Phylogeny of NF-YA trans-activation splicing isoforms in vertebrate evolution.

NF-Y is a trimeric pioneer Transcription Factor (TF) whose target sequence -the CCAAT box- is present in ~25% of mammalian promoters. We reconstruct the phylogenetic history of the regulatory NF-YA subunit in vertebrates. We find that in addition to the remarkable conservation of the subunits-interaction and DNA-binding parts, the Transcriptional Activation Domain (TAD) is also conserved (>90% identity among bony vertebrates). We infer the phylogeny of the alternatively spliced exon-3 and partial splicing events of exon-7 -7N and 7C- revealing independent clade-specific losses of these regions. These isoforms shape the TAD. Absence of exon-3 in basal deuterostomes, cartilaginous fishes and hagfish, but not in lampreys, suggests that the "short" isoform is primordial, with emergence of exon-3 in chordates. Exon 7N was present in the vertebrate common ancestor, while 7C is a molecular innovation of teleost fishes. RNA-seq analysis in several species confirms expression of all these isoforms. We identify 3 blocks of amino acids in the TAD shared across deuterostomes, yet structural predictions and sequence analyses suggest an evolutionary drive for maintenance of an Intrinsically Disordered Region -IDR- within the TAD. Overall, these data help reconstruct the logic for alternative splicing of this essential eukaryotic TF.

On the NF-Y regulome as in ENCODE (2019).

NF-Y is a trimeric Transcription Factor -TF- which binds with high selectivity to the conserved CCAAT element. Individual ChIP-seq analysis as well as ENCODE have progressively identified locations shared by other TFs. Here, we have analyzed data introduced by ENCODE over the last five years in K562, HeLa-S3 and GM12878, including several chromatin features, as well RNA-seq profiling of HeLa cells after NF-Y inactivation. We double the number of sequence-specific TFs and co-factors reported. We catalogue them in 4 classes based on co-association criteria, infer target genes categorizations, identify positional bias of binding sites and gene expression changes. Larger and novel co-associations emerge, specifically concerning subunits of repressive complexes as well as RNA-binding proteins. On the one hand, these data better define NF-Y association with single members of major classes of TFs, on the other, they suggest that it might have a wider role in the control of mRNA production.

NF-Y Overexpression in Liver Hepatocellular Carcinoma (HCC).

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.

A high definition look at the NF-Y regulome reveals genome-wide associations with selected transcription factors.

NF-Y is a trimeric transcription factor (TF), binding the CCAAT box element, for which several results suggest a pioneering role in activation of transcription. In this work, we integrated 380 ENCODE ChIP-Seq experiments for 154 TFs and cofactors with sequence analysis, protein-protein interactions and RNA profiling data, in order to identify genome-wide regulatory modules resulting from the co-association of NF-Y with other TFs. We identified three main degrees of co-association with NF-Y for sequence-specific TFs. In the most relevant one, we found TFs having a significant overlap with NF-Y in their DNA binding loci, some with a precise spacing of binding sites with respect to the CCAAT box, others (FOS, Sp1/2, RFX5, IRF3, PBX3) mostly lacking their canonical binding site and bound to arrays of well spaced CCAAT boxes. As expected, NF-Y binding also correlates with RNA Pol II General TFs and with subunits of complexes involved in the control of H3K4 methylations. Co-association patterns are confirmed by protein-protein interactions, and correspond to specific functional categorizations and expression level changes of target genes following NF-Y inactivation. These data define genome-wide rules for the organization of NF-Y-centered regulatory modules, supporting a model of distinct categorization and synergy with well defined sets of TFs.

NF-YA splice variants have different roles on muscle differentiation.

The heterotrimeric CCAAT-binding factor NF-Y controls the expression of a multitude of genes involved in cell cycle progression. NF-YA is present in two alternatively spliced isoforms, NF-YAs and NF-YAl, differing in 28 aminoacids in the N-terminal Q-rich activation domain. NF-YAs has been identified as a regulator of stemness and proliferation in mouse embryonic cells (mESCs) and human hematopoietic stem cells (hHSCs), whereas the role of NF-YAl is not clear. In the muscle system, NF-YA expression is observed in proliferating cells, but barely detectable in terminally differentiated cells in vitro and adult skeletal muscle in vivo. Here, we show that NF-YA inactivation in mouse myoblasts impairs both proliferation and differentiation. The overexpression of the two NF-YA isoforms differentially affects myoblasts fate: NF-YAs enhance cell proliferation, while NF-YAl boosts differentiation. The molecular mechanisms were investigated by expression profilings, detailing the opposite programs of the two isoforms. Bioinformatic analysis of the regulated promoters failed to detect a significant presence of CCAAT boxes in the regulated genes. NF-YAl activates directly Mef2D, Six genes, and p57kip2 (Cdkn1c), and indirectly the myogenic regulatory factors (MRFs). Specifically, Cdkn1c activation is induced by NF-Y binding to its CCAAT promoter and by reducing the expression of the lncRNA Kcnq1ot1, a negative regulator of Cdkn1c transcription. Overall, our results indicate that NF-YA alternative splicing is an influential muscle cell determinant, through direct regulation of selected cell cycle blocking genes, and, directly and indirectly, of muscle-specific transcription factors.

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.

NF-Y and the transcriptional activation of CCAAT promoters.

The CCAAT box promoter element and NF-Y, the transcription factor (TF) that binds to it, were among the first cis-elements and trans-acting factors identified; their interplay is required for transcriptional activation of a sizeable number of eukaryotic genes. NF-Y consists of three evolutionarily conserved subunits: a dimer of NF-YB and NF-YC which closely resembles a histone, and the "innovative" NF-YA. In this review, we will provide an update on the functional and biological features that make NF-Y a fundamental link between chromatin and transcription. The last 25 years have witnessed a spectacular increase in our knowledge of how genes are regulated: from the identification of cis-acting sequences in promoters and enhancers, and the biochemical characterization of the corresponding TFs, to the merging of chromatin studies with the investigation of enzymatic machines that regulate epigenetic states. Originally identified and studied in yeast and mammals, NF-Y - also termed CBF and CP1 - is composed of three subunits, NF-YA, NF-YB and NF-YC. The complex recognizes the CCAAT pentanucleotide and specific flanking nucleotides with high specificity (Dorn et al., 1997; Hatamochi et al., 1988; Hooft van Huijsduijnen et al, 1987; Kim & Sheffery, 1990). A compelling set of bioinformatics studies clarified that the NF-Y preferred binding site is one of the most frequent promoter elements (Suzuki et al., 2001, 2004; Elkon et al., 2003; Mariño-Ramírez et al., 2004; FitzGerald et al., 2004; Linhart et al., 2005; Zhu et al., 2005; Lee et al., 2007; Abnizova et al., 2007; Grskovic et al., 2007; Halperin et al., 2009; Häkkinen et al., 2011). The same consensus, as determined by mutagenesis and SELEX studies (Bi et al., 1997), was also retrieved in ChIP-on-chip analysis (Testa et al., 2005; Ceribelli et al., 2006; Ceribelli et al., 2008; Reed et al., 2008). Additional structural features of the CCAAT box - position, orientation, presence of multiple Transcriptional Start Sites - were previously reviewed (Dolfini et al., 2009) and will not be considered in detail here.

Expression and function of NF-Y subunits in cancer.

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

The inhibition of inner mitochondrial fusion in hepatocytes reduces non-alcoholic fatty liver and improves metabolic profile during obesity by modulating bile acid conjugation.

AIMS: Mitochondria are plastic organelles that continuously undergo biogenesis, fusion, fission, and mitophagy to control cellular energy metabolism, calcium homeostasis, hormones, sterols, and bile acids (BAs) synthesis. Here, we evaluated how the impairment of mitochondrial fusion in hepatocytes affects diet-induced liver steatosis and obesity. METHODS AND RESULTS: Male mice selectively lacking the key protein involved in inner mitochondrial fusion, optic atrophy 1 (OPA1) (OPA1ΔHep) were fed a high fat diet (HFD) for 20 weeks. OPA1ΔHep mice were protected from the development of hepatic steatosis and obesity because of reduced lipid absorption; a profile which was accompanied by increased respiratory exchange ratio in vivo, suggesting a preference for carbohydrates in OPA1ΔHep compared to controls. At the molecular level, this phenotype emerged as a consequence of poor mitochondria-peroxisome- endoplasmic reticulum (ER) tethering in OPA1 deficient hepatocytes, which impaired BAs conjugation and release in the bile, thus impacting lipid absorption from the diet. Concordantly, the liver of subjects with non-alcoholic fatty liver disease (NAFLD) presented an increased expression of OPA1 and of the network of proteins involved in mitochondrial function when compared with controls. CONCLUSION: Patients with NAFLD present increased expression of proteins involved in mitochondrial fusion in the liver. The selective deficency of OPA1 in hepatocytes protects mice from HFD-induced metabolic dysfunction by reducing BAs secretion and dietary lipids absorption as a consequence of reduced liver mitochondria-peroxisome-ER tethering.

The short isoform of NF-YA belongs to the embryonic stem cell transcription factor circuitry.

Totipotency of embryonic stem cells (ESCs) is controlled at the transcriptional level by a handful of transcription factors (TFs) that promote stemness and prevent differentiation. One of the most enriched DNA elements in promoters and enhancers of genes specifically active in ESCs is the CCAAT box, which is recognized by NF-Y, a trimer with histone-like subunits--NF-YB/NF--YC--and the sequence-specific NF-YA. We show that the levels of the short NF-YA isoform--NF-YAs--is high in mouse ESCs (mESCs) and drops after differentiation; a dominant negative mutant affects expression of important stem cells genes, directly and indirectly. Protein transfections of TAT-NF-YAs stimulate growth and compensate for withdrawal of leukemia inhibitory factor (LIF) in cell cultures. Bioinformatic analysis identifies NF-Y sites as highly enriched in genomic loci of stem TFs in ESCs. Specifically, 30%-50% of NANOG peaks have NF-Y sites and indeed NF-Y-binding is required for NANOG association to DNA. These data indicate that NF-Y belongs to the restricted circle of TFs that govern mESCs, and, specifically, that NF-YAs is the active isoform in these cells.

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.

NF-YAl drives EMT in Claudinlow tumours.

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

The NF-Y splicing signature controls hybrid EMT and ECM-related pathways to promote aggressiveness of colon cancer.

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-YAlhigh) exhibit reduced cell proliferation, rapid single cell amoeboid-like migration, and form irregular spheroids with poor cell-to-cell adhesion. Compared to NF-YAshigh, NF-YAlhigh 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-YAlhigh 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.

MIR205HG/LEADR Long Noncoding RNA Binds to Primed Proximal Regulatory Regions in Prostate Basal Cells Through a Triplex- and Alu-Mediated Mechanism.

Aside serving as host gene for miR-205, MIR205HG transcribes for a chromatin-associated long noncoding RNA (lncRNA) able to restrain the differentiation of prostate basal cells, thus being reannotated as LEADR (Long Epithelial Alu-interacting Differentiation-related RNA). We previously showed the presence of Alu sequences in the promoters of genes modulated upon MIR205HG/LEADR manipulation. Notably, an Alu element also spans the first and second exons of MIR205HG/LEADR, suggesting its possible involvement in target selection/binding. Here, we performed ChIRP-seq to map MIR205HG/LEADR chromatin occupancy at genome-wide level in prostate basal cells. Our results confirmed preferential binding to regions proximal to gene transcription start site (TSS). Moreover, enrichment of triplex-forming sequences was found upstream of MIR205HG/LEADR-bound genes, peaking at -1,500/-500 bp from TSS. Triplexes formed with one or two putative DNA binding sites within MIR205HG/LEADR sequence, located just upstream of the Alu element. Notably, triplex-forming regions of bound genes were themselves enriched in Alu elements. These data suggest, from one side, that triplex formation may be the prevalent mechanism by which MIR205HG/LEADR selects and physically interacts with target DNA, from the other that direct or protein-mediated Alu (RNA)/Alu (DNA) interaction may represent a further functional requirement. We also found that triplex-forming regions were enriched in specific histone modifications, including H3K4me1 in the absence of H3K27ac, H3K4me3 and H3K27me3, indicating that in prostate basal cells MIR205HG/LEADR may preferentially bind to primed proximal regulatory elements. This may underscore the need for basal cells to keep MIR205HG/LEADR target genes repressed but, at the same time, responsive to differentiation cues.

NF-Y subunits overexpression in gastric adenocarcinomas (STAD).

NF-Y is a pioneer transcription factor-TF-formed by the Histone-like NF-YB/NF-YC subunits and the regulatory NF-YA. It binds to the CCAAT box, an element enriched in promoters of genes overexpressed in many types of cancer. NF-YA is present in two major isoforms-NF-YAs and NF-YAl-due to alternative splicing, overexpressed in epithelial tumors. Here we analyzed NF-Y expression in stomach adenocarcinomas (STAD). We completed the partitioning of all TCGA tumor samples (450) according to molecular subtypes proposed by TCGA and ACRG, using the deep learning tool DeepCC. We analyzed differentially expressed genes-DEG-for enriched pathways and TFs binding sites in promoters. CCAAT is the predominant element only in the core group of genes upregulated in all subtypes, with cell-cycle gene signatures. NF-Y subunits are overexpressed, particularly NF-YA. NF-YAs is predominant in CIN, MSI and EBV TCGA subtypes, NF-YAl is higher in GS and in the ACRG EMT subtypes. Moreover, NF-YAlhigh tumors correlate with a discrete Claudinlow cohort. Elevated NF-YB levels are protective in MSS;TP53+ patients, whereas high NF-YAl/NF-YAs ratios correlate with worse prognosis. We conclude that NF-Y isoforms are associated to clinically relevant features of gastric cancer.

NF-Y Subunits Overexpression in HNSCC.

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.

The transcription factor NF-Y participates to stem cell fate decision and regeneration in adult skeletal muscle.

The transcription factor NF-Y promotes cell proliferation and its activity often declines during differentiation through the regulation of NF-YA, the DNA binding subunit of the complex. In stem cell compartments, the shorter NF-YA splice variant is abundantly expressed and sustains their expansion. Here, we report that satellite cells, the stem cell population of adult skeletal muscle necessary for its growth and regeneration, express uniquely the longer NF-YA isoform, majorly associated with cell differentiation. Through the generation of a conditional knock out mouse model that selectively deletes the NF-YA gene in satellite cells, we demonstrate that NF-YA expression is fundamental to preserve the pool of muscle stem cells and ensures robust regenerative response to muscle injury. In vivo and ex vivo, satellite cells that survive to NF-YA loss exit the quiescence and are rapidly committed to early differentiation, despite delayed in the progression towards later states. In vitro results demonstrate that NF-YA-depleted muscle stem cells accumulate DNA damage and cannot properly differentiate. These data highlight a new scenario in stem cell biology for NF-Y activity, which is required for efficient myogenic differentiation.

Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients.

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.

NF-YA overexpression protects from glutamine deprivation.

The heterotrimeric transcription factor NF-Y binds to CCAAT boxes of genes of glutamine metabolism. We set out to study the role of the regulatory NF-YA subunit in this pathway. We produced U2OS and A549 clones stably overexpressing -OE- the two splicing isoforms of NF-YA. NF-YA OE cells show normal growth and colony formation rates, but they become resistant to cell death upon glutamine deprivation. Increased mRNA and protein expression of the key biosynthetic enzyme GLUL in U2OS entails increased production of endogenous glutamine upon deprivation. The use of GLUL inhibitors dampens the NF-YA-mediated effect. NF-YA OE prevents activation of the pro-apoptotic transcription factor CHOP/DDIT3. Elevated basal levels of SERCA1/2, coding for the molecular target of Thapsigargin, correlate with resistance of NF-YA OE cells to the drug. The work represents a proof-of-principle that elevated levels of NF-YA, as found in some tumor types, helps altering cancer metabolic pathways.

Integrating Peak Colocalization and Motif Enrichment Analysis for the Discovery of Genome-Wide Regulatory Modules and Transcription Factor Recruitment Rules.

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) has opened new avenues of research in the genome-wide characterization of regulatory DNA-protein interactions at the genetic and epigenetic level. As a consequence, it has become the de facto standard for studies on the regulation of transcription, and literally thousands of data sets for transcription factors and cofactors in different conditions and species are now available to the scientific community. However, while pipelines and best practices have been established for the analysis of a single experiment, there is still no consensus on the best way to perform an integrated analysis of multiple datasets in the same condition, in order to identify the most relevant and widespread regulatory modules composed by different transcription factors and cofactors. We present here a computational pipeline for this task, that integrates peak summit colocalization, a novel statistical framework for the evaluation of its significance, and motif enrichment analysis. We show examples of its application to ENCODE data, that led to the identification of relevant regulatory modules composed of different factors, as well as the organization on DNA of the binding motifs responsible for their recruitment.