RESUMEN
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.
Asunto(s)
Factor de Unión a CCAAT , Neoplasias , Humanos , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Factores de Transcripción/genética , Neoplasias/genética , Isoformas de Proteínas/genética , Regulación de la Expresión GénicaRESUMEN
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.
Asunto(s)
Factores de Transcripción , Vertebrados , Empalme Alternativo , Animales , Evolución Molecular , Peces/metabolismo , Mamíferos , Filogenia , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Factores de Transcripción/genética , Vertebrados/genéticaRESUMEN
The trimeric CCAAT-binding NF-Y is a "pioneer" Transcription Factor -TF- known to cooperate with neighboring TFs to regulate gene expression. Genome-wide analyses detected a precise stereo-alignment -10/12 bp- of CCAAT with E-box elements and corresponding colocalization of NF-Y with basic-Helix-Loop-Helix (bHLH) TFs. We dissected here NF-Y interactions with USF1 and MAX. USF1, but not MAX, cooperates in DNA binding with NF-Y. NF-Y and USF1 synergize to activate target promoters. Reconstruction of complexes by structural means shows independent DNA binding of MAX, whereas USF1 has extended contacts with NF-Y, involving the USR, a USF-specific amino acid sequence stretch required for trans-activation. The USR is an intrinsically disordered domain and adopts different conformations based on E-box-CCAAT distances. Deletion of the USR abolishes cooperative DNA binding with NF-Y. Our data indicate that the functionality of certain unstructured domains involves adapting to small variation in stereo-alignments of the multimeric TFs sites.
Asunto(s)
ADN/metabolismo , Factores Estimuladores hacia 5'/metabolismo , Regulación de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Dominios ProteicosRESUMEN
The E2F1 transcription factor is a master regulator of cell-cycle progression whose uncontrolled activation contributes to tumor cells growth. E2F1 binds DNA as a heterodimer with DP partners, resulting in a multi-domain quaternary-structure complex composed of DNA binding domains, a coiled coil domain and a marked box domain separated by short linkers. Building on the 3D knowledge of the single domains of E2F and DPs, we characterized the structure and dynamics of the complete E2F1/DP1/DNA complex by a combination of small-angle X-ray scattering and molecular dynamics simulations. It shows an asymmetric contribution of the dynamics of the two proteins. Namely, the coiled-coil domain leans toward the DP1 side of the complex; the DP1 loop between α2 and α3 of the DBD partially populates a helical structure leaning far from the DNA and in the same direction of the coiled-coil domain; and the N-terminal disordered region of DP1, rich in basic residues, contributes to DNA binding stabilization. Intriguingly, tumor mutations in the flexible regions of the complex suggest that perturbation of protein dynamics could affect protein function in a context-dependent way. Our data suggest fundamental contributions of DP proteins in distinct aspects of E2F biology.
Asunto(s)
ADN/química , ADN/metabolismo , Factor de Transcripción E2F1/química , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción DP1/química , Factor de Transcripción DP1/metabolismo , Ciclo Celular , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Conformación de Ácido Nucleico , Fosforilación , Unión Proteica , Conformación ProteicaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Factor de Unión a CCAAT/metabolismo , ADN de Plantas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sitios de Unión , Factor de Unión a CCAAT/química , Factor de Unión a CCAAT/genética , ADN de Plantas/química , Dimerización , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estructura Terciaria de ProteínaRESUMEN
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.
Asunto(s)
Factor de Unión a CCAAT/antagonistas & inhibidores , Factor de Unión a CCAAT/química , Suramina/química , Suramina/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Secuencia de Aminoácidos , Fenómenos Biofísicos , ADN/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Multimerización de Proteína , Relación Estructura-ActividadRESUMEN
Nuclear Factor Y (NF-Y) is an evolutionarily conserved trimer formed by a Histone-Fold Domain (HFD) heterodimeric module shared by core histones, and the sequence-specific NF-YA subunit. In plants, the genes encoding each of the three subunits have expanded in number, giving rise to hundreds of potential trimers. While in mammals NF-Y binds a well-characterized motif, with a defined matrix centered on the CCAAT box, the specificity of the plant trimers has yet to be determined. Here we report that Arabidopsis thaliana NF-Y trimeric complexes, containing two different NF-YA subunits, bind DNA in vitro with similar affinities. We assayed precisely sequence-specificity by saturation mutagenesis, and analyzed genomic DNA sites bound in vivo by selected HFDs. The plant NF-Y CCAAT matrix is different in nucleotides flanking CCAAT with respect to the mammalian matrix, in vitro and in vivo. Our data point to flexible DNA-binding rules by plant NF-Ys, serving the scope of adapting to a diverse audience of genomic motifs.
RESUMEN
Nuclear factor Y (NF-Y) is a transcription factor trimer binding to the functionally important CCAAT box, present in promoters of growth-promoting and cell cycle-regulated genes. The regulatory nuclear factor YA (NF-YA) subunit confers sequence-specificity to the histone-like nuclear factor YB/YC dimer. NF-YA harbors 2 serines-Ser320 and Ser326-shown to be phosphorylated by cyclin-dependent kinase 2. High-throughput proteomics data indicate that they are phosphorylated in vivo. Specifically, Ser320 makes structural contacts with the DNA phosphate backbone; Ser320-P is the major NF-YA phosphorylation isoform following overexpression in HeLa cells, increasing upon mitotic arrest. EMSA with recombinant Ala and Glu mutants confirm a role of Ser320, but not Ser326, in stabilization of DNA binding. Transactivation assays of the CCAAT-dependent MDR1 and RHOB promoters show loss in transcription function for Ser320Glu and Ser320Ala NF-YA mutants. Phylogenetic analysis of NF-YA proteins indicates that Ser320 is indeed evolutionarily conserved. We conclude that phosphorylation of this residue belongs to the core mechanisms of DNA-binding control, possibly driven by the necessity to unfasten binding of or to evict NF-Y from CCAAT sites under specific conditions of growth regulation.-Bernardini, A., Lorenzo, M., Nardini, M., Mantovani, R., Gnesutta, N. The phosphorylatable Ser320 of NF-YA is involved in DNA binding of the NF-Y trimer.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , Serina/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Western Blotting , Factor de Unión a CCAAT/genética , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Fosforilación , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína de Unión al GTP rhoB/metabolismoRESUMEN
Cell Penetrating Peptides -CPPs- are short aminoacidic stretches present in proteins that have the ability to translocate the plasma membrane and facilitate delivery of various molecules. They are usually rich in basic residues, and organized as alpha helices. NF-Y is a transcription factor heterotrimer formed by two Histone Fold Domain -HFD- subunits and the sequence-specific NF-YA. NF-YA possesses two α-helices rich in basic residues. We show that it efficiently enters cells at nanomolar concentrations in the absence of carrier peptides. Mutagenesis identified at least two separate CPPs in the A1 and A2, which overlap with previously identified nuclear localization signals (NLS). The half-life of the transduced protein is short in human cancer cells, longer in mouse C2C12 myoblasts. The internalized NF-YA is capable of trimerization with the HFD subunits and binding to the target CCAAT box. Functionality is further suggested by protein transfection in C2C12 cells, leading to inhibition of differentiation to myotubes. In conclusion, NF-YA contains CPPs, hinting at novel -and unexpected- properties of this subunit.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , Péptidos de Penetración Celular/metabolismo , Secuencia de Aminoácidos , Animales , Factor de Unión a CCAAT/genética , Línea Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Células HCT116 , Células HeLa , Humanos , Ratones , Mioblastos/metabolismo , Señales de Localización Nuclear/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , TransfecciónRESUMEN
CONSTANS (CO) is a master regulator of flowering time, although the mechanisms underlying its role as a transcriptional regulator are not well understood. The DNA-binding domain of CO shares homology with that of NUCLEAR FACTOR YA (NF-YA), a subunit of the CCAAT-binding trimer NF-Y. Recent publications indicate that CO and its rice homolog HEADING DATE 1 (Hd1) form heterotrimers with the histone-fold subunits of NF-Y to efficiently bind promoter elements in the florigen genes. Differences in the DNA-binding specificities of NF-Y and NF-CO can be conceptualized based on our knowledge of the 3D structure of the NF-Y/CCAAT complex. Here we discuss the modes of assembly of NF-Y-like heterotrimers and possible models for their activity as flexible sequence-specific transcriptional regulators.
Asunto(s)
ADN de Plantas/fisiología , Flores/crecimiento & desarrollo , Histonas/metabolismo , ADN de Plantas/genética , Flores/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Histonas/genética , Histonas/fisiología , Fotoperiodo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
Nuclear Factor Y (NF-Y) is a heterotrimeric transcription factor that binds CCAAT elements. The NF-Y trimer is composed of a Histone Fold Domain (HFD) dimer (NF-YB/NF-YC) and NF-YA, which confers DNA sequence specificity. NF-YA shares a conserved domain with the CONSTANS, CONSTANS-LIKE, TOC1 (CCT) proteins. We show that CONSTANS (CO/B-BOX PROTEIN1 BBX1), a master flowering regulator, forms a trimer with Arabidopsis thaliana NF-YB2/NF-YC3 to efficiently bind the CORE element of the FLOWERING LOCUS T promoter. We term this complex NF-CO. Using saturation mutagenesis, electrophoretic mobility shift assays, and RNA-sequencing profiling of co, nf-yb, and nf-yc mutants, we identify CCACA elements as the core NF-CO binding site. CO physically interacts with the same HFD surface required for NF-YA association, as determined by mutations in NF-YB2 and NF-YC9, and tested in vitro and in vivo. The co-7 mutation in the CCT domain, corresponding to an NF-YA arginine directly involved in CCAAT recognition, abolishes NF-CO binding to DNA. In summary, a unifying molecular mechanism of CO function relates it to the NF-YA paradigm, as part of a trimeric complex imparting sequence specificity to HFD/DNA interactions. It is likely that members of the large CCT family participate in similar complexes with At-NF-YB and At-NF-YC, broadening HFD combinatorial possibilities in terms of trimerization, DNA binding specificities, and transcriptional regulation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , ADN de Plantas/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Arabidopsis/genética , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Monóxido de Carbono/metabolismo , Proteínas de Unión al ADN/genética , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
Rice flowering is controlled by changes in the photoperiod that promote the transition to the reproductive phase as days become shorter. Natural genetic variation for flowering time has been largely documented and has been instrumental to define the genetics of the photoperiodic pathway, as well as providing valuable material for artificial selection of varieties better adapted to local environments. We mined genetic variation in a collection of rice varieties highly adapted to European regions and isolated distinct variants of the long day repressor HEADING DATE 1 (Hd1) that perturb its expression or protein function. Specific variants allowed us to define novel features of the photoperiodic flowering pathway. We demonstrate that a histone fold domain scaffold formed by GRAIN YIELD, PLANT HEIGHT AND HEADING DATE 8 (Ghd8) and several NF-YC subunits can accommodate distinct proteins, including Hd1 and PSEUDO RESPONSE REGULATOR 37 (PRR37), and that the resulting OsNF-Y complex containing Hd1 can bind a specific sequence in the promoter of HEADING DATE 3A (Hd3a). Artificial selection has locally favored an Hd1 variant unable to assemble in such heterotrimeric complex. The causal polymorphism was defined as a single conserved lysine in the CCT domain of the Hd1 protein. Our results indicate how genetic variation can be stratified and explored at multiple levels, and how its description can contribute to the molecular understanding of basic developmental processes.
Asunto(s)
Aclimatación/genética , Regulación de la Expresión Génica de las Plantas , Oryza/genética , Productos Agrícolas/genética , Productos Agrícolas/crecimiento & desarrollo , Histonas/genética , Histonas/metabolismo , Oryza/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleótido Simple , Unión Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismoAsunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factor de Unión a CCAAT/química , Factor de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Multimerización de Proteína , Subunidades de Proteína/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Cristalografía por Rayos X , ADN de Plantas/metabolismo , Modelos Moleculares , Unión ProteicaRESUMEN
Photoperiod dependent flowering is one of several mechanisms used by plants to initiate the developmental transition from vegetative growth to reproductive growth. The NUCLEAR FACTOR Y (NF-Y) transcription factors are heterotrimeric complexes composed of NF-YA and histone-fold domain (HFD) containing NF-YB/NF-YC, that initiate photoperiod-dependent flowering by cooperatively interacting with CONSTANS (CO) to drive the expression of FLOWERING LOCUS T (FT). This involves NF-Y and CO binding at distal CCAAT and proximal "CORE" elements, respectively, in the FT promoter. While this is well established for the HFD subunits, there remains some question over the potential role of NF-YA as either positive or negative regulators of this process. Here we provide strong support, in the form of genetic and biochemical analyses, that NF-YA, in complex with NF-YB/NF-YC proteins, can directly bind the distal CCAAT box in the FT promoter and are positive regulators of flowering in an FT-dependent manner.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Factor de Unión a CCAAT/genética , Flores/genética , Secuencia de Aminoácidos/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/biosíntesis , Factor de Unión a CCAAT/biosíntesis , Flores/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells.
Asunto(s)
Factor de Unión a CCAAT/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Redes y Vías Metabólicas/genética , Interferencia de ARN , Factor de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Células HCT116 , Células HeLa , Humanos , Immunoblotting , Células K562 , Modelos Genéticos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The p63 transcription factor, homolog to the p53 tumor suppressor gene, plays a crucial role in epidermal and limb development, as its mutations are associated to human congenital syndromes characterized by skin, craniofacial and limb defects. While limb and skin-specific p63 transcriptional targets are being discovered, little is known of the post-translation modifications controlling ΔNp63α functions. Here we show that the p300 acetyl-transferase physically interacts in vivo with ΔNp63α and catalyzes its acetylation on lysine 193 (K193) inducing ΔNp63α stabilization and activating specific transcriptional functions. Furthermore we show that Fibroblast Growth Factor-8 (FGF8), a morphogenetic signaling molecule essential for embryonic limb development, increases the binding of ΔNp63α to the tyrosine kinase c-Abl as well as the levels of ΔNp63α acetylation. Notably, the natural mutant ΔNp63α-K193E, associated to the Split-Hand/Foot Malformation-IV syndrome, cannot be acetylated by this pathway. This mutant ΔNp63α protein displays promoter-specific loss of DNA binding activity and consequent altered expression of development-associated ΔNp63α target genes. Our results link FGF8, c-Abl and p300 in a regulatory pathway that controls ΔNp63α protein stability and transcriptional activity. Hence, limb malformation-causing p63 mutations, such as the K193E mutation, are likely to result in aberrant limb development via the combined action of altered protein stability and altered promoter occupancy.
Asunto(s)
Anomalías Congénitas/genética , Factor 8 de Crecimiento de Fibroblastos/genética , Proteínas Proto-Oncogénicas c-abl/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Factores de Transcripción p300-CBP/genética , Animales , Línea Celular , Anomalías Congénitas/embriología , Anomalías Congénitas/patología , Proteínas de Unión al ADN/genética , Desarrollo Embrionario/genética , Factor 8 de Crecimiento de Fibroblastos/biosíntesis , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Deformidades Congénitas de las Extremidades/genética , Deformidades Congénitas de las Extremidades/patología , Ratones , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-abl/biosíntesis , Proteínas Proto-Oncogénicas c-abl/metabolismo , Transducción de Señal , Factores de Transcripción/biosíntesis , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/metabolismo , Factores de Transcripción p300-CBP/biosíntesis , Factores de Transcripción p300-CBP/metabolismoRESUMEN
For many plant species, reproductive success relies on the proper timing of flowering, and photoperiod provides a key environmental input. Photoperiod-dependent flowering depends on timely expression of FLOWERING LOCUS T (FT); however, the coordination of various cis-regulatory elements in the FT promoter is not well understood. Here, we provide evidence that long-distance chromatin loops bring distal enhancer elements into close association with the proximal promoter elements bound by CONSTANS (CO). Additionally, we show that NUCLEAR FACTOR Y (NF-Y) binds a CCAAT box in the distal enhancer element and that CCAAT disruption dramatically reduces FT promoter activity. Thus, we propose the recruitment model of photoperiod-dependent flowering where NF-Y complexes, bound at the FT distal enhancer element, help recruit CO to proximal cis-regulatory elements and initiate the transition to reproductive growth.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Factor de Unión a CCAAT/genética , Cromatina/genética , Flores , Regiones Promotoras GenéticasRESUMEN
Core histones are the building block of chromatin and among the most highly conserved proteins in eukaryotes. The related "deviant" histones share the histone-fold domain, and serve various roles in DNA metabolism. We provide here a structural and functional outlook of H2A/H2B-like deviant histones in transcription, replication and remodeling.
Asunto(s)
Cromatina/metabolismo , ADN/metabolismo , Histonas/metabolismo , Secuencia de Aminoácidos , Animales , Factor de Unión a CCAAT/metabolismo , Replicación del ADN , Histonas/química , Humanos , Datos de Secuencia Molecular , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismo , Transcripción Genética , Xenopus/metabolismoRESUMEN
The sequence-specific transcription factor NF-Y binds the CCAAT box, one of the sequence elements most frequently found in eukaryotic promoters. NF-Y is composed of the NF-YA and NF-YB/NF-YC subunits, the latter two hosting histone-fold domains (HFDs). The crystal structure of NF-Y bound to a 25 bp CCAAT oligonucleotide shows that the HFD dimer binds to the DNA sugar-phosphate backbone, mimicking the nucleosome H2A/H2B-DNA assembly. NF-YA both binds to NF-YB/NF-YC and inserts an α helix deeply into the DNA minor groove, providing sequence-specific contacts to the CCAAT box. Structural considerations and mutational data indicate that NF-YB ubiquitination at Lys138 precedes and is equivalent to H2B Lys120 monoubiquitination, important in transcriptional activation. Thus, NF-Y is a sequence-specific transcription factor with nucleosome-like properties of nonspecific DNA binding and helps establish permissive chromatin modifications at CCAAT promoters. Our findings suggest that other HFD-containing proteins may function in similar ways.
Asunto(s)
Factor de Unión a CCAAT/química , Secuencia de Aminoácidos , Animales , Factor de Unión a CCAAT/metabolismo , Cristalografía por Rayos X , ADN/química , ADN/genética , ADN/metabolismo , Proteínas del Choque Térmico HSP72/genética , Histonas/química , Humanos , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Alineación de Secuencia , UbiquitinaciónRESUMEN
BACKGROUND: NF-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. METHODS: We 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. RESULTS: Consistent 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. CONCLUSIONS: Our 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.