Gene expression profiling in neuronal cells identifies a different type of transcriptome modulated by NF-Y.

A heterotrimeric transcription factor NF-Y is crucial for cell-cycle progression in various types of cells. In contrast, studies using NF-YA knockout mice have unveiled its essential role in endoplasmic reticulum (ER) homeostasis in neuronal cells. However, whether NF-Y modulates a different transcriptome to mediate distinct cellular functions remains obscure. Here, we knocked down NF-Y in two types of neuronal cells, neuro2a neuroblastoma cells and mouse brain striatal cells, and performed gene expression profiling. We found that down-regulated genes preferentially contained NF-Y-binding motifs in their proximal promoters, and notably enriched genes related to ER functions rather than those for cell cycle. This contrasts with the profiling data of HeLa and embryonic stem cells in which distinct down-regulation of cell cycle-related genes was observed. Clustering analysis further identified several functional clusters where populations of the down-regulated genes were highly distinct. Further analyses using chromatin immunoprecipitation and RNA-seq data revealed that the transcriptomic difference was not correlated with DNA binding of NF-Y but with splicing of NF-YA. These data suggest that neuronal cells have a different type of transcriptome in which ER-related genes are dominantly modulated by NF-Y, and imply that NF-YA splicing alteration could be involved in this cell type-specific gene modulation.

Nuclear Factor Y (NF-Y) Modulates Encystation in Entamoeba via Stage-Specific Expression of the NF-YB and NF-YC Subunits.

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor composed of three subunits, namely, NF-YA, NF-YB, and NF-YC, which are conserved throughout evolution. In higher eukaryotes, NF-Y plays important roles in several cellular processes (development, cell cycle regulation, apoptosis, and response to growth, stress, and DNA damage) by controlling gene expression through binding to a CCAAT promoter motif. We demonstrated that NF-Y subunits in the protist Entamoeba, while significantly divergent from those of higher eukaryotes, have well-conserved domains important for subunit interactions and DNA binding and that NF-YB and NF-YC are developmentally expressed during encystation. Electrophoretic mobility shift assays confirmed that the NF-Y protein(s) from Entamoeba cysts binds to a CCAAT motif. Consistent with a role as a transcription factor, the NF-Y proteins show nuclear localization during development. Additionally, we demonstrated that NF-YC localizes to the chromatoid body (an RNA processing center) during development, indicating that it may have a role in RNA processing. Finally, silencing of the NF-YC subunit resulted in reduced stability of the NF-Y complex and decreased encystation efficiency. We demonstrated that the NF-Y complex functions at a time point subsequent to the NAD+ flux and expression of the transcription factor encystation regulatory motif-binding protein, both of which are early regulators of Entamoeba development. Taken together, our results demonstrate that the NF-Y complex plays an important role in regulating encystation in Entamoeba and add to our understanding of the transcriptional networks and signals that control this essential developmental pathway in an important human pathogen.IMPORTANCE The human parasite Entamoeba histolytica is an important pathogen with significant global impact and is a leading cause of parasitic death in humans. Since only the cyst form can be transmitted, blocking encystation would prevent new infections, making the encystation pathway an attractive target for the development of new drugs. Identification of the genetic signals and transcriptional regulatory networks that control encystation would be an important advance in understanding the developmental cascade. We show that the Entamoeba NF-Y complex plays a crucial role in regulating the encystation process in Entamoeba.

Epigenetic role of the nuclear factor NF-Y on ID gene family in endometrial tissues of women with endometriosis: a case control study.

BACKGROUND: A predominant difference between endometrial and normal cells is higher proliferation rate in the former cells which is benign. The genes of inhibitor of differentiation (ID) family play a major role in cell proliferation regulation which might be targeted by the nuclear transcription factor Y (NF-Y) for subsequent epigenetic modifications through the CCAAT box regulatory region. The present study was designed to investigate the epigenetic role of NF-Y on ID gene family in endometrial tissue of patients with endometriosis. MATERIALS & METHODS: In this case-control study, 20 patients with endometriosis and 20 normal women were examined for the relative expression of the NF-YA, NF-YB, NF-YC and ID genes by real-time PCR during the proliferative phase. The occupancy of NF-Y on CCAAT box region of ID genes was investigated using chromatin immunoprecipitation (ChIP) followed by real-time PCR. RESULTS: The NF-YA was over-expressed in eutopic endometrium during the proliferative phase. Although the expression level of NF-YB and NF-YC were unchanged in eutopic samples, they were remarkably higher in ectopic group (P<0.05). The ID2 and ID3 genes were up-regulated in ectopic and eutopic tissues, however ID1 and ID4 genes were down-regulated in these samples (P<0.05). The ChIP analysis revealed significant enrichment of NF-Y on regulatory regions of ID2,3 genes in eutopic group, but reduced binding level of NF-Y to the ID1,3 promoters in ectopic specimens (P<0.05). CONCLUSION: The ability of NF-Y to regulate ID genes via CCAAT box region suggests the possible role of NF-Y transcription factor in epigenetic changes in endometrial tissues which may open novel avenues in finding new therapeutic strategies.

Ambient temperature regulates the expression of a small set of sRNAs influencing plant development through NF-YA2 and YUC2.

Plants substantially alter their developmental programme upon changes in the ambient temperature. The 21-24 nt small RNAs (sRNAs) are important gene expression regulators, which play a major role in development and adaptation. However, little is known about how the different sRNA classes respond to changes in the ambient temperature. We profiled the sRNA populations in four different tissues of Arabidopsis thaliana plants grown at 15°C, 21°C, and 27°C. We found that only a small fraction (0.6%) of the sRNA loci are ambient temperature-controlled. We identified thermoresponsive microRNAs and identified their target genes using degradome libraries. We verified that the target of the thermoregulated miR169, NF-YA2, is also ambient temperature-regulated. NF-YA2, as the component of the conserved transcriptional regulator NF-Y complex, binds the promoter of the flowering time regulator FT and the auxin biosynthesis gene YUC2. Other differentially expressed loci include thermoresponsive phased siRNA loci that target various auxin pathway genes and tRNA fragments. Furthermore, a temperature-dependent 24-nt heterochromatic siRNA locus in the promoter of YUC2 may contribute to the epigenetic regulation of auxin homeostasis. This holistic approach facilitated a better understanding of the role of different sRNA classes in ambient temperature adaptation of plants.

Gene structure, expression pattern and interaction of Nuclear Factor-Y family in castor bean (Ricinus communis).

MAIN CONCLUSION: Nuclear Factor-Y transcription factors, which function in regulating seed development (including storage reservoir accumulation) and responding to abiotic stresses, were identified and characterized in castor bean. Nuclear Factor-Y (NF-Y) transcription factors in plants contain three subunits (NF-YA, NF-YB and NF-YC), and function as a heterodimer or heterotrimer complex in regulating plant growth, development and response to stresses. Castor bean (Ricinus communis, Euphorbiaceae) one of the most economically important non-edible oilseed crops, able to grow in diverse soil conditions and displays high tolerance to abiotic stresses. Due to increasing demands for its seed oils, it is necessary to elucidate the molecular mechanism underlying the regulation of growth and development. Based on the available genome data, we identified 25 RcNF-Y members including six RcNF-YAs, 12 RcNF-YBs and seven RcNF-YCs, and characterized their gene structures. Yeast two-hybrid assays confirmed the protein-protein interactions among three subunits. Using transcriptomic data from different tissues, we found that six members were highly or specifically expressed in endosperms (in particular, two LEC1-type members RcNF-YB2 and RcNF-YB12), implying their involvement in regulating seed development and storage reservoir accumulation. Further, we investigated the expression changes of RcNF-Y members in two-week-old seedlings under drought, cold, hot and salt stresses. We found that the expression levels of 20 RcNF-Y members tested were changed and three RcNF-Y members might function in response to abiotic stresses. This study is the first reported on genomic characterization of NF-Y transcription factors in the family Euphorbiaceae. Our results provide the basis for improved understanding of how NF-Y genes function in the regulation of seed development and responses to abiotic stresses in both castor bean and other plants in this family.

Direct non transcriptional role of NF-Y in DNA replication.

NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication.

Epigenetic role of CCAAT box-binding transcription factor NF-Y on ID gene family in human embryonic carcinoma cells.

Nuclear factor Y (NF-Y) is a histone substitute protein that specifically binds to the CCAAT box of the target genes and thereby promotes their regulation. NF-Y transcription factor, with defined CCAAT element-binding activities, target a gene family that encodes a group of basic helix-loop-helix ID factors (ID1-ID4), with or without CCAAT box at their promoter region. In this study, the expressions of NF-Y in mRNA and protein level were evaluated in a human embryonic carcinoma cell line, named NTera2, before and after 7 days induction of differentiation. We also looked into expression levels of ID genes in NTera2 cells during differentiation because of their critical role in development. By using chromatin immunoprecipitation coupled with real-time polymerase chain reaction, NF-Y incorporation and acetylation/dimethylation of histone H3 at lysine 9 (H3K9ac/me2) was quantitatively evaluated on the regulatory regions of considered genes to monitor the changes in epigenetic markers at ID gene promoters throughout differentiation. The results demonstrated a marked down-regulation of ID1, ID2, and ID3 genes, parallel to a loss of NF-Y binding to the promoters of these genes. The data show that although the genes encoding NF-Y complex remained expressed at mRNA level, NF-YC is lost at the protein level onset of differentiation. Additionally, the epigenetic marks of H3K9ac and H3K9me2 at the target gene promoters decreased and increased, respectively, after 1 day of differentiation. It is suggested that, in the absence of NF-Y binding, the corresponding regions adopt a heterochromatic nature, whereas when NF-Y comes back after 7 days of differentiation, the ID1-3 promoters become again converted into active chromatin. The ID4 gene, lacking a CCAAT box, behaves differently and does not show any incorporation. This experiment implies for the first time that the presence of NF-Y transcription factor plays a pivotal role in transcriptional regulation of ID genes in development.

DNA methylation and NF-Y regulate Piwil1 expression during chicken spermatogenesis.

The P-element induced wimpy testis (Piwi) protein family, a subfamily of the Argonaute protein family, is involved in gene silencing and shows specific expression in spermatogenic cells. To reveal the transcriptional regulatory mechanisms of Piwil1 in chickens, we cloned sequences of the chicken Piwil1 promoter region and performed luciferase reporter and electrophoretic mobility shift assays to analyze the transcriptional activity and identify important transcriptional regulatory elements. The results showed that the region from -90 to -43 in the 5'-flanking region of Piwil1 contains a transcriptional regulatory CCAAT box that was necessary for the transcriptional activity of the Piwil1 promoter. Moreover, the transcription factor nuclear factor Y (NF-Y) was bound to the Piwil1 promoter CCAAT box specifically in germ cells. In addition, bisulfite sequencing to determine the methylation profile of the Piwil1 promoter CpG island in different spermatogenic and non-germ cell populations was performed. Compared with germ cells, non-germ cells showed increased methylation of the promoter region containing the CCAAT box, loss of NF-Y binding, and silencing of the Piwil1 locus. It is demonstrated that the specific expression of Piwil1 in chicken germ cells is regulated by the transcription factor NF-Y and differential CpG island methylation.

The gene pair PRR11 and SKA2 shares a NF-Y-regulated bidirectional promoter and contributes to lung cancer development.

Head-to-head gene pairs represent a unique feature of gene organization in eukaryotes, accounting for >10% of genes in the human genome. Identification and functional analysis of such gene pairs is only in its infancy. Recently, we identified PRR11 as a novel cancer-related gene that is implicated in cell cycle and lung cancer. Here we demonstrate that PRR11 is oriented in a head-to-head configuration with its neighboring gene, SKA2. 5'-RACE assay revealed that the intergenic spacer region between the two genes is <500 bp. Serial luciferase reporter assays demonstrated that a minimal 80-bp intergenic region functions as a core bidirectional promoter to drive basal transcription in both the PRR11 and SKA2 orientations. EMSA and ChIP assays demonstrated that NF-Y binds to and directly transactivates the PRR11-SKA2 bidirectional promoter. SiRNA-mediated NF-Y depletion significantly downregulated PRR11 and SKA2 expression. Expression of both PRR11 and SKA2 was significantly upregulated in lung cancer. Expression of the two genes was highly correlated with each other and with NF-Y expression. Remarkably, high expression of both PRR11 and SKA2 was associated with poorer prognosis in lung cancer patients compared with high expression of one gene or low expression of both genes. Knockdown of PRR11 and/or SKA2 remarkably reduced cell proliferation, migration, and invasion in lung cancer cells. Thus, the PRR11-SKA2 bidirectional transcription unit, which is a novel direct target of NF-Y, is essential for the accelerated proliferation and motility of lung cancer cells and may represent a potential target in the diagnosis and/or treatment of human lung cancer.

MicroRNA-193b Controls Adiponectin Production in Human White Adipose Tissue.

CONTEXT: MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression. In white adipose tissue (WAT), recent studies suggest that miRNA levels are altered in various metabolic diseases, including obesity. OBJECTIVE: The objective of the study was to determine whether adipocyte-expressed miRNAs altered by obesity can regulate adiponectin expression/secretion in fat cells. DESIGN: Eleven miRNAs previously shown to be altered in obese human WAT were overexpressed in human in vitro-differentiated adipocytes followed by assessments of adiponectin levels in conditioned media. SETTING: This was cohort study (n = 56) in an academic hospital. PATIENTS: Subcutaneous WAT was obtained from nonobese and obese individuals. INTERVENTIONS: There were no interventions in this study. MAIN OUTCOME MEASURE(S): Protein and mRNA levels of adiponectin were measured. RESULTS: Of the 11 investigated miRNAs, three (miR-193b/-126/-26a) increased adiponectin secretion when overexpressed in human adipocytes. However, in human WAT only miR-193b expression correlated with adiponectin gene expression and homeostasis model assessment of insulin resistance. Moreover, quantitative PCR of miR-193b in both WAT and isolated adipocytes showed a significant association with serum adiponectin levels. Overexpression of miR-193b altered the gene expression of seven known adiponectin regulators. 3'-untranslated region reporter assays confirmed binding to cAMP-responsive element binding protein 5, nuclear receptor interacting protein 1, and nuclear transcription factor Yα. The effects of miR-193b on nuclear transcription factor Yα expression were confirmed at the protein level. Transfection with individual miRNA target protectors selective for nuclear transcription factor Yα and nuclear receptor interacting protein 1 abolished the stimulatory effect of miR-193b on adiponectin secretion. CONCLUSIONS: In human adipocytes, miR-193b controls adiponectin production via pathways involving nuclear transcription factor Yα and possibly nuclear receptor interacting protein 1.