NF-Y Subunits Research Articles

Characterization of the human mitochondrial thiamine pyrophosphate transporter SLC25A19 minimal promoter: A role for NF-Y in regulating basal transcription

Transcriptional regulation of expression of the human mitochondrial thiamine pyrophosphate transporter (the product of the SLC25A19 gene) is unknown. To understand this regulation, we cloned and characterized the 5′-regulatory region of the SLC25A19 gene (1080bp). The cloned fragment was found to possess promoter activity in transiently transfected human-derived liver HepG2 cells. 5′- and 3′-deletion analysis has identified the minimal region required for basal SLC25A19 promoter activity to be between −131 and +20 (using the distal transcriptional start site as +1). The minimal promoter lacks typical TATA motif and contains two inverted CCAAT boxes (binding sites for NF-Y transcriptional factor). By means of mutational analysis, the critical role of both the upstream and downstream CCAAT boxes in basal SLC25A19 promoter activity was established; however, each of these boxes alone was found to be unable to support promoter activity. EMSA and supershift EMSA (with the use of specific antibodies against NF-Y subunits) studies, as well as chromatin immunoprecipitation assay, demonstrated the binding of NF-Y to both CCAAT boxes in vitro and in vivo, respectively. The requirement for NF-Y in SLC25A19 promoter activity in vivo was directly confirmed by the use of a dominant negative NF-YA mutant in transiently transfected HepG2 cells. These studies report for the first time the characterization of the SLC25A19 promoter and demonstrate an essential role for NF-Y in its basal activity.

NF-YA Underlies EZH2 Upregulation and Is Essential for Proliferation of Human Epithelial Ovarian Cancer Cells

Epithelial ovarian cancer (EOC) accounts for the most gynecologic malignancy-associated deaths in the United States. Enhancer of zeste homolog 2 (EZH2), which silences gene expression through generating trimethylation on lysine 27 residue of histone H3 (H3K27Me3), is often overexpressed in EOCs and has been suggested as a therapeutic target. However, the mechanism underlying EZH2 overexpression in EOCs is unknown. Here, we show that EZH2 is upregulated at the transcription level, and two CCAAT boxes in the proximal regions of the human EZH2 gene promoter are critical for its transcription in EOC cells. Indeed, NF-YA, the regulatory subunit of the CCAAT-binding transcription factor NF-Y, is expressed at higher levels in human EOCs than in primary human ovarian surface epithelial (HOSE) cells. In addition, there is a positive correlation between expression of NF-YA and EZH2 in EOCs. Notably, high NF-YA expression predicts shorter overall survival in patients with EOCs. The association of NF-YA with the promoter of the human EZH2 gene is enhanced in human EOC cells compared with primary HOSE cells. Significantly, knockdown of NF-YA downregulates EZH2, decreases H3K27Me3 levels, and suppresses the growth of human EOC cells both in vitro and in a xenograft mouse model. Notably, NF-YA knockdown induces apoptosis of EOC cells and ectopic EZH2 expression partially rescues apoptosis induced by NF-YA knockdown. Together, these data reveal that NF-Y is a key regulator of EZH2 expression and is required for EOC cell proliferation, thus representing a novel target for developing EOC therapeutics.

Highlights of This Issue

Acute myeloid leukemia (AML) is generally amenable to first-line chemotherapeutics that typically induce a remission. However, at least 40% of AML patients will experience a relapse, suggesting that AML cells either acquire resistance or originate from a stem cell population. Leukemia-initiating cells (LIC) are believed to be important for AML growth and are thought to be responsible for AML relapses. Therefore, strategies specifically targeting LICs are of translational interest. Weidenaar and colleagues evaluated a receptor tyrosine kinase inhibitor, PTK787/ZK222584 (PTK/ZK), using long-term cocultures of pediatric CD34+ AML cells. Critically, PTK/ZK inhibited outgrowth and self-renewal of LICs when cultured up to 10 weeks. Proteome profiler kinase array analysis of downstream signaling systems revealed that PTK/ZK reduced activation of phosphoinositide 3-kinase/Akt kinase signaling. Treatment of AML cells with an Akt inhibitor showed similar results, whereas antibodies against VEGFA and VEGF receptors did not. These data suggest that targeting of multiple kinase pathways with PTK/ZK could be a promising approach to improve AML outcomes.The histone methyltransferase enhancer of zeste homolog 2 (EZH2) is upregulated in many cancer types and is considered a putative therapeutic target. Garipov and colleagues report that EZH2 is upregulated in ovarian cell lines and in expression data from patients with ovarian cancer. They observed that transcriptional activation of EZH2 was dependent on 2 NF-Y binding sites in the proximal region of the EZH2 promoter. Moreover, elevated expression of NF-YA, the regulatory subunit of NF-Y, was correlated with increased EZH2 expression and EZH2 was predictive of shorter patient survival. Mechanistically, depletion of NF-YA was sufficient to decrease EZH2 levels, which phenocopy EZH2 inhibition and result in apoptosis of cancer cells. This study establishes NF-Y as a key regulator of EZH2 expression and implies that targeting NF-Y represents an alternative strategy to inhibiting EZH2 activity in cancer.The origin recognition complex (ORC), composed of multiple DNA binding proteins that associate with origins of replication, is highly regulated throughout the cell cycle to avoid re-replication and potential genomic instability. In tumorderived cells, these ORC proteins are often expressed at high levels to accommodate the stress of replication. During replication, only a fraction of origins are used, while others are thought to remain dormant, and these “dormant” replication origins have been shown to enhance recovery from replication stress. Zimmerman and colleagues show that depletion of ORC1, ORC6, or CDC6 to levels that do not affect viability, which was considered optimal for the depletion of dormant origins without affecting normal replication, hypersensitized tumor-derived cells, but not immortalized, nontransformed cells, to replication stress. Additional study revealed that altering p53 or MYC affected the sensitivity of immortalized, nontransformed cells. These findings suggest that tumor cells have a greater reliance on origin licensing capacity, revealing ORC proteins as potential targets for therapeutic intervention.Prostate cancer is a leading cause of cancer associated death in men, due largely to disseminated disease. Unfortunately, the mechanisms that promote prostate cancer metastasis are not well studied, nor does an effective treatment exist for disseminated disease. To this end, the current study focused on the expression and role of the adhesion protein, β1 integrin. Lee and colleagues determined that β1 integrin and downstream phospho-FAK were increased in both primary prostate cancer and lymph node metastases. Moreover, highly metastatic prostate cancer cells were found to have higher constitutively active β1 integrin that correlated with resistance to anoikis. Antibody blockade of β1 integrin resulted in inhibition of phenotypes, signaling, and survival processes associated with metastatic potential. Finally, systemic delivery of a neutralizing antibody to β1 integrin suppressed metastasis to lymph nodes and to bone following orthotopic and intracardiac injections, respectively. Thus, constitutively active β1 integrin is a critical effector of prostate cancer metastasis, and these preclinical findings identify a potential target for metastasis prevention.

Interactions and CCAAT-Binding of Arabidopsis thaliana NF-Y Subunits

BackgroundNF-Y is a transcription factor that recognizes with high specificity and affinity the widespread CCAAT box promoter element. It is formed by three subunits: NF-YA and the NF-YB/NF-YC- heterodimer containing histone fold domains (HFDs). We previously identified a large NF-Y gene family in Arabidopsis thaliana, composed of 29 members, and characterized their expression patterns in various plant tissues.MethodsWe used yeast Two-hybrids assays (Y2H), pull-down and Electrophoretic Mobility Shift Assay (EMSA) in vitro experiments with recombinant proteins to dissect AtNF-YB/AtNF-YC interactions and DNA-binding with different AtNF-YAs.ResultsConsistent with robust conservation within HFDs, we show that heterodimerization is possible among all histone-like subunits, including the divergent and related LEC1/AtNF-YB9 and L1L/AtNF-YB6 required for embryo development. DNA-binding to a consensus CCAAT box was investigated with specific AtNF-YB/AtNF-YC combinations and observed with some, but not all AtNF-YA subunits.ConclusionsOur results highlight (i) the conserved heterodimerization capacity of AtNF-Y histone-like subunits, and (ii) the different affinities of AtNF-YAs for the CCAAT sequence. Because of the general expansion of NF-Y genes in plants, these results most likely apply to other species.

Studies on Differential Nuclear Translocation Mechanism and Assembly of the Three Subunits of the Arabidopsis thaliana Transcription Factor NF-Y

The eukaryotic transcription factor NF-Y consists of three subunits (A, B, and C), which are encoded in Arabidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, all potential combinations of the subunits are possible for the assembly of the heterotrimeric complex. We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y. The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished. By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated. Using GFP fusion constructs, NF-YA and NF-YC localization in the nucleus was demonstrated, while NF-YB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC. This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms. Based on a peptide structure model of the histone-fold-motifs, disulfide bonding among intramolecular conserved cysteine residues of NF-YB, which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans, can be excluded for Arabidopsis NF-YB.

The CCAAT box-binding transcription factor NF-Y regulates basal expression of human proteasome genes

Protein degradation by the proteasome plays an important role in all major cellular pathways. Aberrant proteasome activity is associated with numerous human diseases including cancer and neurological disorders, but the underlying mechanism is virtually unclear. At least part of the reason for this is due to lack of understanding of the regulation of human proteasome genes. In this study, we found that a large set of human proteasome genes carry the CCAAT box in their promoters. We further demonstrated that the basal expression of these CCAAT box-containing proteasome genes is regulated by the transcription factor NF-Y. Knockdown of NF-YA, an essential subunit of NF-Y, reduced proteasome gene expression and compromised the cellular proteasome activity. In addition, we showed that knockdown of NF-YA sensitized breast cancer cells to the proteasome inhibitor MG132. This study unveils a new role for NF-Y in the regulation of human proteasome genes and suggests that NF-Y may be a potential target for cancer therapy.

Altered expression of NFY-C and RORA in colorectal adenocarcinomas

NFY-C, a subunit of the transcription factor NFY, binds to the promoters of several eukaryotic genes, including cell cycle-related genes. RORA is a steroid hormone receptor implicated in a range of important cellular processes. We evaluated the expression of NFY-C and RORA in colorectal adenocarcinomas and normal colonic tissue. NFY-C expression was elevated in adenocarcinomas. Moreover, NFY-C mRNA levels correlated with time to disease progression, while NFY-C protein expression was significantly higher in metastatic disease. RORA expression was downregulated in CRC adenocarcinomas compared to normal controls and correlated with time to disease progression. The role of NFY-C and RORA in CRC merits further investigation.

Multiple NF‐Y‐binding CCAAT boxes are essential for transcriptional regulation of the human C7orf24 gene, a novel tumor‐associated gene

Human chromosome 7 ORF 24 (C7orf24) has been identified as a tumor-related protein, and shown to be a γ-glutamyl cyclotransferase. In the current study, we characterized the promoter region of the human C7orf24 gene to explore the transcriptional regulation of the gene. We revealed that the human C7orf24 promoter is a TATA-less promoter, containing five CCAAT boxes aligned in a forward orientation. By performing a luciferase reporter assay with 5'-deleted and site-directed mutated constructs in HeLa, MCF-7 and IMR-90 cells, we found that three proximal CCAAT boxes are important for basal transcription. Electrophoretic mobility gel shift assay and chromatin immunoprecipitation assay demonstrated that NF-Y specifically bound to all three CCAAT boxes. In addition, the mRNA and protein expression levels of C7orf24 were significantly reduced in HeLa cells depleted of NF-YB, a subunit of NF-Y. These results suggested that NF-Ys bound to the three proximal CCAAT boxes play a central role in the transcription of the gene. Furthermore, as in the case of other genes transcribed under the control of multiple NF-Ys, such as human E2f1 and cyclin b1, the C7orf24 gene expression profile oscillated during the cell cycle, implying that C7orf24 is a novel cell cycle-associated gene.

NF-Y Recruits Ash2L to Impart H3K4 Trimethylation on CCAAT Promoters

BackgroundDifferent histone post-translational modifications (PTMs) are crucial in the regulation of chromatin, including methylations of H3 at Lysine 4 by the MLL complex. A relevant issue is how this is causally correlated to the binding of specific transcription factors (TFs) in regulatory regions. NF-Y is a TF that regulates 30% of mammalian promoters containing the widespread CCAAT element. We and others established that the presence of H3K4me3 is dependent upon the binding of NF-Y. Here, we investigate the mechanisms of H3K4me3 deposition by NF-Y.MethodsWe employed Chromatin Immunoprecipitation in cells in which Ash2L and NF-Y subunits were knocked down by RNAi, to monitor the presence of histones PTMs and components of the MLL complex. We performed gene expression profiling of Ash2L-knocked down cells and analyzed the regulated genes. We performed ChIPs in leukemic cells in which MLL1 is devoid of the methyltransferase domain and fused to the AF4 gene.ResultsKnock down of the Ash2L subunit of MLL leads to a decrease in global H3K4me3 with a concomitant increase in H3K79me2. Knock down of NF-Y subunits prevents promoter association of Ash2L, but not MLL1, nor WDR5, and H3K4me3 drops dramatically. Endogenous NF-Y and Ash2L specifically interact in vivo. Analysis of the promoters of Ash2L regulated genes, identified by transcriptional profiling, suggests that a handful TF binding sites are moderately enriched, among which the CCAAT box. Finally, leukemic cells carrying the MLL-AF4 translocation show a decrease of H3K4me3, absence of Ash2L and increase in H3K79me2, while NF-Y binding was not significantly affected.ConclusionsThree types of conclusions are reached: (i) H3K4 methylation is not absolutely required for NF-Y promoter association. (ii) NF-Y acts upstream of H3K4me3 deposition by recruiting Ash2L. (iii) There is a general cross-talk between H3K4me3 and H3K79me2 which is independent from the presence of MLL oncogenic fusions.

Specific inhibition of NF-Y subunits triggers different cell proliferation defects

Regulated gene expression is essential for a proper progression through the cell cycle. The transcription factor NF-Y has a fundamental function in transcriptional regulation of cell cycle genes, particularly of G2/M genes. In order to investigate common and distinct functions of NF-Y subunits in cell cycle regulation, NF-YA, NF-YB and NF-YC have been silenced by shRNAs in HCT116 cells. NF-YA loss led to a delay in S-phase progression, DNA damage and apoptosis: we showed the activation of the replication checkpoint, through the recruitment of Δp53 and of the replication proteins PCNA and Mcm7 to chromatin. Differently, NF-YB depletion impaired cells from exiting G2/M, but did not interfere with S-phase progression. Gene expression analysis of NF-YA and NF-YB inactivated cells highlighted a common set of hit genes, as well as a plethora of uncommon genes, unveiling a different effect of NF-Y subunits loss on NF-Y binding to its target genes. Chromatin extracts and ChIP analysis showed that NF-YA depletion was more effective than NF-YB in hitting NF-Y recruitment to CCAAT-promoters. Our data suggest a critical role of NF-Y expression, highlighting that the lack of the single subunits are differently perceived by the cells, which activate diverse cell cycle blocks and signaling pathways.

A C Subunit of the Plant Nuclear Factor NF-Y Required for Rhizobial Infection and Nodule Development Affects Partner Selection in the Common Bean–Rhizobium etliSymbiosis

Legume plants are able to interact symbiotically with soil bacteria to form nitrogen-fixing root nodules. Although specific recognition between rhizobia and legume species has been extensively characterized, plant molecular determinants that govern the preferential colonization by different strains within a single rhizobium species have received little attention. We found that the C subunit of the heterotrimeric nuclear factor NF-Y from common bean (Phaseolus vulgaris) NF-YC1 plays a key role in the improved nodulation seen by more efficient strains of rhizobia. Reduction of NF-YC1 transcript levels by RNA interference (RNAi) in Agrobacterium rhizogenes-induced hairy roots leads to the arrest of nodule development and defects in the infection process with either high or low efficiency strains. Induction of three G2/M transition cell cycle genes in response to rhizobia was impaired or attenuated in NF-YC1 RNAi roots, suggesting that this transcription factor might promote nodule development by activating cortical cell divisions. Furthermore, overexpression of this gene has a positive impact on nodulation efficiency and selection of Rhizobium etli strains that are naturally less efficient and bad competitors. Our findings suggest that this transcription factor might be part of a mechanism that links nodule organogenesis with an early molecular dialogue that selectively discriminates between high- and low-quality symbiotic partners, which holds important implications for optimizing legume performance.

TAT-mediated transduction of NF-Ya peptide induces the ex vivo proliferation and engraftment potential of human hematopoietic progenitor cells

AbstractRetroviral overexpression of NF-Ya, the regulatory subunit of the transcription factor NF-Y, activates the transcription of multiple genes implicated in hematopoietic stem cell (HSC) self-renewal and differentiation and directs HSCs toward self-renewal. We asked whether TAT-NF-Ya fusion protein could be used to transduce human CD34+ cells as a safer, more regulated alternative approach to gene therapy. Here we show that externally added recombinant protein was able to enter the cell nucleus and activate HOXB4, a target gene of NF-Ya, using real-time polymerase chain reaction RNA and luciferase-based protein assays. After TAT-NF-Ya transduction, the proliferation of human CD34+ cells in the presence of myeloid cytokines was increased 4-fold. Moreover, TAT-NF-Ya-treated human primary bone marrow cells showed a 4-fold increase in the percentage of huCD45+ cells recovered from the bone marrow of sublethally irradiated, transplanted NOD-Scid IL2Rγnull mice. These data demonstrate that TAT-peptide therapies are an alternative approach to retroviral stem cell therapies and suggest that NF-Ya peptide delivery should be further evaluated as a tool for HSC/progenitors ex vivo expansion and therapy.

BZIP28 and NF-Y Transcription Factors Are Activated by ER Stress and Assemble into a Transcriptional Complex to Regulate Stress Response Genes in Arabidopsis

Stress agents known to elicit the unfolded protein response in Arabidopsis thaliana upregulate the expression of a constellation of genes dependent on the membrane-associated basic domain/leucine zipper (bZIP) transcription factor, bZIP28. Among the stress-activated genes, a consensus promoter sequence corresponding to the endoplasmic reticulum (ER) stress-responsive element I (ERSE-I), CCAAT-N10-CACG, was identified. Disruption of either the CCAAT or CACG subelement in ERSE-I resulted in reduction of the transcriptional response to ER stress. bZIP28 forms homo- and heterodimers with other bZIP TF family members (in subgroup D) and interacts with CCAAT box binding factors, heterotrimeric factors composed of NF-Y subunits. Arabidopsis encodes 36 NF-Y subunits, and it was found that subunits NF-YB3 and -YC2 interact with bZIP28 and NF-YA4, respectively, in a yeast three-hybrid system. A transcriptional complex containing bZIP28 and the above-mentioned three NF-Y subunits was assembled in vitro on DNA containing ERSE-I. bZIP28, on its own, binds to the CACG subelement in ERSE-I to form a smaller complex I, and in combination with the NF-Y subunits above, bZIP28 assembles into a larger transcriptional complex (complex II). bZIP28 was shown to interact with NF-Y subunits in vivo in bimolecular fluorescence complementation analyses and in coimmunoprecipitation assays. Treatment of seedlings with ER stress agents led to the upregulation of NF-YC2 and the relocation of NF-YB3 from the cytoplasm to the nucleus. Thus, in response to ER stress, bZIP28 is mobilized by proteolysis and recruits NF-Y subunits to form a transcriptional complex that upregulates the expression of ER stress-induced genes.

NF-YC Functions as a Corepressor of Agonist-bound Mineralocorticoid Receptor

The role of aldosterone has been implicated in the metabolic syndrome and cardiovascular diseases. The biological actions of aldosterone are mediated through mineralocorticoid receptor (MR). Nuclear receptor-mediated gene expression is regulated by dynamic and coordinated recruitment of coactivators and corepressors. To identify new coregulators of the MR, full-length MR was used as bait in yeast two-hybrid screening. We isolated NF-YC, one of the subunits of heterotrimeric transcription factor NF-Y. Specific interaction between MR and NF-YC was confirmed by yeast two-hybrid, mammalian two-hybrid, coimmunoprecipitation assays, and fluorescence subcellular imaging. Transient transfection experiments in COS-7 cells demonstrated that NF-YC repressed MR transactivation in a hormone-sensitive manner. Moreover, reduction of NF-YC protein levels by small interfering RNA potentiated hormonal activation of endogenous target genes in stably MR-expressing cells, indicating that NF-YC functions as an agonist-dependent MR corepressor. The corepressor function of NF-YC is selective for MR, because overexpression of NF-YC did not affect transcriptional activity mediated by androgen, progesterone, or glucocorticoid receptors. Chromatin immunoprecipitation experiments showed that endogenous MR and steroid receptor coactivator-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner, and endogenous NF-YC was sequentially recruited to the same element. Immunohistochemistry showed that endogenous MR and NF-YC were colocalized within the mouse kidney. Although aldosterone induces interaction of the N and C termini of MR, NF-YC inhibited the N/C interaction. These findings indicate that NF-YC functions as a new corepressor of agonist-bound MR via alteration of aldosterone-induced MR conformation.

NF-YC Complexity Is Generated by Dual Promoters and Alternative Splicing

The CCAAT box is a DNA element present in the majority of human promoters, bound by the trimeric NF-Y, composed of NF-YA, NF-YB, and NF-YC subunits. We describe and characterize novel isoforms of one of the two histone-like subunits, NF-YC. The locus generates a minimum of four splicing products, mainly located within the Q-rich activation domain. The abundance of each isoform is cell-dependent; only one major NF-YC isoform is present in a given cell type. The 37- and 50-kDa isoforms are mutually exclusive, and preferential pairings with NF-YA isoforms possess different transcriptional activities, with specific combinations being more active on selected promoters. The transcriptional regulation of the NF-YC locus is also complex, and mRNAs arise from the two promoters P1 and P2. Transient transfections, chromatin immunoprecipitations, and reverse transcription-PCRs indicate that P1 has a robust housekeeping activity; P2 possesses a lower basal activity, but it is induced in response to DNA damage in a p53-dependent way. Alternative promoter usage directly affects NF-YC splicing, with the 50-kDa transcript being excluded from P2. Specific functional inactivation of the 37-kDa isoform affects the basal levels of G(1)/S blocking and pro-apoptotic genes but not G(2)/M promoters. In summary, our data highlight an unexpected degree of complexity and regulation of the NF-YC gene, demonstrating the existence of a discrete cohort of NF-Y trimer subtypes resulting from the functional diversification of Q-rich transactivating subunits and a specific role of the 37-kDa isoform in suppression of the DNA damage-response under growing conditions.

NF-Ya Is Essential for Hematopoieic Stem Cell Proliferation.

Abstract 1507Poster Board I-530Proliferation, self-renewal and differentiation of hematopoietic stem cells (HSCs) must be tightly regulated in order to sustain hematopoiesis over a lifetime and to prevent uncontrolled expansion of cells. Several genes have been implicated in the regulation of HSC behavior, such as HoxB4, Notch1, Lef-1 and others. Previous studies from our and other laboratories have demonstrated that the heterotrimeric transcription factor NF-Y is a potent inducer of many of these regulatory genes by over-expressing NF-Ya, the regulatory subunit of NF-Y. Furthermore, Bhattarchaya et al. showed that the deletion of NF-Ya in mice leads to lethality before day E8.5, highlighting its importance in mouse development. While there is no doubt that NF-Y plays a role in the progression of the cell cycle in vitro, different groups – targeting different subunits for deletion or silencing – have obtained different results. In order to comprehensively investigate the in vivo function of NF-Y in the hematopoietic system, we utilized a conditional knockout mouse model. We found that the bone marrow (BM) cellularity decreases sharply starting as soon as one day after the ablation of NF-Ya. Our data indicate that the cell loss can be attributed to a combination of cell cycle arrest in G2/M-phase of the cell cycle and apoptosis at 24 hours after the gene deletion.Since NF-Y has been identified as a master regulator of genes involved in HSCs behavior, we focused on the effects of the NF-Ya deletion within the HSC compartment. We found a down regulation of HoxB4, Notch-1, Lef-1 and Bmi-1 following NF-Ya deletion. However, 24h after induction of NF-Ya deletion the HSC population appeared unaffected and their numbers remained stable, likely due to their predominantly quiescent status.To investigate the capability of stem cells to progress though the cell cycle, we activated HSCs using the interferon inducer poly IC and observed that, once activated, also HSCs accumulate in the G2/M-phase of the cell cycle. Finally, to test whether the deletion of NF-Ya impairs or absolutely abrogates HSC function, we performed long term experiments including competitive BM transplantation and colony formation assays that demonstrate that NF-Y activity is absolutely essential for HSC function.Altogether, our data identify NF-Y plays a pivotal role in the survival of hematopoietic cells and the progression of cells though the G2/M-phase of the cell cycle in vivo. Additionally, while we found that NF-Y ablation leads to reduced expression of many genes important for HSC behavior, this had no immediate effects on the maintenance of these cells due to their quiescent nature. DisclosuresNo relevant conflicts of interest to declare.

Arabidopsis NF‐YB subunits LEC1 and LEC1‐LIKE activate transcription by interacting with seed‐specific ABRE‐binding factors

LEAFY COTYLEDON 1 (LEC1) plays vital roles in the regulation of seed maturation in Arabidopsis. LEC1 encodes a homolog of yeast HAP3 or mammalian NF-YB/CBF-A subunit of trimeric CCAAT binding factor (CBF). Among the nine paralogs of NF-YB in Arabidopsis, LEC1-LIKE (L1L) is most closely related to LEC1, and can complement the lec1 mutation when expressed under the control of the LEC1 promoter. Although the nature of the B3-type seed maturation regulators as transcription factors have been investigated, knowledge of the molecular action of LEC1 is limited. When co-expressed with NF-YC2 in the presence of ABA, we found that LEC1 or L1L, but not other NF-YBs, activated the promoter of CRUCIFERIN C (CRC), which encodes a seed storage protein. However, additional expression of an NF-YA subunit interfered with the activation. The LEC1/L1L-[NF-YC2] activation depended on ABA-response elements present in the promoter, which led to the finding that LEC1/L1L-[NF-YC2] can strongly activate the CRC promoter in the absence of ABA when co-expressed with a seed-specific ABA-response element (ABRE)-binding factor, bZIP67. Functional coupling of LEC1/L1L-[AtNF-YC2] and bZIP67 was also observed in the regulation of sucrose synthase 2 (SUS2). Immunoprecipitation experiments revealed that L1L and bZIP67 formed a protein complex in vivo. These results demonstrate a novel plant-specific mechanism for NF-Y subunit function that enables LEC1 and L1L to regulate a defined developmental network.

Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling

Dentin sialophosphoprotein (DSPP), an important odontoblast differentiation marker, is necessary for tooth development and mineralization. Bone morphogenetic protein 2 (BMP2) plays a vital role in odontoblast function via diverse signal transduction systems. We hypothesize that BMP2 regulates DSPP gene transcription and thus odontoblast differentiation. Here we report that expression of BMP2 and DSPP is detected during mouse odontogenesis by in situ hybridization assay, and BMP2 up-regulates DSPP mRNA and protein expression as well as DSPP-luciferase promoter activity in mouse preodontoblasts. By sequentially deleting fragments of the mouse DSPP promoter, we show that a BMP2-response element is located between nucleotides -97 and -72. By using antibody and oligonucleotide competition assays in electrophoretic mobility shift analysis and chromatin immunoprecipitation experiments, we show that the heterotrimeric transcription factor Y (NF-Y) complex physically interacts with the inverted CCAAT box within the BMP2-response element. BMP2 induces NF-Y accumulation into the nucleus increasing its recruitment to the mouse DSPP promoter in vivo. Furthermore, forced overexpression of NF-Y enhances promoter activity and increases endogenous DSPP protein levels. In contrast, mutations in the NF-Y-binding motif reduce BMP2-induced DSPP transcription. Moreover, inhibiting BMP2 signaling by Noggin, a BMP2 antagonist, results in significant inhibition of DSPP gene expression in preodontoblasts. Taken together, these results indicate that BMP2 mediates DSPP gene expression and odontoblast differentiation via NF-Y signaling during tooth development.

An NF-Y-Dependent Switch of Positive and Negative Histone Methyl Marks on CCAAT Promoters

BackgroundHistone tails have a plethora of different post-translational modifications, which are located differently in “open” and “closed” parts of genomes. H3K4me3/H3K79me2 and H4K20me3 are among the histone marks associated with the early establishment of active and inactive chromatin, respectively. One of the most widespread promoter elements is the CCAAT box, bound by the NF-Y trimer. Two of NF-Y subunits have an H2A-H2B-like structure.Principal findingsWe established the causal relationship between NF-Y binding and positioning of methyl marks, by ChIP analysis of mouse and human cells infected with a dominant negative NF-YA: a parallel decrease in NF-Y binding, H3K4me3, H3K79me2 and transcription was observed in promoters that are dependent upon NF-Y. On the contrary, changes in the levels of H3K9-14ac were more subtle. Components of the H3K4 methylating MLL complex are not recruited in the absence of NF-Y. As for repressed promoters, NF-Y removal leads to a decrease in the H4K20me3 mark and deposition of H3K4me3.ConclusionsTwo relevant findings are reported: (i) NF-Y gains access to its genomic locations independently from the presence of methyl histone marks, either positive or negative; (ii) NF-Y binding has profound positive or negative consequences on the deposition of histone methyl marks. Therefore NF-Y is a fundamental switch at the heart of decision between gene activation and repression in CCAAT regulated genes.

Determination of the Role and Mechanism of the Transcription Factor NF-Y in Murine Hematopoietic Stem Cell Biology.

Previous studies from our and other laboratories have demonstrated that the trimeric transcription factor NF-Y is a potent inducer of many of the genes implicated in hematopoietic stem cell (HSC) self-renewal, suggesting that NF-Y functions as a dominant regulator of genes controlling the balance between self-renewal and differentiation of stem cells. Furthermore, over-expression of NF-Ya, the regulatory subunit of NF-Y, was shown to increase HSC potency in vivo through increased expression of a whole series of genes playing central roles in stem cell function including HoxB4 and Notch-1. The importance of the NF-Y transcription factor for mammalian development is further highlighted by a study demonstrating that a loss of function mutation of NF-Ya in mice, leads to lethality before day E8.5 (Bhattarcharya A. et al. 2003). Therefore, it can be reasoned that the NF-Y transcription factor might act as a master gene among the network of genes involved in early development regulating self-renewal and differentiation of (hematopoietic) stem cells. A concept in cancer biology, well-established in chronic myelogenous leukemia (CML), is that a rare population of cancer stem cells (CSSs) exists that is capable of extensive self-renewal, while most tumor cells have a limited proliferative capacity. Many studies suggest that the similar behavior of HSCs and CSSs are due to similar, yet not identical, molecular mechanisms determining whether a stem cell self-renews or differentiates. Knowledge about the regulatory mechanisms underlying cellular NF-Y abundance and activity and thereby NF-Y-mediated fate decision of SCs would open an elegant way to manipulate SCs, including leukemic stem cells (LSC), for therapeutic use. In this study, we have determined NFYa to have an essential role in murine HSC biology. To circumvent embryonic lethality, we generated bone marrow (BM) chimeric mice in which the deletion of functional NF-Ya can be induced selectively in the hematopoietic system. The analysis of lineage committed cells of the BM, spleen and thymus ten weeks after the disruption of the NF-Ya gene revealed an essential role for NF-Y activity in the hematopoietic system. Furthermore, BM cells from wild type, heterozygous and NF-Ya mutant BM chimera were subjected to colony formation assays, clearly demonstrating the indispensability of NF-Y function for hematopoietic stem and precursor cells. At that time not a single colony deficient for NF-Ya could be found, highlighting the absolute necessity of NF-Y activity for HPCs and HSCs. To explain these deleterious defects mechanistically, the role of NF-Y in regulating potential target genes that in turn control hematopoietic stem cell behavior is comprehensively addressed. Our approach is to ectopically express these downstream genes, such as HoxB4, Notch-1, Bmi-1 and Lef-1 in vivo and subsequently delete NF-Y activity. The results from these assays reveal information about the mechanistic interplay and the position within different pathways of these proteins in HSC behavior. Since the loss of NF-Y has lethal consequences for normal HSCs we are currently testing the effects of NF-Y deletion on LSCs in vivo using an established mouse model for CML. If LSCs and normal HSCs share a common machinery of fate-regulation, it is expected that NF-Ya is essential for LSC survival. If it turns out though, that LSCs and HSCs can be discriminated upon their dependence on NF-Y activity, this would harbor tremendous therapeutic possibilities with medical relevance extending into cancer therapy.