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1.
Biochim Biophys Acta ; 1863(4): 673-85, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26732297

RESUMEN

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.


Asunto(s)
Factor de Unión a CCAAT/fisiología , Replicación del ADN/genética , Animales , Factor de Unión a CCAAT/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , ADN/metabolismo , Células HCT116 , Humanos , Regiones Promotoras Genéticas , Fase S/genética , Elongación de la Transcripción Genética , Transcripción Genética , Xenopus laevis
2.
Biochim Biophys Acta ; 1859(4): 627-38, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26921500

RESUMEN

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.


Asunto(s)
Factor de Unión a CCAAT/genética , Diferenciación Celular/genética , Desarrollo de Músculos/genética , Músculo Esquelético/crecimiento & desarrollo , Isoformas de Proteínas/genética , Animales , Factor de Unión a CCAAT/biosíntesis , Proliferación Celular/genética , Ciclina B/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Mioblastos/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética
3.
Genome Res ; 23(8): 1195-209, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23595228

RESUMEN

NF-Y, a trimeric transcription factor (TF) composed of two histone-like subunits (NF-YB and NF-YC) and a sequence-specific subunit (NF-YA), binds to the CCAAT motif, a common promoter element. Genome-wide mapping reveals 5000-15,000 NF-Y binding sites depending on the cell type, with the NF-YA and NF-YB subunits binding asymmetrically with respect to the CCAAT motif. Despite being characterized as a proximal promoter TF, only 25% of NF-Y sites map to promoters. A comparable number of NF-Y sites are located at enhancers, many of which are tissue specific, and nearly half of the NF-Y sites are in select subclasses of HERV LTR repeats. Unlike most TFs, NF-Y can access its target DNA motif in inactive (nonmodified) or polycomb-repressed chromatin domains. Unexpectedly, NF-Y extensively colocalizes with FOS in all genomic contexts, and this often occurs in the absence of JUN and the AP-1 motif. NF-Y also coassociates with a select cluster of growth-controlling and oncogenic TFs, consistent with the abundance of CCAAT motifs in the promoters of genes overexpressed in cancer. Interestingly, NF-Y and several growth-controlling TFs bind in a stereo-specific manner, suggesting a mechanism for cooperative action at promoters and enhancers. Our results indicate that NF-Y is not merely a commonly used proximal promoter TF, but rather performs a more diverse set of biological functions, many of which are likely to involve coassociation with FOS.


Asunto(s)
Factor de Unión a CCAAT/metabolismo , Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Secuencia de Bases , Sitios de Unión , Cromatina/genética , Cromatina/metabolismo , Secuencia de Consenso , Regulación de la Expresión Génica , Ontología de Genes , Genoma Humano , Células HeLa , Humanos , Células K562 , Anotación de Secuencia Molecular , Especificidad de Órganos , Unión Proteica , Transporte de Proteínas , Secuencias Repetidas Terminales , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción
4.
Nucleic Acids Res ; 39(13): 5356-68, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21415014

RESUMEN

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.


Asunto(s)
Factor de Unión a CCAAT/antagonistas & inhibidores , Proliferación Celular , Ciclo Celular , Línea Celular Tumoral , Daño del ADN , Replicación del ADN , Expresión Génica , Silenciador del Gen , Humanos , Subunidades de Proteína/antagonistas & inhibidores , Fase S , Proteína p53 Supresora de Tumor/metabolismo
5.
J Biol Chem ; 284(49): 34189-200, 2009 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-19690168

RESUMEN

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


Asunto(s)
Empalme Alternativo , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/fisiología , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular Tumoral , Biología Computacional/métodos , Daño del ADN , Dimerización , Células HeLa , Histonas/química , Humanos , Datos de Secuencia Molecular , Isoformas de Proteínas , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transcripción Genética
6.
Nucleic Acids Res ; 36(5): 1415-28, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18187512

RESUMEN

The transcription factor NF-Y is a trimer with histone-like subunits that binds and activates CCAAT-containing promoters. NF-Y controls the expression of several key regulators of the cell cycle. In this study, we examined the functional and molecular effects of NF-YB knockdown. Cell cycle progression is affected with a G2/M-specific depletion. This is due to the inability of activation of G2/M-specific genes, as evidenced by expression profiling, RT-PCR and ChIP data. Surprisingly, apoptosis is also observed, with Caspase 3/7/8 cleavage. A role of p53 and Bcl-2 family members is important. NF-YB inactivation is sufficient to functionally activate p53, in the absence of DNA damage. Failure to maintain a physiologic level of CCAAT-dependent transcription of anti-apoptotic genes contributes to impairment of Bax/Bcl-2 and Bax/Bcl-X(L) ratios. Our data highlight the importance of fine balancing the NF-Y-p53 duo for cell survival by (i) maintaining transcription of anti-apoptotic genes and (ii) preventing p53 activation that triggers the apoptotic cascade.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Apoptosis/genética , Factor de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Factor de Unión a CCAAT/antagonistas & inhibidores , Factor de Unión a CCAAT/genética , Caspasas/metabolismo , Ciclo Celular/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Humanos , Interferencia de ARN , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Transcripción Genética
7.
Oncotarget ; 7(29): 45901-45915, 2016 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-27323853

RESUMEN

The expression of the high risk HPV18 E6 and E7 oncogenic proteins induces the transformation of epithelial cells, through the disruption of p53 and Rb function. The binding of cellular transcription factors to cis-regulatory elements in the viral Upstream Regulatory Region (URR) stimulates E6/E7 transcription. Here, we demonstrate that the CCAAT-transcription factor NF-Y binds to a non-canonical motif within the URR and activates viral gene expression. In addition, NF-Y indirectly up-regulates HPV18 transcription through the transactivation of multiple cellular transcription factors. NF-YA depletion inhibits the expression of E6 and E7 genes and re-establishes functional p53. The activation of p53 target genes in turn leads to apoptotic cell death. Finally, we show that NF-YA loss sensitizes HPV18-positive cells toward the DNA damaging agent Doxorubicin, via p53-mediated transcriptional response.


Asunto(s)
Factor de Unión a CCAAT/metabolismo , Transformación Celular Viral/fisiología , Proteínas de Unión al ADN/biosíntesis , Regulación Viral de la Expresión Génica/fisiología , Proteínas Oncogénicas Virales/biosíntesis , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Células Epiteliales/metabolismo , Células Epiteliales/virología , Células HeLa , Papillomavirus Humano 18 , Humanos , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/metabolismo , Activación Transcripcional
8.
Oncotarget ; 7(2): 1633-50, 2016 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-26646448

RESUMEN

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 Inversa
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