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1.
Cell ; 172(5): 897-909.e21, 2018 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-29474918

RESUMEN

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders.


Asunto(s)
Trastornos Distónicos/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Genoma Humano , Transcriptoma/genética , Empalme Alternativo/genética , Elementos Alu/genética , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Estudios de Cohortes , Familia , Femenino , Sitios Genéticos , Haplotipos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Intrones/genética , Masculino , Repeticiones de Minisatélite/genética , Modelos Genéticos , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Nucleótido Esparcido Corto , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo
2.
Mol Cell ; 84(13): 2553-2572.e19, 2024 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-38917794

RESUMEN

CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression.


Asunto(s)
Exones , Humanos , Exones/genética , Sistemas CRISPR-Cas , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo , Aptitud Genética , Células HEK293 , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Sitios de Empalme de ARN , Mutación , Regulación de la Expresión Génica , Empalme Alternativo
3.
Annu Rev Biochem ; 84: 355-79, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25494299

RESUMEN

Members of the FET protein family, consisting of FUS, EWSR1, and TAF15, bind to RNA and contribute to the control of transcription, RNA processing, and the cytoplasmic fates of messenger RNAs in metazoa. FET proteins can also bind DNA, which may be important in transcription and DNA damage responses. FET proteins are of medical interest because chromosomal rearrangements of their genes promote various sarcomas and because point mutations in FUS or TAF15 can cause neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar dementia. Recent results suggest that both the normal and pathological effects of FET proteins are modulated by low-complexity or prion-like domains, which can form higher-order assemblies with novel interaction properties. Herein, we review FET proteins with an emphasis on how the biochemical properties of FET proteins may relate to their biological functions and to pathogenesis.


Asunto(s)
Proteína FUS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Transporte Activo de Núcleo Celular , Animales , Reparación del ADN , Humanos , Neoplasias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Procesamiento Postranscripcional del ARN , Proteína FUS de Unión a ARN/química , Proteínas de Unión al ARN/química , Factores Asociados con la Proteína de Unión a TATA/química , Transcripción Genética
4.
Nature ; 625(7994): 345-351, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38057661

RESUMEN

Frontotemporal lobar degeneration (FTLD) causes frontotemporal dementia (FTD), the most common form of dementia after Alzheimer's disease, and is often also associated with motor disorders1. The pathological hallmarks of FTLD are neuronal inclusions of specific, abnormally assembled proteins2. In the majority of cases the inclusions contain amyloid filament assemblies of TAR DNA-binding protein 43 (TDP-43) or tau, with distinct filament structures characterizing different FTLD subtypes3,4. The presence of amyloid filaments and their identities and structures in the remaining approximately 10% of FTLD cases are unknown but are widely believed to be composed of the protein fused in sarcoma (FUS, also known as translocated in liposarcoma). As such, these cases are commonly referred to as FTLD-FUS. Here we used cryogenic electron microscopy (cryo-EM) to determine the structures of amyloid filaments extracted from the prefrontal and temporal cortices of four individuals with FTLD-FUS. Surprisingly, we found abundant amyloid filaments of the FUS homologue TATA-binding protein-associated factor 15 (TAF15, also known as TATA-binding protein-associated factor 2N) rather than of FUS itself. The filament fold is formed from residues 7-99 in the low-complexity domain (LCD) of TAF15 and was identical between individuals. Furthermore, we found TAF15 filaments with the same fold in the motor cortex and brainstem of two of the individuals, both showing upper and lower motor neuron pathology. The formation of TAF15 amyloid filaments with a characteristic fold in FTLD establishes TAF15 proteinopathy in neurodegenerative disease. The structure of TAF15 amyloid filaments provides a basis for the development of model systems of neurodegenerative disease, as well as for the design of diagnostic and therapeutic tools targeting TAF15 proteinopathy.


Asunto(s)
Degeneración Lobar Frontotemporal , Factores Asociados con la Proteína de Unión a TATA , Humanos , Amiloide/química , Amiloide/metabolismo , Amiloide/ultraestructura , Tronco Encefálico/metabolismo , Tronco Encefálico/patología , Microscopía por Crioelectrón , Demencia Frontotemporal/etiología , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Degeneración Lobar Frontotemporal/complicaciones , Degeneración Lobar Frontotemporal/metabolismo , Degeneración Lobar Frontotemporal/patología , Corteza Motora/metabolismo , Corteza Motora/patología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factores Asociados con la Proteína de Unión a TATA/ultraestructura , Lóbulo Temporal/metabolismo , Lóbulo Temporal/patología
5.
Cell ; 155(5): 1049-1060, 2013 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-24267890

RESUMEN

The low-complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS), and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here, we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state and released for elongation following phosphorylation of the CTD.


Asunto(s)
ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Activación Transcripcional , Células HeLa , Humanos , Hidrogeles/química , Hidrogeles/metabolismo , Repeticiones de Microsatélite , Fosforilación , Polimerizacion , Estructura Terciaria de Proteína , Proteína EWS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/metabolismo
6.
Mol Cell ; 77(5): 1143-1152.e7, 2020 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-31866147

RESUMEN

In eukaryotes, gene expression is performed by three RNA polymerases that are targeted to promoters by molecular complexes. A unique common factor, the TATA-box binding protein (TBP), is thought to serve as a platform to assemble pre-initiation complexes competent for transcription. Here, we describe a novel molecular mechanism of nutrient regulation of gene transcription by dynamic O-GlcNAcylation of TBP. We show that O-GlcNAcylation at T114 of TBP blocks its interaction with BTAF1, hence the formation of the B-TFIID complex, and its dynamic cycling on and off of DNA. Transcriptomic and metabolomic analyses of TBPT114A CRISPR/Cas9-edited cells showed that loss of O-GlcNAcylation at T114 increases TBP binding to BTAF1 and directly impacts expression of 408 genes. Lack of O-GlcNAcylation at T114 is associated with a striking reprogramming of cellular metabolism induced by a profound modification of the transcriptome, leading to gross alterations in lipid storage.


Asunto(s)
Glucosa/metabolismo , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIID/metabolismo , Animales , Cromatina/genética , Cromatina/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Regulación de la Expresión Génica , Glicosilación , Células HEK293 , Células HeLa , Humanos , Metabolismo de los Lípidos/genética , Masculino , Complejos Multiproteicos , Ratas Sprague-Dawley , Transducción de Señal , Factores Asociados con la Proteína de Unión a TATA/genética , Proteína de Unión a TATA-Box/genética , Factores de Tiempo , Factor de Transcripción TFIID/genética , Transcripción Genética , Transcriptoma
7.
Genes Dev ; 34(19-20): 1287-1303, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-33004486

RESUMEN

There are many large protein complexes involved in transcription in a chromatin context. However, recent studies on the SAGA coactivator complex are generating new paradigms for how the components of these complexes function, both independently and in concert. This review highlights the initial discovery of the canonical SAGA complex 23 years ago, our evolving understanding of its modular structure and the relevance of its modular nature for its coactivator function in gene regulation.


Asunto(s)
Regulación de la Expresión Génica/fisiología , Transactivadores/química , Transactivadores/metabolismo , Animales , Histona Acetiltransferasas/metabolismo , Complejos Multiproteicos/metabolismo , Péptido Hidrolasas/metabolismo , Subunidades de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , Factores Asociados con la Proteína de Unión a TATA/metabolismo
8.
Mol Cell ; 74(6): 1148-1163.e7, 2019 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-31005419

RESUMEN

Self-renewal and pluripotency of the embryonic stem cell (ESC) state are established and maintained by multiple regulatory networks that comprise transcription factors and epigenetic regulators. While much has been learned regarding transcription factors, the function of epigenetic regulators in these networks is less well defined. We conducted a CRISPR-Cas9-mediated loss-of-function genetic screen that identified two epigenetic regulators, TAF5L and TAF6L, components or co-activators of the GNAT-HAT complexes for the mouse ESC (mESC) state. Detailed molecular studies demonstrate that TAF5L/TAF6L transcriptionally activate c-Myc and Oct4 and their corresponding MYC and CORE regulatory networks. Besides, TAF5L/TAF6L predominantly regulate their target genes through H3K9ac deposition and c-MYC recruitment that eventually activate the MYC regulatory network for self-renewal of mESCs. Thus, our findings uncover a role of TAF5L/TAF6L in directing the MYC regulatory network that orchestrates gene expression programs to control self-renewal for the maintenance of mESC state.


Asunto(s)
Células Madre Embrionarias/metabolismo , Redes Reguladoras de Genes , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Animales , Sistemas CRISPR-Cas , Ciclo Celular/genética , Proliferación Celular , Reprogramación Celular , Embrión de Mamíferos , Células Madre Embrionarias/citología , Epigénesis Genética , Fibroblastos/citología , Fibroblastos/metabolismo , Edición Génica , Regulación de la Expresión Génica , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Cultivo Primario de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Factores Asociados con la Proteína de Unión a TATA/metabolismo
9.
Proc Natl Acad Sci U S A ; 121(33): e2401217121, 2024 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-39102544

RESUMEN

X-linked dystonia-parkinsonism (XDP) is a severe neurodegenerative disorder resulting from an inherited intronic SINE-Alu-VNTR (SVA) retrotransposon in the TAF1 gene that causes dysregulation of TAF1 transcription. The specific mechanism underlying this dysregulation remains unclear, but it is hypothesized to involve the formation of G-quadruplexes (G4) structures within the XDP-SVA that impede transcription. In this study, we show that ZNF91, a critical repressor of SVA retrotransposons, specifically binds to G4-forming DNA sequences. Further, we found that genetic deletion of ZNF91 exacerbates the molecular phenotype associated with the XDP-SVA insertion in patient cells, while no difference was observed when ZNF91 was deleted from isogenic control cells. Additionally, we observed a significant age-related reduction in ZNF91 expression in whole blood and brain, indicating a progressive loss of repression of the XDP-SVA in XDP. These findings indicate that ZNF91 plays a crucial role in controlling the molecular phenotype associated with XDP. Since ZNF91 binds to G4-forming DNA sequences in SVAs, this suggests that interactions between ZNF91 and G4-forming sequences in the XDP-SVA minimize the severity of the molecular phenotype. Our results showing that ZNF91 expression levels significantly decrease with age provide a potential explanation for the age-related progressive neurodegenerative character of XDP. Collectively, our study provides important insights into the protective role of ZNF91 in XDP pathogenesis and suggests that restoring ZNF91 expression, destabilization of G4s, or targeted repression of the XDP-SVA could be future therapeutic strategies to prevent or treat XDP.


Asunto(s)
Trastornos Distónicos , Enfermedades Genéticas Ligadas al Cromosoma X , Fenotipo , Humanos , Trastornos Distónicos/genética , Trastornos Distónicos/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , G-Cuádruplex , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Masculino , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Retroelementos/genética , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo
10.
Nature ; 577(7792): 711-716, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31969704

RESUMEN

SAGA (Spt-Ada-Gcn5-acetyltransferase) is a 19-subunit complex that stimulates transcription via two chromatin-modifying enzymatic modules and by delivering the TATA box binding protein (TBP) to nucleate the pre-initiation complex on DNA, a pivotal event in the expression of protein-encoding genes1. Here we present the structure of yeast SAGA with bound TBP. The core of the complex is resolved at 3.5 Å resolution (0.143 Fourier shell correlation). The structure reveals the intricate network of interactions that coordinate the different functional domains of SAGA and resolves an octamer of histone-fold domains at the core of SAGA. This deformed octamer deviates considerably from the symmetrical analogue in the nucleosome and is precisely tuned to establish a peripheral site for TBP, where steric hindrance represses binding of spurious DNA. Complementary biochemical analysis points to a mechanism for TBP delivery and release from SAGA that requires transcription factor IIA and whose efficiency correlates with the affinity of DNA to TBP. We provide the foundations for understanding the specific delivery of TBP to gene promoters and the multiple roles of SAGA in regulating gene expression.


Asunto(s)
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Pichia , Regiones Promotoras Genéticas/genética , Proteína de Unión a TATA-Box/metabolismo , Transactivadores/química , Transactivadores/metabolismo , Sitios de Unión , ADN de Hongos/química , ADN de Hongos/metabolismo , Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Histonas/química , Histonas/metabolismo , Modelos Moleculares , Pichia/química , Pichia/genética , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/química , Factor de Transcripción TFIIA/química , Factor de Transcripción TFIIA/metabolismo , Factor de Transcripción TFIID/química , Factor de Transcripción TFIID/metabolismo
11.
Dev Biol ; 511: 53-62, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38593904

RESUMEN

Early embryonic development is a finely orchestrated process that requires precise regulation of gene expression coordinated with morphogenetic events. TATA-box binding protein-associated factors (TAFs), integral components of transcription initiation coactivators like TFIID and SAGA, play a crucial role in this intricate process. Here we show that disruptions in TAF5, TAF12 and TAF13 individually lead to embryonic lethality in the mouse, resulting in overlapping yet distinct phenotypes. Taf5 and Taf12 mutant embryos exhibited a failure to implant post-blastocyst formation, and Taf5 mutants have aberrant lineage specification within the inner cell mass. In contrast, Taf13 mutant embryos successfully implant and form egg-cylinder stages but fail to initiate gastrulation. Strikingly, we observed a depletion of pluripotency factors in TAF13-deficient embryos, including OCT4, NANOG and SOX2, highlighting an indispensable role of TAF13 in maintaining pluripotency. Transcriptomic analysis revealed distinct gene targets affected by the loss of TAF5, TAF12 and TAF13. Thus, we propose that TAF5, TAF12 and TAF13 convey locus specificity to the TFIID complex throughout the mouse genome.


Asunto(s)
Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Factores Asociados con la Proteína de Unión a TATA , Animales , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Ratones , Desarrollo Embrionario/genética , Factor de Transcripción TFIID/metabolismo , Factor de Transcripción TFIID/genética , Femenino , Blastocisto/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Gastrulación/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXB1/genética , Proteína Homeótica Nanog/metabolismo , Proteína Homeótica Nanog/genética , Embrión de Mamíferos/metabolismo
12.
Hum Mol Genet ; 32(1): 104-121, 2023 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-35925837

RESUMEN

Eukaryotic RNA polymerase I (Pol I) products play fundamental roles in ribosomal assembly, protein synthesis, metabolism and cell growth. Abnormal expression of both Pol I transcription-related factors and Pol I products causes a range of diseases, including ribosomopathies and cancers. However, the factors and mechanisms governing Pol I-dependent transcription remain to be elucidated. Here, we report that transcription factor IIB-related factor 1 (BRF1), a subunit of transcription factor IIIB required for RNA polymerase III (Pol III)-mediated transcription, is a nucleolar protein and modulates Pol I-mediated transcription. We showed that BRF1 can be localized to the nucleolus in several human cell types. BRF1 expression correlates positively with Pol I product levels and tumour cell growth in vitro and in vivo. Pol III transcription inhibition assays confirmed that BRF1 modulates Pol I-directed transcription in an independent manner rather than through a Pol III product-to-45S pre-rRNA feedback mode. Mechanistically, BRF1 binds to the Pol I transcription machinery components and can be recruited to the rDNA promoter along with them. Additionally, alteration of BRF1 expression affects the recruitment of Pol I transcription machinery components to the rDNA promoter and the expression of TBP and TAF1A. These findings indicate that BRF1 modulates Pol I-directed transcription by controlling the expression of selective factor 1 subunits. In summary, we identified a novel role of BRF1 in Pol I-directed transcription, suggesting that BRF1 can independently regulate both Pol I- and Pol III-mediated transcription and act as a key coordinator of Pol I and Pol III.


Asunto(s)
Neoplasias , Factores Asociados con la Proteína de Unión a TATA , Humanos , ADN Ribosómico/genética , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción/genética , Transcripción Genética
13.
Development ; 149(3)2022 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-35043944

RESUMEN

Establishment of a healthy ovarian reserve is contingent upon numerous regulatory pathways during embryogenesis. Previously, mice lacking TBP-associated factor 4b (Taf4b) were shown to exhibit a diminished ovarian reserve. However, potential oocyte-intrinsic functions of TAF4b have not been examined. Here, we use a combination of gene expression profiling and chromatin mapping to characterize TAF4b-dependent gene regulatory networks in mouse oocytes. We find that Taf4b-deficient oocytes display inappropriate expression of meiotic, chromatin modification/organization, and X-linked genes. Furthermore, dysregulated genes in Taf4b-deficient oocytes exhibit an unexpected amount of overlap with dysregulated genes in oocytes from XO female mice, a mouse model of Turner Syndrome. Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we observed TAF4b enrichment at genes involved in chromatin remodeling and DNA repair, some of which are differentially expressed in Taf4b-deficient oocytes. Interestingly, TAF4b target genes were enriched for Sp/Klf family and NFY target motifs rather than TATA-box motifs, suggesting an alternative mode of promoter interaction. Together, our data connect several gene regulatory nodes that contribute to the precise development of the mammalian ovarian reserve.


Asunto(s)
Redes Reguladoras de Genes/genética , Oogénesis , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética , Animales , Reparación del ADN , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Células Germinativas/citología , Células Germinativas/metabolismo , Meiosis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oocitos/citología , Oocitos/metabolismo , Regiones Promotoras Genéticas , Factores Asociados con la Proteína de Unión a TATA/deficiencia , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/deficiencia , Factor de Transcripción TFIID/metabolismo , Cromosoma X/genética , Cromosoma X/metabolismo
14.
FASEB J ; 38(1): e23376, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-38112167

RESUMEN

Male germ cell development is dependent on the orchestrated regulation of gene networks. TATA-box binding protein associated factors (TAFs) facilitate interactions of TATA-binding protein with the TATA element, which is known to coordinate gene transcription during organogenesis. TAF7 like (Taf7l) is situated on the X chromosome and has been implicated in testis development. We examined the biology of TAF7L in testis development using the rat. Taf7l was prominently expressed in preleptotene to leptotene spermatocytes. To study the impact of TAF7L on the testis we generated a global loss-of-function rat model using CRISPR/Cas9 genome editing. Exon 3 of the Taf7l gene was targeted. A founder was generated possessing a 110 bp deletion within the Taf7l locus, which resulted in a frameshift and the premature appearance of a stop codon. The mutation was effectively transmitted through the germline. Deficits in TAF7L did not adversely affect pregnancy or postnatal survival. However, the Taf7l disruption resulted in male infertility due to compromised testis development and failed sperm production. Mutant germ cells suffer meiotic arrest at late zygotene/early pachynema stages, with defects in sex body formation. This testis phenotype was more pronounced than previously described for the subfertile Taf7l null mouse. We conclude that TAF7L is essential for male germ cell development in the rat.


Asunto(s)
Semen , Espermatogénesis , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Animales , Femenino , Masculino , Embarazo , Ratas , Diferenciación Celular , Meiosis , Semen/metabolismo , Espermatocitos/metabolismo , Espermatogénesis/fisiología , Espermatozoides/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Testículo/metabolismo , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo
15.
Mol Cell ; 68(1): 130-143.e5, 2017 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-28918903

RESUMEN

Prior studies suggested that SAGA and TFIID are alternative factors that promote RNA polymerase II transcription, with about 10% of genes in S. cerevisiae dependent on SAGA. We reassessed the role of SAGA by mapping its genome-wide location and role in global transcription in budding yeast. We find that SAGA maps to the UAS elements of most genes, overlapping with Mediator binding and irrespective of previous designations of SAGA- or TFIID-dominated genes. Disruption of SAGA through mutation or rapid subunit depletion reduces transcription from nearly all genes, measured by newly synthesized RNA. We also find that the acetyltransferase Gcn5 synergizes with Spt3 to promote global transcription and that Spt3 functions to stimulate TBP recruitment at all tested genes. Our data demonstrate that SAGA acts as a general cofactor required for essentially all RNA polymerase II transcription and is not consistent with the previous classification of SAGA- and TFIID-dominated genes.


Asunto(s)
Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/genética , ARN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteína de Unión a TATA-Box/genética , Transactivadores/genética , Factores de Transcripción/genética , Eliminación de Gen , Histona Acetiltransferasas/metabolismo , Regiones Promotoras Genéticas , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Polimerasa II/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética
16.
Mol Cell ; 67(4): 594-607.e4, 2017 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-28735899

RESUMEN

Pervasive transcription initiates from cryptic promoters and is observed in eukaryotes ranging from yeast to mammals. The Set2-Rpd3 regulatory system prevents cryptic promoter function within expressed genes. However, conserved systems that control pervasive transcription within intergenic regions have not been well established. Here we show that Mot1, Ino80 chromatin remodeling complex (Ino80C), and NC2 co-localize on chromatin and coordinately suppress pervasive transcription in S. cerevisiae and murine embryonic stem cells (mESCs). In yeast, all three proteins bind subtelomeric heterochromatin through a Sir3-stimulated mechanism and to euchromatin via a TBP-stimulated mechanism. In mESCs, the proteins bind to active and poised TBP-bound promoters along with promoters of polycomb-silenced genes apparently lacking TBP. Depletion of Mot1, Ino80C, or NC2 by anchor away in yeast or RNAi in mESCs leads to near-identical transcriptome phenotypes, with new subtelomeric transcription in yeast, and greatly increased pervasive transcription in both yeast and mESCs.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Células Madre Embrionarias/enzimología , Fosfoproteínas/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/genética , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN , Eucromatina/genética , Eucromatina/metabolismo , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Genotipo , Heterocromatina/genética , Heterocromatina/metabolismo , Fenotipo , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , Proteínas Represoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIID , Factores de Transcripción/genética , Transfección
17.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-35115407

RESUMEN

Plant root growth is indeterminate but continuously responds to environmental changes. We previously reported on the severe root growth defect of a double mutant in bZIP17 and bZIP28 (bz1728) modulating the unfolded protein response (UPR). To elucidate the mechanism by which bz1728 seedlings develop a short root, we obtained a series of bz1728 suppressor mutants, called nobiro, for rescued root growth. We focused here on nobiro6, which is defective in the general transcription factor component TBP-ASSOCIATED FACTOR 12b (TAF12b). The expression of hundreds of genes, including the bZIP60-UPR regulon, was induced in the bz1728 mutant, but these inductions were markedly attenuated in the bz1728nobiro6 mutant. In view of this, we assigned transcriptional cofactor activity via physical interaction with bZIP60 to NOBIRO6/TAF12b. The single nobiro6/taf12b mutant also showed an altered sensitivity to endoplasmic reticulum stress for both UPR and root growth responses, demonstrating that NOBIRO6/TAF12b contributes to environment-responsive root growth control through UPR.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factor XII/metabolismo , Raíces de Plantas/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Respuesta de Proteína Desplegada/fisiología , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Plantones/metabolismo , Transducción de Señal/fisiología
18.
Genes Dev ; 31(21): 2162-2174, 2017 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-29203645

RESUMEN

TFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-binding protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-associated factor (TAF) subunits recognize downstream promoter elements, act as coactivators, and interact with nucleosomes. In yeast nuclear extracts, transcription induces stable TAF binding to downstream promoter DNA, promoting subsequent activator-independent transcription reinitiation. In vivo, promoter responses to TAF mutations correlate with the level of downstream, rather than overall, Taf1 cross-linking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.


Asunto(s)
Regiones Promotoras Genéticas/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Transcripción Genética/genética , Acetilación , Histonas/metabolismo , Mutación/genética , Unión Proteica , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factores de Transcripción/metabolismo
19.
J Biol Chem ; 299(7): 104873, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37257820

RESUMEN

Dysregulation of long noncoding RNAs (lncRNAs) contributes to tumorigenesis by modulating specific cancer-related pathways, but the roles of N6-methyladenosine (m6A)-enriched lncRNAs and underlying mechanisms remain elusive in nasopharyngeal carcinoma (NPC). Here, we reanalyzed the previous genome-wide analysis of lncRNA profiles in 18 pairs of NPC and normal tissues as well as in ten paired samples from NPC with or without post-treatment metastases. We discerned that an oncogenic m6A-enriched lncRNA, LINC00839, which was substantially upregulated in NPC and correlated with poor clinical prognosis, promoted NPC growth and metastasis both in vitro and in vivo. Mechanistically, by using RNA pull-down assay combined with mass spectrometry, we found that LINC00839 interacted directly with the transcription factor, TATA-box binding protein associated factor (TAF15). Besides, chromatin immunoprecipitation and dual-luciferase report assays demonstrated that LINC00839 coordinated the recruitment of TAF15 to the promoter region of amine oxidase copper-containing 1 (AOC1), which encodes a secreted glycoprotein playing vital roles in various cancers, thereby activating AOC1 transcription in trans. In this study, potential effects of AOC1 in NPC progression were first proposed. Moreover, ectopic expression of AOC1 partially rescued the inhibitory effect of downregulation of LINC00839 in NPC. Furthermore, we showed that silencing vir-like m6A methyltransferase-associated (VIRMA) and insulin-like growth factor 2 mRNA-binding proteins 1 (IGF2BP1) attenuated the expression level and RNA stability of LINC00839 in an m6A-dependent manner. Taken together, our study unveils a novel oncogenic VIRMA/IGF2BP1-LINC00839-TAF15-AOC1 axis and highlights the significance and prognostic value of LINC00839 expression in NPC carcinogenesis.


Asunto(s)
Neoplasias Nasofaríngeas , ARN Largo no Codificante , Factores Asociados con la Proteína de Unión a TATA , Humanos , Aminas , Carcinogénesis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Carcinoma Nasofaríngeo/patología , Neoplasias Nasofaríngeas/patología , Oxidorreductasas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo
20.
J Biol Chem ; 299(3): 102945, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36707053

RESUMEN

Deregulation of transcription factor AP2 alpha (TFAP2A) and RNA polymerase III (Pol III) products is associated with tumorigenesis. However, the mechanism underlying this event is not fully understood and the connection between TFAP2A and Pol III-directed transcription has not been investigated. Here, we report that TFAP2A functions as a positive factor in the regulation of Pol III-directed transcription and cell proliferation. We found TFAP2A is also required for the activation of Pol III transcription induced by the silencing of filamin A, a well-known cytoskeletal protein and an inhibitor in Pol III-dependent transcription identified previously. Using a chromatin immunoprecipitation technique, we showed TFAP2A positively modulates the assembly of Pol III transcription machinery factors at Pol III-transcribed gene loci. We found TFAP2A can activate the expression of Pol III transcription-related factors, including BRF1, GTF3C2, and c-MYC. Furthermore, we demonstrate TFAP2A enhances expression of MDM2, a negative regulator of tumor suppressor p53, and also inhibits p53 expression. Finally, we found MDM2 overexpression can rescue the inhibition of Pol III-directed transcription and cell proliferation caused by TFAP2A silencing. In summary, we identified that TFAP2A can activate Pol III-directed transcription by controlling multiple pathways, including general transcription factors, c-MYC and MDM2/p53. The findings from this study provide novel insights into the regulatory mechanisms of Pol III-dependent transcription and cancer cell proliferation.


Asunto(s)
Neoplasias , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción AP-2 , Humanos , Proliferación Celular , ARN Polimerasa III/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción AP-2/genética , Factor de Transcripción AP-2/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo
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