2. Taken together, our results suggest that the interactions between CCT and NF-Y proteins play an important role in the integration of the vernalization and photoperiod seasonal signals, and provide a flexible combinatorial system to integrate multiple developmental and environmental signals in the regulation of flowering initiation in the temperate cereals.

Key message: The NF-Y gene family was identified and characterized in Populus trichocarpa by genome-wide analysis. Expression of PtoNF-Y was analyzed during floral bud development in Populus tomentosa. Nuclear factor Y (NF-Y) is a transcription factor widely found in eukaryotes. It is composed of three subunits: NF-YA, NF-YB, and NF-YC. NF-Y has been identified as a key regulator of multiple pathways that control responses to developmental, biotic, and abiotic factors. Although the NF-Y gene has been reported in many plants, it has not been comprehensively analyzed in poplar. In this study, a total of 46 NF-Y gene family members in the P. trichocarpa genome were accessed using NF-Y amino acid sequences from Arabidopsis thaliana as a probe. In addition, bioinformatic characterization of NF-Y gene family members in P. trichocarpa, including gene structure, chromosome location, and phylogenetic relationships, was conducted. The results of chromosome distribution showed that the 46 PtNF-Y genes were distributed among 16 chromosomes of P. trichocarpa at varying frequencies. Gene-structure analysis showed that seven members have no introns. Phylogenetic analysis indicated that the NF-Y protein may have similar functions in Arabidopsis and P. trichocarpa. We used transcriptome data from different tissues to analyze the expression of the NF-Y genes in Populus tomentosa, and verified the results by qRT-PCR. The results indicated that some PtoNF-Y genes play significant roles in the floral bud development of P. tomentosa. [ABSTRACT FROM AUTHOR]

Main conclusion: OsNF-YB9 controls heading by affecting expression of regulators of flowering. It affects the development of the reproductive meristem by interacting with MADS1 and controlling expression of hormone-related genes. Nuclear Factor-Y (NF-Y) family of transcription factors takes part in many aspects of growth and development in eukaryotes. They have been classified into three subunit classes, namely, NF-YA, NF-YB and NF-YC. In plants, this transcription factor family is much diverged and takes part in several developmental processes and stress. We investigated NF-Y subunit genes of rice (Oryza sativa) and found OsNF-YB9 as the closest homologue of LEAFY COTYLEDON1. OsNF-YB9 delayed the heading date when ectopically expressed in rice. Expression of several heading date regulating genes such as Hd1, Ehd1, Hd3a and RFT1 were altered. OsNF-YB9 overexpression also resulted in morphological defects in the reproductive organs and led to pseudovivipary. OsNF-YB9 interacted with MADS1, a key regulator of floral development. This NF-Y subunit acted upstream to several transcription factors as well as signalling proteins involved in brassinosteroid and gibberellic acid metabolism and cell cycle. OsNF-YB9 and OsNF-YC12 interacted in planta and the latter also delayed heading in rice upon overexpression suggesting its involvement in a similar pathway. Our data provide new insights into the rice heading date pathway integrating these OsNF-Y subunit members to the network. These features can be exploited to improve vegetative growth and yield of rice plants in future. [ABSTRACT FROM AUTHOR]

Knowledge of all binding sites for transcriptional activators and repressors is essential for computationally aided identification of transcriptional networks. The techniques developed for defining the binding sites of transcription factors tend to be cumbersome and not adaptable to high throughput. We refined a versatile yeast strategy to rapidly and efficiently identify genomic targets of DNA-binding proteins. Yeast expressing a transcription factor is mated to yeast containing a library of genomic fragments cloned upstream of the reporter gene URA3. DNA fragments with target-binding sites are identified by growth of yeast clones in media lacking uracil. The experimental approach was validated with the tumor suppressor protein p53 and the forkhead protein FoxI1 using genomic libraries for zebrafish and mouse generated by shotgun cloning of short genomic fragments. Computational analysis of the genomic fragments recapitulated the published consensus-binding site for each protein. Identified fragments were mapped to identify the genomic context of each binding site. Our yeast screening strategy, combined with bioinformatics approaches, will allow both detailed and high-throughput characterization of transcription factors, scalable to the analysis of all putative DNA-binding proteins.

NF-Y is the CCAAT-binding trimer formed by the histone fold domain (HFD), NF-YB/NF-YC and NF-YA. The CCAAT box is generally prevalent in promoters of “cancer” genes. We reported the overexpression of NF-YA in BRCA, LUAD and LUSC, and of all subunits in HCC. Altered splicing of NF-YA was found in breast and lung cancer. We analyzed RNA-seq datasets of TCGA and cell lines of head and neck squamous cell carcinomas (HNSCC). We partitioned all TCGA data into four subtypes, deconvoluted single-cell RNA-seq of tumors and derived survival curves. The CCAAT box was enriched in the promoters of overexpressed genes. The “short” NF-YAs was overexpressed in all subtypes and the “long” NF-YAl in Mesenchymal. The HFD subunits are overexpressed, except Basal (NF-YB) and Atypical (NF-YC); NF-YAl is increased in p53 mutated tumors. In HPV-positive tumors, high levels of NF-YAs, p16 and ΔNp63 correlate with better prognosis. Deconvolution of single cell RNA-seq (scRNA-seq) found a correlation of NF-YAl with Cancer Associated Fibroblasts (CAFs) and p-EMT cells, a population endowed with metastatic potential. We conclude that overexpression of HFD subunits and NF-YAs is protective in HPV-positive tumors; expression of NF-YAl is largely confined to mutp53 tumors and malignant p-EMT cells.

Nuclear factor Y (NF-Y) is a ubiquitous transcription factor that regulates important physiological and developmental processes. In this study, we identified 34 OsNF-Y genes in rice, including 6 newly identified genes. Expression profile analysis covering the whole life cycle revealed that transcripts of OsNF-Y differentially accumulated in a tissue-specific, preferential or constitutive manner. In addition, gene duplication studies and expression analyses were performed to determine the evolutionary origins of the OsNF-Y gene family. Nine OsNF-Y genes were differentially expressed after treatment of seedlings with one or more abiotic stresses such as drought, salt and cold. Analysis of expression correlation and Gene Ontology annotation suggested that OsNF-Y genes were co-expressed with genes that participated in stress, accumulation of seed storage reserves, and plant development. Co-expression analysis also revealed that OsNF-Y genes might interact with each other, suggesting that NF-Y subunits formed complexes that take part in transcriptional regulation. These results provide useful information for further elucidating the function of the NF-Y family and their regulatory pathways.

Abstract Cell Penetrating Peptides -CPPs- are short aminoacidic stretches present in proteins that have the ability to translocate the plasma membrane and facilitate delivery of various molecules. They are usually rich in basic residues, and organized as alpha helices. NF-Y is a transcription factor heterotrimer formed by two Histone Fold Domain –HFD- subunits and the sequence-specific NF-YA. NF-YA possesses two α-helices rich in basic residues. We show that it efficiently enters cells at nanomolar concentrations in the absence of carrier peptides. Mutagenesis identified at least two separate CPPs in the A1 and A2, which overlap with previously identified nuclear localization signals (NLS). The half-life of the transduced protein is short in human cancer cells, longer in mouse C2C12 myoblasts. The internalized NF-YA is capable of trimerization with the HFD subunits and binding to the target CCAAT box. Functionality is further suggested by protein transfection in C2C12 cells, leading to inhibition of differentiation to myotubes. In conclusion, NF-YA contains CPPs, hinting at novel -and unexpected- properties of this subunit. Highlights • The NF-YA subunit of the CCAAT binding transcription factor NF-Y has Cell penetrating capacity. • Mutagenesis identified two separate Cell Penetrating Peptides (CPPs). • The NF-YA CPPs are alpha elices rich in arginines. • The transduced protein is functional in trimerization and DNAbinding. [ABSTRACT FROM AUTHOR]

If a transgene is effectively delivered to a cell, its expression may still be limited by epigenetic mechanisms that silence the transgene. Indeed, once the transgene reaches the nucleus, it may be bound by histone proteins and condensed into heterochromatin or associated with repressor proteins that block transcription. In this study, we sought to enhance transgene expression by adding binding motifs for several different epigenetic enzymes either upstream or downstream of two promoters (CMV and EF1α). Screening these plasmids revealed that luciferase expression was enhanced 10‐fold (10.4 ± 5.8) by the addition of a CCAAT box just upstream of the EF1α promoter to recruit nuclear transcription factor Y (NF‐Y), while inserting a CCCTC‐binding factor (CTCF) motif downstream of the EF1α promoter enhanced expression at least 14‐fold (14.03 ± 6.54). ChIP assays confirmed that NF‐Y and CTCF bound to the motifs that were added to each plasmid, but the presence of NF‐Y and CTCF did not significantly affect the levels of histone acetylation (H3K9ac) or methylation (H3K9me3). Overall, these results show that transgene expression from the EF1α promoter can be significantly increased with motifs that recruit NF‐Y or CTCF. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1581–1588, 2018 [ABSTRACT FROM AUTHOR]

NF-Y is a pioneer trimeric transcription factor formed by the Histone Fold Domain (HFD) NF-YB/NF-YC subunits and NF-YA. Three subunits are required for DNA binding. CCAAT-specificity resides in NF-YA and transactivation resides in Q-rich domains of NF-YA and NF-YC. They are involved in alternative splicing (AS). We recently showed that NF-YA is overexpressed in breast and lung carcinomas. We report here on the overexpression of all subunits in the liver hepatocellular carcinoma (HCC) TCGA database, specifically the short NF-YAs and NF-YC2 (37 kDa) isoforms. This is observed at all tumor stages, in viral-infected samples and independently from the inflammatory status. Up-regulation of NF-YAs and NF-YC, but not NF-YB, is associated to tumors with mutant p53. We used a deep-learning-based method (DeepCC) to extend the partitioning of the three molecular clusters to all HCC TCGA tumors. In iCluster3, CCAAT is a primary matrix found in promoters of up-regulated genes, and cell-cycle pathways are enriched. Finally, clinical data indicate that, globally, only NF-YAs, but not HFD subunits, correlate with the worst prognosis; in iCluster1 patients, however, all subunits correlate. The data show a difference with other epithelial cancers, in that global overexpression of the three subunits is reported and clinically relevant in a subset of patients; yet, they further reinstate the regulatory role of the sequence-specific subunit.

NF-Y is a transcription factor (TF) comprising three subunits (NF-YA, NF-YB, NF-YC) that binds with high specificity to the CCAAT sequence, a widespread regulatory element in gene promoters of prosurvival, cell-cycle-promoting, and metabolic genes. Tumor cells undergo “metabolic rewiring” through overexpression of genes involved in such pathways, many of which are under NF-Y control. In addition, NF-YA appears to be overexpressed in many tumor types. Thus, limiting NF-Y activity may represent a desirable anti-cancer strategy, which is an ongoing field of research. With virtual-screening docking simulations on a library of pharmacologically active compounds, we identified suramin as a potential NF-Y inhibitor. We focused on suramin given its high water-solubility that is an important factor for in vitro testing, since NF-Y is sensitive to DMSO. By electrophoretic mobility shift assays (EMSA), isothermal titration calorimetry (ITC), STD NMR, X-ray crystallography, and molecular dynamics (MD) simulations, we showed that suramin binds to the histone fold domains (HFDs) of NF-Y, preventing DNA-binding. Our analyses, provide atomic-level detail on the interaction between suramin and NF-Y and reveal a region of the protein, nearby the suramin-binding site and poorly conserved in other HFD-containing TFs, that may represent a promising starting point for rational design of more specific and potent inhibitors with potential therapeutic applications.

NF-YA, the regulatory subunit of the trimeric transcription factor (TF) NF-Y, is regulated by alternative splicing (AS) generating two major isoforms, “long” (NF-YAl) and “short” (NF-YAs). Muscle cells express NF-YAl. We ablated exon 3 in mouse C2C12 cells by a four-guide CRISPR/Cas9n strategy, obtaining clones expressing exclusively NF-YAs (C2-YAl-KO). C2-YAl-KO cells grow normally, but are unable to differentiate. Myogenin and—to a lesser extent, MyoD— levels are substantially lower in C2-YAl-KO, before and after differentiation. Expression of the fusogenic Myomaker and Myomixer genes, crucial for the early phases of the process, is not induced. Myomaker and Myomixer promoters are bound by MyoD and Myogenin, and Myogenin overexpression induces their expression in C2-YAl-KO. NF-Y inactivation reduces MyoD and Myogenin, but not directly: the Myogenin promoter is CCAAT-less, and the canonical CCAAT of the MyoD promoter is not bound by NF-Y in vivo. We propose that NF-YAl, but not NF-YAs, maintains muscle commitment by indirectly regulating Myogenin and MyoD expression in C2C12 cells. These experiments are the first genetic evidence that the two NF-YA isoforms have functionally distinct roles.

The endoplasmic reticulum (ER) is a morphologically dynamic organelle containing different membrane subdomains with distinct cellular functions. Numerous observations have revealed that ER stress response induced by disturbed ER homeostasis is linked to various neurological/neurodegenerative disorders. In contrast, recent findings unveil that ER structural derangements are linked to the progression of several neurological diseases. The derangements involve two distinct, and likely opposing pathways. One is dysfunction of ER dynamics machinery, leading to disruption of ER network organization. Another one is facilitation of pre-existing machinery, leading to generation of markedly-ordered de novo membranous structure. Restoring the ER network can be the effective way toward the cure of ER-deranged neurological disorders.

Objective: Leptin gene expression is highly correlated with cellular lipid content in adipocytes but the transcriptional mechanisms controlling leptin expression in vivo are poorly understood. In this report, we set out to identify cis- and trans-regulatory elements controlling leptin expression. Methods: Leptin-BAC luciferase transgenic mice combining with other computational and molecular techniques were used to identify transcription regulatory elements including a CCAAT-binding protein Nuclear Factor Y (NF-Y). The function of NF-Y in adipocyte was studied in vitro with 3T3-L1 cells and in vivo with adipocyte-specific knockout of NF-Y. Results: Using Leptin-BAC luciferase mice, we showed that DNA sequences between −22 kb and +8.8 kb can confer quantitative expression of a leptin reporter. Computational analysis of sequences and gel shift assays identified a 32 bp sequence (chr6: 28993820–2899385) consisting a CCAAT binding site for Nuclear Factor Y (NF-Y) and this was confirmed by a ChIP assay in vivo. A deletion of this 32 bp sequence in the −22 kb to +8.8 kb leptin-luciferase BAC reporter completely abrogates luciferase reporter activity in vivo. RNAi mediated knockdown of NF-Y interfered with adipogenesis in vitro and adipocyte-specific knockout of NF-Y in mice reduced expression of leptin and other fat specific genes in vivo. Further analyses of the fat specific NF-Y knockout revealed that these animals develop a moderately severe lipodystrophy that is remediable with leptin therapy. Conclusions: These studies advance our understanding of leptin gene expression and show that NF-Y controls the expression of leptin and other adipocyte genes and identifies a new form of lipodystrophy.

Aberrant splicing events are associated with colorectal cancer (CRC) and provide new opportunities for tumor diagnosis and treatment. The expression of the splice variants of NF-YA, the DNA binding subunit of the transcription factor NF-Y, is deregulated in multiple cancer types compared to healthy tissues. NF-YAs and NF-YAl isoforms differ in the transactivation domain, which may result in distinct transcriptional programs. In this study, we demonstrated that the NF-YAl transcript is higher in aggressive mesenchymal CRCs and predicts shorter patients' survival. In 2D and 3D conditions, CRC cells overexpressing NF-YAl (NF-YAl high ) exhibit reduced cell proliferation, rapid single cell amoeboid-like migration, and form irregular spheroids with poor cell-to-cell adhesion. Compared to NF-YAs high , NF-YAl high cells show changes in the transcription of genes involved in epithelial-mesenchymal transition, extracellular matrix and cell adhesion. NF-YAl and NF-YAs bind similarly to the promoter of the E-cadherin gene, but oppositely regulate its transcription. The increased metastatic potential of NF-YAl high cells in vivo was confirmed in zebrafish xenografts. These results suggest that the NF-YAl splice variant could be a new CRC prognostic factor and that splice-switching strategies may reduce metastatic CRC progression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Summary: Many plants sense the seasonal cues, day length or photoperiod changes, to align the timing of the developmental transition to flowering with changing seasons for reproductive success. Inductive day lengths through the photoperiod pathway induce the expression of FLOWERING LOCUS T (FT) or FT relatives that encode a major mobile florigen to promote flowering. In Arabidopsis thaliana, under inductive long days the photoperiod pathway output CONSTANS (CO) accumulates toward the end of the day, and associates with the B and C subunits of Nuclear Factor Y (NF‐Y) to form the NF‐CO complex that acts to promote FT expression near dusk, whereas Polycomb group (PcG) proteins function to silence FT expression. How NF‐CO acts to antagonize the function of PcG proteins to regulate FT expression remains unclear. Here, we show that the NF‐CO complex bound to the proximal FT promoter, through chromatin looping, acts in concert with an NF‐Y complex bound to a distal enhancer to reduce the levels of PcG proteins, including both Polycomb repressive complex 1 (PRC1) and PRC2 at the FT promoter, leading to a relieving of Polycomb silencing and thus FT de‐repression near dusk. Thus, our study provides molecular insights on how the ‘active’ photoperiod pathway and the ‘repressive’ Polycomb silencing system interact to control temporal FT expression, conferring the long‐day induction of flowering in Arabidopsis. [ABSTRACT FROM AUTHOR]

The heterotrimeric NF-Y complex is a pioneer factor that binds to CCAAT-genes and regulates their transcription. NF-Y cooperates with multiple transcription factors and co-regulators in order to positively or negatively influence gene transcription. The recruitment of NF-Y to CCAAT box is significantly enriched in cancer-associated gene promoters loci and positively correlates with malignancy. NF-Y subunits, in particular the DNA-binding subunit NF-YA and the histone-fold subunit NF-YC, appear overexpressed in specific types of cancer. Here we demonstrate that NF-Y subunits expression is finely regulated through transcriptional and post-translational mechanisms thus allowing control over basal expression levels. NF-Y negatively regulates the transcription of the genes encoding for its subunits. DNA pull-down/affinity purification assay coupled with Mass Spectrometry identified putative co-regulators, such as Lamin A, involved in NF-YA gene transcription level. We also evidentiate how the stability of the complex is severely affected by the absence of one subunit. Our results identified for the first time one of the mechanisms responsible for NF-Y expression, which may be involved in the aberrant expression and activity observed in tumor cells and other pathological conditions. [ABSTRACT FROM AUTHOR]

The cardiac sarco/endoplasmic reticulum Ca2+-ATPase-2a (SERCA2a) is vital for the correct handling of calcium concentration in cardiomyocytes. Recent studies showed that the induction of endoplasmic reticulum (ER) stress (ERS) with the SERCA2 inhibitor Thapsigargin (Tg) increases the mRNA and protein levels of SERCA2a. The SERCA2 gene promoter contains an ERS response element (ERSE) at position −78 bp that is conserved among species and might transcriptionally regulate SERCA2 gene expression. However, its involvement in SERCA2 basal and calcium-mediated transcriptional activation has not been elucidated. In this work, we show that in cellular cultures of neonatal rat ventricular myocytes, the treatment with Tg or the calcium ionophore A23187 increases the SERCA2a mRNA and protein abundance, as well as the transcriptional activity of two chimeric human SERCA2 gene constructs, containing −254 and −2579 bp of 5′-regulatory region cloned in the pGL3-basic vector and transiently transfected in cultured cardiomyocytes. We found that the ERSE present in the SERCA2 proximal promoter contains a CCAAT box that is involved in basal and ERS-mediated hSERCA2 transcriptional activation. The EMSA results showed that the CCAAT box present in the ERSE recruits the NF-Y transcription factor. Additionally, by ChIP assays, we confirmed in vivo binding of NF-Y and C/EBPβ transcription factors to the SERCA2 gene proximal promoter. [ABSTRACT FROM AUTHOR]

CONSTANS (CO) is a master regulator of flowering time, although the mechanisms underlying its role as a transcriptional regulator are not well understood. The DNA-binding domain of CO shares homology with that of NUCLEAR FACTOR YA (NF-YA), a subunit of the CCAAT -binding trimer NF-Y. Recent publications indicate that CO and its rice homolog HEADING DATE 1 (Hd1) form heterotrimers with the histone-fold subunits of NF-Y to efficiently bind promoter elements in the florigen genes. Differences in the DNA-binding specificities of NF-Y and NF-CO can be conceptualized based on our knowledge of the 3D structure of the NF-Y/ CCAAT complex. Here we discuss the modes of assembly of NF-Y-like heterotrimers and possible models for their activity as flexible sequence-specific transcriptional regulators. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) is a morphologically dynamic organelle containing different membrane subdomains with distinct cellular functions. Numerous observations have revealed that ER stress response induced by disturbed ER homeostasis is linked to various neurological/neurodegenerative disorders. In contrast, recent findings unveil that ER structural derangements are linked to the progression of several neurological diseases. The derangements involve two distinct, and likely opposing pathways. One is dysfunction of ER dynamics machinery, leading to disruption of ER network organization. Another one is facilitation of pre-existing machinery, leading to generation of markedly- ordered de novo membranous structure. Restoring the ER network can be the effective way toward the cure of ER-deranged neurological disorders. [ABSTRACT FROM AUTHOR]

Myostatin (MSTN) is a strong inhibitor of skeletal muscle growth in human and other vertebrates. Its transcription is controlled by a proximal promoter/enhancer ( Mstn P/E) containing a TATA box besides CREB, NF-Y, MEIS1 and FXR transcription factor binding sites (TFBSs), which are conserved throughout evolution. The aim of this work was to investigate the role of these TFBSs on Mstn P/E activity and evaluate the potential of their putative ligands as Mstn trans regulators. Mstn P/E mutant constructs were used to establish the role of conserved TFBSs using dual-luciferase assays. Expression analyses were performed by RT-PCR and in situ hybridization in C2C12 myoblasts and E10.5 mouse embryos, respectively. Our results revealed that CREB, NF-Y and MEIS1 sites are required to balance Mstn P/E activity, keeping Mstn transcription within basal levels during myoblast proliferation. Furthermore, our data showed that NF-Y site is essential, although not sufficient, to mediate Mstn P/E transcriptional activity. In turn, CREB and MEIS1 binding sites seem to depend on the presence of NF-Y site to induce Mstn P/E. FXR appears not to confer any effect on Mstn P/E activity, except in the absence of all other conserved TFBS. Accordingly, expression studies pointed to CREB, NF-Y and MEIS1 but not to FXR factors as possible regulators of Mstn transcription in the myogenic context. Altogether, our findings indicated that CREB, NF-Y and MEIS1 conserved sites are essential to control basal Mstn transcription during early myogenesis, possibly by interacting with these or other related factors. [ABSTRACT FROM AUTHOR]

The organic cation transporter 1 (OCT1, SLC22A1) is strongly expressed in the human liver and facilitates the hepatic uptake of drugs such as morphine, metformin, tropisetron, sumatriptan and fenoterol and of endogenous substances such as thiamine. OCT1 expression is inter-individually highly variable. Here, we analyzed SNPs in the OCT1 promoter concerning their potential contribution to the variability in OCT1 expression. Using electrophoretic mobility shift and luciferase reporter gene assays in HepG2, Hep3B, and Huh7 cell lines, we identified the SNPs −1795G>A (rs6935207) and −201C>G (rs58812592) as having effects on transcription factor binding and/or promoter activity. The A-allele of the −1795G>A SNP showed allele-specific binding of the transcription factor NF-Y leading to 2.5-fold increased enhancer activity of the artificial SV40 promoter. However, the −1795G>A SNP showed no significant effects on the native OCT1 promoter activity. Furthermore, the −1795G>A SNP was not associated with the pharmacokinetics of metformin, fenoterol, sumatriptan and proguanil in healthy individuals or tropisetron efficacy in patients undergoing chemotherapy. Allele-dependent differences in USF1/2 binding and nearly total loss in OCT1 promoter activity were detected for the G-allele of −201C>G, but the SNP is apparently very rare. In conclusion, common OCT1 promoter SNPs have only minor effects on OCT1 expression.

Nuclear factor Y (NF-Y) is one of the most ubiquitous transcription factors (TFs), comprising NF-YA, NF-YB and NF-YC subunits, and has been identified and reported in various aspects of development for plants and animals. In this work, StNF-YB3.1 , a putative potato NF-YB subunit encoding gene, was isolated from Solanum tuberosum by rapid amplification of cDNA ends (RACE). Overexpression of StNF-YB3.1 in potato (cv. Atlantic) resulted in accelerated onset of flowering, and significant increase in leaf chlorophyll content in field trials. However, transgenic potato plants overexpressing StNF-YB3.1 (OEYB3.1) showed significant decreases in photosynthetic rate and stomatal conductance both at tuber initiation and bulking stages. OEYB3.1 lines were associated with significantly fewer tuber numbers and yield reduction. Guard cell size and stomatal density were not changed in OEYB3.1 plants, whereas ABA-mediated stomatal closure was accelerated compared to that of wild type plants because of the up-regulation of genes for ABA signaling, such as StCPK10-like , StSnRK2.6/OST1-like , StSnRK2.7-like and StSLAC1-like . We speculate that the acceleration of stomatal closure was a possible reason for the significantly decreased stomatal conductance and photosynthetic rate. [ABSTRACT FROM AUTHOR]

Background The two control points of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase ( HMGCR ) and squalene monooxygenase ( SQLE ) are known targets of the transcription factor sterol-regulatory element binding protein-2 (SREBP-2). Yet the location of the sterol-regulatory elements (SREs) and cofactor binding sites, nuclear factor-Y (NF-Y) and specificity protein 1 (Sp1), have not been satisfactorily mapped in the human SQLE promoter, or at all in the human HMGCR promoter. Methods We used luciferase reporter assays to screen the sterol-responsiveness of a library of predicted SRE, Sp1 and NF-Y site mutants and hence identify bone fide binding sites. We confirmed SREs via an electrophoretic mobility shift assay (EMSA) and ChIP-PCR. Results We identified two SREs in close proximity in both the human HMGCR and SQLE promoters, as well as one NF-Y site in HMGCR and two in SQLE . In addition, we found that HMGCR expression is highly activated only when SREBP-2 levels are very high, in contrast to the low density lipoprotein receptor ( LDLR ), a result reflected in mouse models used in other studies. Conclusions Both HMGCR and SQLE promoters have two SREs that may act as a homing region to attract a single SREBP-2 homodimer, with HMGCR being activated only when there is absolute need for cholesterol synthesis. This ensures preferential uptake of exogenous cholesterol via LDLR, thereby conserving energy. General Significance We provide the first comprehensive investigation of SREs and NF-Ys in the human HMGCR and SQLE promoters, increasing our fundamental understanding of the transcriptional regulation of cholesterol synthesis. [ABSTRACT FROM AUTHOR]

The recently determined crystal structures of the sequence-specific transcription factor NF-Y have illuminated the structural mechanism underlying transcription at the CCAAT box. NF-Y is a trimeric protein complex composed by the NF-YA, NF-YB, and NF-YC subunits. NF-YB and NF-YC contain a histone-like domain and assemble on a head-to-tail fashion to form a dimer, which provides the structural scaffold for the DNA sugar-phosphate backbone binding (mimicking the nucleosome H2A/H2B–DNA assembly) and for the interaction with NF-YA. The NF-YA subunit hosts two structurally extended α-helices; one is involved in NF-YB/NF-YC binding and the other inserts deeply into the DNA minor groove, providing exquisite sequence-specificity for recognition and binding of the CCAAT box. The analysis of these structural data is expected to serve as a powerful guide for future experiments aimed at understanding the role of post-translational modification at NF-Y regulation sites and to unravel the three-dimensional architecture of higher order complexes formed between NF-Y and other transcription factors that act synergistically for transcription activation. Moreover, these structures represent an excellent starting point to challenge the formation of a stable hybrid nucleosome between NF-Y and core histone proteins, and to rationalize the fine molecular details associated with the wide combinatorial association of plant NF-Y subunits. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani. [ABSTRACT FROM AUTHOR]

The CCAAT-binding factor CBF/NF-Y is needed for cell proliferation and early embryonic development. NF-Y can regulate the expression of different cell type-specific genes that are activated by various physiological signaling pathways. Dysregulation of NF-Y was observed in pathogenic conditions in humans such as scleroderma, neurodegenerative disease, and cancer. Conditional inactivation of the NF-YA gene in mice demonstrated that NF-Y activity is essential for normal tissue homeostasis, survival, and metabolic function. Altogether, NF-Y is an essential transcription factor that plays a critical role in mammalian development, from the early stages to adulthood, and in human pathogenesis. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani. [ABSTRACT FROM AUTHOR]

Both Drosophila melanogaster and Caenorhabditis elegans ( C. elegans ) are useful model organisms to study in vivo roles of NF-Y during development. Drosophila NF-Y (dNF-Y) consists of three subunits dNF-YA, dNF-YB and dNF-YC. In some tissues, dNF-YC-related protein Mes4 may replace dNF-YC in dNF-Y complex. Studies with eye imaginal disc-specific dNF-Y-knockdown flies revealed that dNF-Y positively regulates the sevenless gene encoding a receptor tyrosine kinase, a component of the ERK pathway and negatively regulates the Sensless gene encoding a transcription factor to ensure proper development of R7 photoreceptor cells together with proper R7 axon targeting. dNF-Y also controls the Drosophila Bcl-2 ( debcl ) to regulate apoptosis. In thorax development, dNF-Y is necessary for both proper Drosophila JNK ( basket ) expression and JNK signaling activity that is responsible for thorax development. Drosophila p53 gene was also identified as one of the dNF-Y target genes in this system. C. elegans contains two forms of NF-YA subunit, CeNF-YA1 and CeNF-YA2. C. elegans NF-Y (CeNF-Y) therefore consists of CeNF-YB, CeNF-YC and either CeNF-YA1 or CeNF-YA2. CeNF-Y negatively regulates expression of the Hox gene egl-5 (ortholog of Drosophila Abdominal-B ) that is involved in tail patterning. CeNF-Y also negatively regulates expression of the tbx-2 gene that is essential for development of the pharyngeal muscles, specification of neural cell fate and adaptation in olfactory neurons. Negative regulation of the expression of egl-5 and tbx-2 by CeNF-Y provides new insight into the physiological meaning of negative regulation of gene expression by NF-Y during development. In addition, studies on NF-Y in platyhelminths are also summarized. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani. [ABSTRACT FROM AUTHOR]

CCAAT boxes are motifs found within the proximal promoter of many genes, including the human globin genes. The highly conserved nature of CCAAT box motifs within the promoter region of both α-like and β-like globin genes emphasises the functional importance of the CCAAT sequence in globin gene regulation. Mutations within the β-globin CCAAT box result in β-thalassaemia, while mutations within the distal γ-globin CCAAT box cause the Hereditary Persistence of Foetal Haemoglobin, a benign condition which results in continued γ-globin expression during adult life. Understanding the transcriptional regulation of the globin genes is of particular interest, as reactivating the foetal γ-globin gene alleviates the symptoms of β-thalassaemia and sickle cell anaemia. NF-Y is considered to be the primary activating transcription factor which binds to globin CCAAT box motifs. Here we review recruitment of NF-Y to globin CCAAT boxes and the role NF-Y plays in regulating globin gene expression. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani. [ABSTRACT FROM AUTHOR]

Background Sequence specific polyamide HxIP 1 , targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα ( topo IIα ) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3 ) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole. Methods DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18 h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®. Results Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5′-TACGAT-3′ on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1–2 μM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage. Conclusions Polyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents. General significance Pharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani. [ABSTRACT FROM AUTHOR]

Nuclear Factor Y (NF-Y) was first described as one of the CCAAT binding factors. Although CCAAT motifs were found to be present in various genes, NF-Y attracted a lot of interest early on, due to its role in Major Histocompatibility Complex ( MHC ) gene regulation. MHC genes are crucial in immune response and show peculiar expression patterns. Among other conserved elements on MHC promoters, an NF-Y binding CCAAT box was found to contribute to MHC transcriptional regulation. NF-Y along with other DNA binding factors assembles in a stereospecific manner to form a multiprotein scaffold, the MHC enhanceosome, which is necessary but not sufficient to drive transcription. Transcriptional activation is achieved by the recruitment of yet another factor, the class II transcriptional activator (CIITA). In this review, we briefly discuss basic findings on MHCII transcription regulation and we highlight NF-Y different modes of function in MHCII gene activation. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani. [ABSTRACT FROM AUTHOR]

NF-Y is a ubiquitous heterotrimeric transcription factor with a binding affinity for the CCAAT consensus motif, one of the most common cis-acting element in the promoter and enhancer regions of eukaryote genes in direct (CCAAT) or reverse (ATTGG) orientation. NF-Y consists of three subunits, NF-YA, the regulatory subunit of the trimer, NF-YB, and NF-YC, all required for CCAAT binding. Growing evidence in cells and animal models support the notion that NF-Y, driving transcription of a plethora of cell cycle regulatory genes, is a key player in the regulation of proliferation. Proper control of cellular growth is critical for cancer prevention and uncontrolled proliferation is a hallmark of cancer cells. Indeed, during cell transformation aberrant molecular pathways disrupt mechanisms controlling proliferation and many growth regulatory genes are altered in tumors. Here, we review bioinformatics, molecular and functional evidence indicating the involvement of the cell cycle regulator NF-Y in cancer-associated pathways. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani. [ABSTRACT FROM AUTHOR]