ABSTRACT
Transcription factors (TFs) play a central role in gene regulation, and their malfunction can result in a plethora of severe diseases. TFs are therefore interesting therapeutic targets, but their involvement in protein-protein interaction networks and the frequent lack of well-defined binding pockets render them challenging targets for classical small molecules. As an alternative, peptide-based scaffolds have proven useful, in particular with an α-helical active conformation. Peptide-based strategies often require extensive structural optimization efforts, which could benefit from a more detailed understanding of the dynamics in inhibitor/protein interactions. In this study, we investigate how truncated stapled α-helical peptides interact with the transcription factor Nuclear Factor-Y (NF-Y). We identified a 13-mer minimal binding core region, for which two crystal structures with an altered C-terminal peptide conformation when bound to NF-Y were obtained. Subsequent molecular dynamics simulations confirmed that the C-terminal part of the stapled peptide is indeed relatively flexible while still showing defined interactions with NF-Y. Our findings highlight the importance of flexibility in the bound state of peptides, which can contribute to overall binding affinity.
Subject(s)
CCAAT-Binding Factor , Molecular Dynamics Simulation , Peptides , Protein Binding , Peptides/chemistry , Peptides/metabolism , CCAAT-Binding Factor/metabolism , CCAAT-Binding Factor/chemistry , Binding Sites , Humans , Crystallography, X-Ray , Amino Acid SequenceABSTRACT
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.
Subject(s)
CCAAT-Binding Factor/genetics , Gene Expression Regulation, Neoplastic , Protein Subunits/genetics , Stomach Neoplasms/genetics , CCAAT-Binding Factor/chemistry , CCAAT-Binding Factor/metabolism , Cell Line, Tumor , Computational Biology/methods , Gene Expression Profiling , Humans , Prognosis , Protein Binding , Protein Interaction Domains and Motifs/genetics , Protein Isoforms/genetics , Protein Subunits/metabolism , Signal Transduction , Stomach Neoplasms/metabolism , Stomach Neoplasms/mortality , Stomach Neoplasms/pathology , TranscriptomeABSTRACT
Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor playing crucial roles in various biological process in plant. However, thorough research on NF-Y gene family of Tartary buckwheat (Fagopyrum tataricum) is little. In this study, 38 FtNF-Y genes (12 FtNF-YAs, 17 FtNF-YBs, and 9 FtNF-YCs) were identified and renamed on the basis of their subfamily and chromosomal location. Their gene structure, genomic mapping, motif composition, conserved domain, phylogenetic relationships, cis-acting elements and gene expression were investigated. Illustration of gene structures and conserved domains of FtNF-Ys revealed their functional conservation and specificity. Construction of phylogenetic trees of NF-Ys in Tartary buckwheat, Arabidopsis, tomato, rice and banana, allowed us to predict functional similarities among NF-Ys from different species. Gene expression analysis displayed that twenty-four FtNF-Ys were expressed in all the tissues and the transcript levels of them were different, suggesting their function varieties. Moreover, expression profiles of twenty FtNF-Ys along five different fruit development stages acquired by real-time quantitative PCR (RT-qPCR) demonstrated distinct abundance diversity at different stages, providing some clues of potential fruit development regulators. Our study could provide helpful reference information for further function characterization of FtNF-Ys and for the fruit quality enhancement of Tartary buckwheat.
Subject(s)
CCAAT-Binding Factor/genetics , Fagopyrum/genetics , Fruit/growth & development , Fruit/genetics , Genome, Plant , Multigene Family , Plant Proteins/genetics , Amino Acid Motifs , Amino Acid Sequence , CCAAT-Binding Factor/chemistry , Chromosomes, Plant/genetics , Conserved Sequence , Evolution, Molecular , Gene Duplication/genetics , Gene Expression Profiling , Gene Expression Regulation, Plant , Organ Specificity/genetics , Phylogeny , Plant Proteins/chemistry , Promoter Regions, Genetic/geneticsABSTRACT
With the explosive growth of protein sequences, large-scale automated protein function prediction (AFP) is becoming challenging. A protein is usually associated with dozens of gene ontology (GO) terms. Therefore, AFP is regarded as a problem of large-scale multi-label classification. Under the learning to rank (LTR) framework, our previous NetGO tool integrated massive networks and multi-type information about protein sequences to achieve good performance by dealing with all possible GO terms (>44 000). In this work, we propose the updated version as NetGO 2.0, which further improves the performance of large-scale AFP. NetGO 2.0 also incorporates literature information by logistic regression and deep sequence information by recurrent neural network (RNN) into the framework. We generate datasets following the critical assessment of functional annotation (CAFA) protocol. Experiment results show that NetGO 2.0 outperformed NetGO significantly in biological process ontology (BPO) and cellular component ontology (CCO). In particular, NetGO 2.0 achieved a 12.6% improvement over NetGO in terms of area under precision-recall curve (AUPR) in BPO and around 2.6% in terms of $\mathbf {F_{max}}$ in CCO. These results demonstrate the benefits of incorporating text and deep sequence information for the functional annotation of BPO and CCO. The NetGO 2.0 web server is freely available at http://issubmission.sjtu.edu.cn/ng2/.
Subject(s)
Proteins/physiology , Software , CCAAT-Binding Factor/chemistry , CCAAT-Binding Factor/metabolism , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/metabolism , High-Throughput Nucleotide Sequencing , Neural Networks, Computer , Protein Domains , Proteins/classification , Proteins/metabolism , Sequence Analysis, ProteinABSTRACT
BCL11A, the major regulator of fetal hemoglobin (HbF, α2γ2) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, α2ß2). To uncover how BCL11A initiates repression, we used CRISPR-Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)-globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the >50-kb ß-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.
Subject(s)
CCAAT-Binding Factor/chemistry , Fetal Hemoglobin/genetics , Hemoglobin A/genetics , Promoter Regions, Genetic , Repressor Proteins/chemistry , gamma-Globins/chemistry , Base Sequence , Binding Sites , Binding, Competitive , CCAAT-Binding Factor/genetics , CCAAT-Binding Factor/metabolism , Erythropoiesis/genetics , Fetal Hemoglobin/metabolism , Gene Editing/methods , Gene Expression Regulation , HEK293 Cells , Hemoglobin A/metabolism , Humans , Models, Molecular , Primary Cell Culture , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Repressor Proteins/genetics , Repressor Proteins/metabolism , Stem Cells , beta-Globins/chemistry , beta-Globins/genetics , beta-Globins/metabolism , gamma-Globins/genetics , gamma-Globins/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , CCAAT-Binding Factor/metabolism , DNA, Plant/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Binding Sites , CCAAT-Binding Factor/chemistry , CCAAT-Binding Factor/genetics , DNA, Plant/chemistry , Dimerization , Oryza/genetics , Oryza/metabolism , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Structure, TertiaryABSTRACT
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.
Subject(s)
CCAAT-Binding Factor/genetics , Carcinoma, Hepatocellular/genetics , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , Biomarkers, Tumor , CCAAT-Binding Factor/chemistry , CCAAT-Binding Factor/metabolism , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Gene Expression Profiling , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Neoplasm Staging , Promoter Regions, Genetic , Protein Isoforms , Protein Subunits/genetics , Protein Subunits/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Protein p53/geneticsABSTRACT
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.
Subject(s)
CCAAT-Binding Factor/antagonists & inhibitors , CCAAT-Binding Factor/chemistry , Suramin/chemistry , Suramin/pharmacology , Transcription Factors/antagonists & inhibitors , Transcription Factors/chemistry , Amino Acid Sequence , Biophysical Phenomena , DNA/metabolism , Humans , Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Protein Multimerization , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Bees/genetics , Bees/physiology , CCAAT-Binding Factor/metabolism , Gene Expression Profiling , Stress, Physiological/genetics , Amino Acid Motifs , Animals , Antioxidants/pharmacology , Bees/drug effects , CCAAT-Binding Factor/chemistry , CCAAT-Binding Factor/genetics , Drosophila/genetics , Gene Expression Regulation/drug effects , Genome, Insect , Honey , Models, Biological , Oxidative Stress/drug effects , Phylogeny , Protein Binding/drug effects , Protein Structure, Tertiary , Subcellular Fractions/metabolism , Time FactorsABSTRACT
Oral squamous cell carcinoma (OSCC) has been reported to be the most common oral carcinoma. Emerging evidence has revealed the key role that long noncoding RNAs (lncRNAs) play in numerous malignancies, including OSCC. LncRNA small nucleolar RNA host gene 3 (SNHG3) has been reported as an oncogenic factor in some cancers. Nonetheless, the role of SNHG3 in OSCC has never been clarified. In this study, we analyzed the expression patterns of SNHG3 in OSCC through quantitative real-time polymerase chain reaction. It was revealed that the expression level of SNHG3 was remarkably elevated in OSCC cell lines compared with the nontumor cell line. It was demonstrated by functional experiments that SNHG3 knockdown notably inhibited cell proliferation and migration in OSCC. RNA immunoprecipitation, RNA pull down, and messenger RNA (mRNA) stability test verified that SNHG3 decoyed ELAV like RNA-binding protein 1 (ELAVL1) and therefore stabilized nuclear transcription factor Y subunit gamma (NFYC) mRNA to upregulate the expression levels of NFYC in OSCC cells. At last, it was confirmed by rescue experiments that the inhibiting impacts of SNHG3 knockdown on OSCC cell proliferation and migration could be partly revived by NFYC overexpression. Besides, we validated that Wnt/ß-catenin pathway was also involved in SNHG3-regulated OSCC progression. In conclusion, SNHG3 might serve as a novel biomarker for OSCC.
Subject(s)
Biomarkers, Tumor/metabolism , CCAAT-Binding Factor/metabolism , Carcinoma, Squamous Cell/pathology , ELAV-Like Protein 1/metabolism , Gene Expression Regulation, Neoplastic , Mouth Neoplasms/pathology , RNA, Long Noncoding/genetics , Apoptosis , Biomarkers, Tumor/genetics , CCAAT-Binding Factor/chemistry , CCAAT-Binding Factor/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Cell Movement , Cell Proliferation , ELAV-Like Protein 1/genetics , Humans , Mouth Neoplasms/genetics , Mouth Neoplasms/metabolism , RNA Stability , Tumor Cells, Cultured , Wnt Signaling Pathway , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
Protein complex formation depends on the interplay between preorganization and flexibility of the binding epitopes involved. The design of epitope mimetics typically focuses on stabilizing a particular bioactive conformation, often without considering conformational dynamics, which limits the potential of peptidomimetics against challenging targets such as transcription factors. We developed a peptide-derived inhibitor of the NF-Y transcription factor by first constraining the conformation of an epitope through hydrocarbon stapling and then fine-tuning its flexibility. In the initial set of constrained peptides, a single non-interacting α-methyl group was observed to have a detrimental effect on complex stability. Biophysical characterization revealed how this methyl group affects the conformation of the peptide in its bound state. Adaption of the methylation pattern resulted in a peptide that inhibits transcription factor assembly and subsequent recruitment to the target DNA.
Subject(s)
CCAAT-Binding Factor/chemistry , Peptides/chemistry , Protein Multimerization/drug effects , Base Sequence , Binding Sites , Cross-Linking Reagents/chemistry , Crystallization , DNA/chemistry , Epitopes/chemistry , Humans , Macrocyclic Compounds/chemistry , Methylation , Molecular Dynamics Simulation , Peptidomimetics , Protein Binding , Protein Conformation , ThermodynamicsABSTRACT
Different transcription factors operate together at promoters and enhancers to regulate gene expression. Transcription factors either bind directly to their target DNA or are tethered to it by other proteins. The transcription factor Sp2 serves as a paradigm for indirect genomic binding. It does not require its DNA-binding domain for genomic DNA binding and occupies target promoters independently of whether they contain a cognate DNA-binding motif. Hence, Sp2 is strikingly different from its closely related paralogs Sp1 and Sp3, but how Sp2 recognizes its targets is unknown. Here, we sought to gain more detailed insights into the genomic targeting mechanism of Sp2. ChIP-exo sequencing in mouse embryonic fibroblasts revealed genomic binding of Sp2 to a composite motif where a recognition sequence for TALE homeoproteins and a recognition sequence for the trimeric histone-fold domain protein nuclear transcription factor Y (Nf-y) are separated by 11 bp. We identified a complex consisting of the TALE homeobox protein Prep1, its partner PBX homeobox 1 (Pbx1), and Nf-y as the major partners in Sp2-promoter interactions. We found that the Pbx1:Prep1 complex together with Nf-y recruits Sp2 to co-occupied regulatory elements. In turn, Sp2 potentiates binding of Pbx1:Prep1 and Nf-y. We also found that the Sp-box, a short sequence motif close to the Sp2 N terminus, is crucial for Sp2's cofactor function. Our findings reveal a mechanism by which the DNA binding-independent activity of Sp2 potentiates genomic loading of Pbx1:Prep1 and Nf-y to composite motifs present in many promoters of highly expressed genes.
Subject(s)
CCAAT-Binding Factor/metabolism , Genomics , Homeodomain Proteins/metabolism , Pre-B-Cell Leukemia Transcription Factor 1/metabolism , Sp2 Transcription Factor/metabolism , Animals , CCAAT-Binding Factor/chemistry , Cell Line , Histones/metabolism , Mice , Nucleotide Motifs , Protein Binding , Protein Transport , Sp2 Transcription Factor/chemistry , Zinc FingersSubject(s)
Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , CCAAT-Binding Factor/chemistry , CCAAT-Binding Factor/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Histones/metabolism , Protein Multimerization , Protein Subunits/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Crystallography, X-Ray , DNA, Plant/metabolism , Models, Molecular , Protein BindingABSTRACT
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.
Subject(s)
CCAAT-Binding Factor/chemistry , Protein Multimerization , Response Elements , Animals , CCAAT-Binding Factor/genetics , CCAAT-Binding Factor/metabolism , Humans , Protein Structure, Quaternary , Protein Structure, SecondaryABSTRACT
ChIP-seq experiments detect the chromatin occupancy of known transcription factors in a genome-wide fashion. The comparisons of several species-specific ChIP-seq libraries done for different transcription factors have revealed a complex combinatorial and context-specific co-localization behavior for the identified binding regions. In this study we have investigated human derived ChIP-seq data to identify common cis-regulatory principles for the human transcription factor c-Fos. We found that in four different cell lines, c-Fos targeted proximal and distal genomic intervals show prevalences for either AP-1 motifs or CCAAT boxes as known binding motifs for the transcription factor NF-Y, and thereby act in a mutually exclusive manner. For proximal regions of co-localized c-Fos and NF-YB binding, we gathered evidence that a characteristic configuration of repeating CCAAT motifs may be responsible for attracting c-Fos, probably provided by a nearby AP-1 bound enhancer. Our results suggest a novel regulatory function of NF-Y in gene-proximal regions. Specific CCAAT dimer repeats bound by the transcription factor NF-Y define this novel cis-regulatory module. Based on this behavior we propose a new enhancer promoter interaction model based on AP-1 motif defined enhancers which interact with CCAAT-box characterized promoter regions.
Subject(s)
CCAAT-Binding Factor/chemistry , CCAAT-Binding Factor/metabolism , Promoter Regions, Genetic , Proto-Oncogene Proteins c-fos/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Cell Line, Tumor , Dimerization , Humans , Models, Molecular , Proto-Oncogene Proteins c-fos/chemistry , Transcription Factor AP-1/metabolism , p300-CBP Transcription Factors/metabolismABSTRACT
Endogenous viral elements (EVE) in animal genomes are the fossil records of ancient viruses and provide invaluable information on the origin and evolution of extant viruses. Extant hepadnaviruses include avihepadnaviruses of birds and orthohepadnaviruses of mammals. The core promoter (Cp) of hepadnaviruses is vital for viral gene expression and replication. We previously identified in the budgerigar genome two EVEs that contain the full-length genome of an ancient budgerigar hepadnavirus (eBHBV1 and eBHBV2). Here, we found eBHBV1 Cp and eBHBV2 Cp were active in several human and chicken cell lines. A region from nt -85 to -11 in eBHBV1 Cp was critical for the promoter activity. Bioinformatic analysis revealed a putative binding site of nuclear factor Y (NF-Y), a ubiquitous transcription factor, at nt -64 to -50 in eBHBV1 Cp. The NF-Y core binding site (ATTGG, nt -58 to -54) was essential for eBHBV1 Cp activity. The same results were obtained with eBHBV2 Cp and duck hepatitis B virus Cp. The subunit A of NF-Y (NF-YA) was recruited via the NF-Y core binding site to eBHBV1 Cp and upregulated the promoter activity. Finally, the NF-Y core binding site is conserved in the Cps of all the extant avihepadnaviruses but not of orthohepadnaviruses. Interestingly, a putative and functionally important NF-Y core binding site is located at nt -21 to -17 in the Cp of human hepatitis B virus. In conclusion, our findings have pinpointed an evolutionary conserved and functionally critical NF-Y binding element in the Cps of avihepadnaviruses.
Subject(s)
CCAAT-Binding Factor/genetics , DNA Transposable Elements , DNA, Viral/genetics , Genome , Hepadnaviridae/genetics , Hepatocytes/metabolism , Animals , Binding Sites , Biological Evolution , Bird Diseases/virology , CCAAT-Binding Factor/chemistry , CCAAT-Binding Factor/metabolism , Cell Line , Cell Line, Tumor , Chick Embryo , Chickens , Conserved Sequence , DNA, Viral/metabolism , Extinction, Biological , Fibroblasts/metabolism , Fibroblasts/virology , Fossils , HEK293 Cells , Hepadnaviridae/classification , Hepadnaviridae/metabolism , Hepadnaviridae Infections/veterinary , Hepadnaviridae Infections/virology , Hepatitis B virus/genetics , Hepatitis B virus/metabolism , Hepatocytes/virology , Host-Pathogen Interactions , Humans , Melopsittacus , Phylogeny , Promoter Regions, Genetic , Protein BindingABSTRACT
MOTIVATION: miRNAs are potent regulators of gene expression and modulate multiple cellular processes in physiology and pathology. Deregulation of miRNAs expression has been found in various cancer types, thus, miRNAs may be potential targets for cancer therapy. However, the mechanisms through which miRNAs are regulated in cancer remain unclear. Therefore, the identification of transcriptional factor-miRNA crosstalk is one of the most update aspects of the study of miRNAs regulation. RESULTS: In the present study we describe the development of a fast and user-friendly software, named infinity, able to find the presence of DNA matrices, such as binding sequences for transcriptional factors, on ~65kb (kilobase) of 939 human miRNA genomic sequences, simultaneously. Of note, the power of this software has been validated in vivo by performing chromatin immunoprecipitation assays on a subset of new in silico identified target sequences (CCAAT) for the transcription factor NF-Y on colon cancer deregulated miRNA loci. Moreover, for the first time, we have demonstrated that NF-Y, through its CCAAT binding activity, regulates the expression of miRNA-181a, -181b, -21, -17, -130b, -301b in colon cancer cells. CONCLUSIONS: The infinity software that we have developed is a powerful tool to underscore new TF/miRNA regulatory networks. AVAILABILITY AND IMPLEMENTATION: Infinity was implemented in pure Java using Eclipse framework, and runs on Linux and MS Windows machine, with MySQL database. The software is freely available on the web at https://github.com/bio-devel/infinity. The website is implemented in JavaScript, PHP and HTML with all major browsers supported.
Subject(s)
Colonic Neoplasms/genetics , Computational Biology/methods , MicroRNAs/genetics , Software , Algorithms , CCAAT-Binding Factor/chemistry , Cell Line, Tumor , DNA/chemistry , Gene Expression Regulation, Neoplastic , Genome, Human , Humans , Programming Languages , Promoter Regions, Genetic , Protein BindingABSTRACT
Nuclear factor-Y (NF-Y), is a plant heterotrimeric transcription factor constituted by NF-YA, NF-YB and NF-YC subunits. The function of many NF-Y subunits, mostly of the A and B type, has been studied in plants, but knowledge regarding the C subunit remains fragmentary. Here, a water stress-induced NF-YC gene from Amaranthus hypochondriacus (AhNF-YC) was further characterized by its overexpression in transgenic Arabidospis thaliana plants. A role in development was inferred from modified growth rates in root, rosettes and inflorescences recorded in AhNF-YC overexpressing Arabidopsis plants, in addition to a delayed onset of flowering. Also, the overexpression of AhNF-YC caused increased seedling sensitivity to abscisic acid (ABA), and influenced the expression of several genes involved in secondary metabolism, development and ABA-related responses. An altered expression of the latter in water stressed and recovered transgenic plants, together with the observed increase in ABA sensitivity, suggested that their increased water stress resistance was partly ABA-dependent. An untargeted metabolomic analysis also revealed an altered metabolite pattern, both in normal and water stress/recovery conditions. These results suggest that AhNF-YC may play an important regulatory role in both development and stress, and represents a candidate gene for the engineering of abiotic stress resistance in commercial crops.
Subject(s)
Amaranthus/genetics , Arabidopsis/physiology , CCAAT-Binding Factor/genetics , Ectopic Gene Expression , Gene Expression Regulation, Plant , Plant Proteins/genetics , Stress, Physiological/genetics , Amaranthus/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/growth & development , CCAAT-Binding Factor/chemistry , CCAAT-Binding Factor/metabolism , Droughts , Oligonucleotide Array Sequence Analysis , Phylogeny , Plant Proteins/chemistry , Plant Proteins/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Sequence Alignment , Sequence Analysis, DNAABSTRACT
The NF-YB transcription factor gene family encodes a subunit of the CCAAT box-binding factor (CBF), a highly conserved trimeric activator that strongly binds to the CCAAT box promoter element. Studies on model plants have shown that NF-YB proteins participate in important developmental and physiological processes, but little is known about NF-YB proteins in trees. Here, we identified seven NF-YB transcription factor-encoding genes in Vernicia fordii, an important oilseed tree in China. A phylogenetic analysis separated the genes into two groups; non-LEC1 type (VfNF-YB1, 5, 7, 9, 11, 13) and LEC1-type (VfNF-YB 14). A gene structure analysis showed that VfNF-YB 5 has three introns and the other genes have no introns. The seven VfNF-YB sequences contain highly conserved domains, a disordered region at the N terminus, and two long helix structures at the C terminus. Phylogenetic analyses showed that VfNF-YB family genes are highly homologous to GmNF-YB genes, and many of them are closely related to functionally characterized NF-YBs. In expression analyses of various tissues (root, stem, leaf, and kernel) and the root during pathogen infection, VfNF-YB1, 5, and 11 were dominantly expressed in kernels, and VfNF-YB7 and 9 were expressed only in the root. Different VfNF-YB family genes showed different responses to pathogen infection, suggesting that they play different roles in the pathogen response. Together, these findings represent the first extensive evaluation of the NF-YB family in tung tree and provide a foundation for dissecting the functions of VfNF-YB genes in seed development, stress adaption, fatty acid synthesis, and pathogen response.
Subject(s)
CCAAT-Binding Factor/genetics , Euphorbiaceae/genetics , Genes, Plant , Amino Acid Sequence , CCAAT-Binding Factor/chemistry , Euphorbiaceae/metabolism , Molecular Sequence Data , Phylogeny , RNA, Messenger/metabolism , Sequence Homology, Amino AcidABSTRACT
NFYA5 is an important drought-stress inducible transcription factor gene that is targeted by miR169 in Arabidopsis. We show here that the cis-natural antisense transcript gene of NFYA5, NFYA5 Enhancing RING FINGER (NERF), can produce siRNAs from their overlapping region (OR) and affect NFYA5 transcripts by functioning together with miR169. The NERF protein functions as an E3 ligase for ubiquitination. Overexpression of NERF or OR cDNA leads to siRNANERF accumulation, miR169 repression, and NFYA5 transcript enhancement; knock-down of NERF transcripts by an artificial miRNA enhances miR169 abundance and reduces NFYA5 transcripts. Overexpression of NFYA5 does not affect the NERF mRNA level. Deep sequencing of the small RNA library from 35S::OR plants identifies 960 sequences representing 323 unique siRNAs that originate from OR; the sequences of some siRNANERF are similar/complementary to those of miR169. Overexpression of the 195- to 280-bp OR cDNA-containing siRNAs similar/complementary to miR169 also leads to the accumulation of NFYA5 transcripts. Analysis of NERF knock-down plants and NERF overexpression lines showed that, like NFYA5, NERF is important for controlling stomatal aperture and drought resistance. This regulatory model might apply to other natural antisense transcripts with positively correlated expression patterns.