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1.
Mol Cell ; 74(2): 268-283.e5, 2019 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-30902546

RESUMEN

Linker histone H1 has been correlated with transcriptional inhibition, but the mechanistic basis of the inhibition and its reversal during gene activation has remained enigmatic. We report that H1-compacted chromatin, reconstituted in vitro, blocks transcription by abrogating core histone modifications by p300 but not activator and p300 binding. Transcription from H1-bound chromatin is elicited by the H1 chaperone NAP1, which is recruited in a gene-specific manner through direct interactions with activator-bound p300 that facilitate core histone acetylation (by p300) and concomitant eviction of H1 and H2A-H2B. An analysis in B cells confirms the strong dependency on NAP1-mediated H1 eviction for induction of the silent CD40 gene and further demonstrates that H1 eviction, seeded by activator-p300-NAP1-H1 interactions, is propagated over a CCCTC-binding factor (CTCF)-demarcated region through a distinct mechanism that also involves NAP1. Our results confirm direct transcriptional inhibition by H1 and establish a gene-specific H1 eviction mechanism through an activator→p300→NAP1→H1 pathway.


Asunto(s)
Factor de Unión a CCCTC/genética , Proteína p300 Asociada a E1A/genética , Proteínas/genética , Transcripción Genética , Acetilación , Linfocitos B/química , Sitios de Unión , Factor de Unión a CCCTC/química , Antígenos CD40/genética , Cromatina/química , Cromatina/genética , Proteína p300 Asociada a E1A/química , Código de Histonas , Histonas/química , Histonas/genética , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Nucleosomas/química , Nucleosomas/genética , Regiones Promotoras Genéticas , Unión Proteica/genética , Proteínas/química , ARNt Metiltransferasas
2.
Mol Cell ; 75(4): 683-699.e7, 2019 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-31399344

RESUMEN

Transcriptional regulation in eukaryotes occurs at promoter-proximal regions wherein transcriptionally engaged RNA polymerase II (Pol II) pauses before proceeding toward productive elongation. The role of chromatin in pausing remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 binds to Pol II and prevents the release of the negative elongation factor (NELF), thus stabilizing Pol II promoter-proximal pausing. Genetic depletion of SIRT6 or its chromatin deficiency upon glucose deprivation causes intragenic enrichment of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of cyclin-dependent kinase 9 (CDK9)-that phosphorylates NELF and the carboxyl terminal domain of Pol II-and enrichment of the positive transcription elongation factors MYC, BRD4, PAF1, and the super elongation factors AFF4 and ELL2. These events lead to increased expression of genes involved in metabolism, protein synthesis, and embryonic development. Our results identified SIRT6 as a Pol II promoter-proximal pausing-dedicated histone deacetylase.


Asunto(s)
Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Sirtuinas/metabolismo , Elongación de la Transcripción Genética , Acetilación , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Eliminación de Gen , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Polimerasa II/genética , Sirtuinas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismo
3.
EMBO Rep ; 24(10): e57108, 2023 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-37535603

RESUMEN

The H3K4 methyltransferase SETD1A plays a crucial role in leukemia cell survival through its noncatalytic FLOS domain-mediated recruitment of cyclin K and regulation of DNA damage response genes. In this study, we identify a functional nuclear localization signal in and interaction partners of the FLOS domain. Our screen for FLOS domain-binding partners reveals that the SETD1A FLOS domain binds mitosis-associated proteins BuGZ/BUB3. Inhibition of both cyclin K and BuGZ/BUB3-binding motifs in SETD1A shows synergistic antileukemic effects. BuGZ/BUB3 localize to SETD1A-bound promoter-TSS regions and SETD1A-negative H3K4me1-positive enhancer regions adjacent to SETD1A target genes. The GLEBS motif and intrinsically disordered region of BuGZ are required for both SETD1A-binding and leukemia cell proliferation. Cell-cycle-specific SETD1A restoration assays indicate that SETD1A expression at the G1/S phase of the cell cycle promotes both the expression of DNA damage response genes and cell cycle progression in leukemia cells.


Asunto(s)
Leucemia , Mitosis , Humanos , Mitosis/genética , Ciclinas/genética , Ciclinas/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Leucemia/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética
4.
Nature ; 567(7749): 535-539, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30867594

RESUMEN

Chemical modifications of histones can mediate diverse DNA-templated processes, including gene transcription1-3. Here we provide evidence for a class of histone post-translational modification, serotonylation of glutamine, which occurs at position 5 (Q5ser) on histone H3 in organisms that produce serotonin (also known as 5-hydroxytryptamine (5-HT)). We demonstrate that tissue transglutaminase 2 can serotonylate histone H3 tri-methylated lysine 4 (H3K4me3)-marked nucleosomes, resulting in the presence of combinatorial H3K4me3Q5ser in vivo. H3K4me3Q5ser displays a ubiquitous pattern of tissue expression in mammals, with enrichment observed in brain and gut, two organ systems responsible for the bulk of 5-HT production. Genome-wide analyses of human serotonergic neurons, developing mouse brain and cultured serotonergic cells indicate that H3K4me3Q5ser nucleosomes are enriched in euchromatin, are sensitive to cellular differentiation and correlate with permissive gene expression, phenomena that are linked to the potentiation of TFIID4-6 interactions with H3K4me3. Cells that ectopically express a H3 mutant that cannot be serotonylated display significantly altered expression of H3K4me3Q5ser-target loci, which leads to deficits in differentiation. Taken together, these data identify a direct role for 5-HT, independent from its contributions to neurotransmission and cellular signalling, in the mediation of permissive gene expression.


Asunto(s)
Regulación de la Expresión Génica , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Serotonina/metabolismo , Factor de Transcripción TFIID/metabolismo , Animales , Diferenciación Celular , Línea Celular , Femenino , Proteínas de Unión al GTP/metabolismo , Glutamina/química , Glutamina/metabolismo , Humanos , Metilación , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Proteína Glutamina Gamma Glutamiltransferasa 2 , Neuronas Serotoninérgicas/citología , Transglutaminasas/metabolismo
5.
Mol Cell ; 67(2): 308-321.e6, 2017 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-28732206

RESUMEN

Enhancer activation is a critical step for gene activation. Here we report an epigenetic crosstalk at enhancers between the UTX (H3K27 demethylase)-MLL4 (H3K4 methyltransferase) complex and the histone acetyltransferase p300. We demonstrate that UTX, in a demethylase activity-independent manner, facilitates conversion of inactive enhancers in embryonic stem cells to an active (H3K4me1+/H3K27ac+) state by recruiting and coupling the enzymatic functions of MLL4 and p300. Loss of UTX leads to attenuated enhancer activity, characterized by reduced levels of H3K4me1 and H3K27ac as well as impaired transcription. The UTX-MLL4 complex enhances p300-dependent H3K27 acetylation through UTX-dependent stimulation of p300 recruitment, while MLL4-mediated H3K4 monomethylation, reciprocally, requires p300 function. Importantly, MLL4-generated H3K4me1 further enhances p300-dependent transcription. This work reveals a previously unrecognized cooperativity among enhancer-associated chromatin modulators, including a unique function for UTX, in establishing an "active enhancer landscape" and defines a detailed mechanism for the joint deposition of H3K4me1 and H3K27ac.


Asunto(s)
Cromatina/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Células Madre Embrionarias/enzimología , Elementos de Facilitación Genéticos , Histona Demetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Transcripción Genética , Activación Transcripcional , Animales , Cromatina/genética , Ensamble y Desensamble de Cromatina , Proteína p300 Asociada a E1A/genética , Retroalimentación Fisiológica , Redes Reguladoras de Genes , Células HEK293 , Histona Demetilasas/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Humanos , Masculino , Metilación , Ratones , Interferencia de ARN , Transfección
6.
Nucleic Acids Res ; 50(8): 4450-4463, 2022 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-35394046

RESUMEN

Mediator activates RNA polymerase II (Pol II) function during transcription, but it remains unclear whether Mediator is able to travel with Pol II and regulate Pol II transcription beyond the initiation and early elongation steps. By using in vitro and in vivo transcription recycling assays, we find that human Mediator 1 (MED1), when phosphorylated at the mammal-specific threonine 1032 by cyclin-dependent kinase 9 (CDK9), dynamically moves along with Pol II throughout the transcribed genes to drive Pol II recycling after the initial round of transcription. Mechanistically, MED31 mediates the recycling of phosphorylated MED1 and Pol II, enhancing mRNA output during the transcription recycling process. Importantly, MED1 phosphorylation increases during prostate cancer progression to the lethal phase, and pharmacological inhibition of CDK9 decreases prostate tumor growth by decreasing MED1 phosphorylation and Pol II recycling. Our results reveal a novel role of MED1 in Pol II transcription and identify phosphorylated MED1 as a targetable driver of dysregulated Pol II recycling in cancer.


Asunto(s)
Neoplasias , ARN Polimerasa II , Animales , Humanos , Masculino , Mamíferos/genética , Complejo Mediador/metabolismo , Subunidad 1 del Complejo Mediador/genética , Neoplasias/genética , Fosforilación , ARN Polimerasa II/metabolismo , Transcripción Genética
7.
Proc Natl Acad Sci U S A ; 118(35)2021 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-34426493

RESUMEN

Cellular senescence causes a dramatic alteration of chromatin organization and changes the gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies through the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained by the CCCTC-binding factor (CTCF); however, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains unclear. We discovered that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impairs the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin and activates the transcription of SASP-like inflammatory genes, promoting malignant transformation. Notably, pericentromeric ncRNA was transferred into surrounding cells via small extracellular vesicles acting as a tumorigenic SASP factor. Because CTCF blocks the expression of pericentromeric ncRNA in young cells, the down-regulation of CTCF during cellular senescence triggers the up-regulation of this ncRNA and SASP-related inflammatory gene expression. In this study, we show that pericentromeric ncRNA provokes chromosomal alteration by inhibiting CTCF, leading to a SASP-like inflammatory response in a cell-autonomous and non-cell-autonomous manner and thus may contribute to the risk of tumorigenesis during aging.


Asunto(s)
Envejecimiento/genética , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Inflamación/genética , ARN no Traducido/fisiología , Fenotipo Secretor Asociado a la Senescencia/genética , Animales , Senescencia Celular/genética , Centrómero , ADN de Neoplasias/metabolismo , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias , Unión Proteica/genética
8.
Br J Cancer ; 128(7): 1208-1222, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36725920

RESUMEN

BACKGROUND: Oestrogen receptor (ER) signalling-dependent cancer cell growth is one of the major features of ER-positive breast cancer (BC). Inhibition of ER function is a standard and effective treatment for ER-positive tumours; however, ~20% of patients with ER-positive BC experience early or late recurrence. In this study, we examined intertumour heterogeneity from an epigenetic perspective based on the hypothesis that the intrinsic difference in epigenetic states around ER signalling pathway underlies endocrine therapy resistance. METHODS: We performed transposase-accessible chromatin sequencing (ATAC-seq) analysis of 42 BC samples, including 35 ER-positive(+) human epidermal growth factor receptor 2 (HER2)-negative(-) and 7 triple-negative tumours. We also reanalysed ATAC-seq data of 45 ER + /HER2 - tumours in the Cancer Genome Atlas (TCGA) BC cohort to validate our observations. RESULTS: We conducted a comprehensive analysis of cis-regulatory elements (CREs) using ATAC-seq, identifying three subgroups based on chromatin accessibility profiles. We identified a subgroup of ER-positive BCs with a distinctive chromatin accessibility pattern including reduced accessibility to ER-responsive elements (EREs). The same subgroup was also observed in TCGA BC cohort. Despite the reduced accessibility to EREs, the expression of ER and potential ER target genes were not decreased in these tumours. CONCLUSION: Our findings highlight the existence of a subset of ER-positive BCs with unchanged ER expression but reduced EREs accessibility that cannot be distinguished by conventional immunostaining for ER. Future studies should determine whether these tumours are associated with resistance to endocrine therapy.


Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Cromatina/genética , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Resultado del Tratamiento , Transducción de Señal
9.
Mol Biol Rep ; 50(2): 1209-1220, 2023 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-36436079

RESUMEN

BACKGROUND: Estrogen receptor alpha (ERα) is a frequently mutated gene in breast cancer (BC). While many studies have investigated molecular dysregulation by hotspot mutations at Y537 and D538, which exhibit an estrogen-independent constitutively active phenotype, the functional abnormalities of other mutations remain obscure. The K303R mutation in primary invasive BC has been implicated with endocrine resistance, tumor size, and lymph node positivity. However, the impact of the K303R mutation on the cell epigenome is yet unknown. METHODS AND RESULTS: We introduced the K303R ERα mutant in ERα-negative MDA-MB-453 cells to monitor ERα-dependent transactivation and to perform epigenomic analyses. ATAC-seq and ChIP-Seq analyses indicated that both wild-type (WT) and the K303R mutant associated with Forkhead box (Fox) protein family motif regions at similar rates, even without an ERα-binding sequence, but only the K303R mutant induced chromatin opening at those regions. Biochemical analyses demonstrated that the WT and the K303R mutant can be tethered on DNA by FoxA1 indirectly, but only the K303R/FoxA1/DNA complex can induce associations with the nuclear receptor cofactor 2 (NCOA2). CONCLUSIONS: These findings suggest that the K303R mutant induces chromatin opening at the Fox binding region through the FoxA1-dependent associations of the K303R mutant to NCOA2 and then probably disrupts the regulation of Fox-target genes, resulting in K303R-related BC events.


Asunto(s)
Cromatina , Receptor alfa de Estrógeno , Factores de Transcripción Forkhead , Factor Nuclear 3-alfa del Hepatocito , Humanos , Línea Celular Tumoral , Cromatina/genética , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Estrógenos/farmacología , Factores de Transcripción Forkhead/genética , Regulación Neoplásica de la Expresión Génica , Mutación/genética , Unión Proteica , Factor Nuclear 3-alfa del Hepatocito/metabolismo
10.
Genes Dev ; 29(3): 308-21, 2015 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-25644605

RESUMEN

PR domain-containing 16 (PRDM16) induces expression of brown fat-specific genes in brown and beige adipocytes, although the underlying transcription-related mechanisms remain largely unknown. Here, in vitro studies show that PRDM16, through its zinc finger domains, directly interacts with the MED1 subunit of the Mediator complex, is recruited to the enhancer of the brown fat-specific uncoupling protein 1 (Ucp1) gene through this interaction, and enhances thyroid hormone receptor (TR)-driven transcription in a biochemically defined system in a Mediator-dependent manner, thus providing a direct link to the general transcription machinery. Complementary cell-based studies show that upon forskolin treatment, PRDM16 induces Ucp1 expression in undifferentiated murine embryonic fibroblasts, that this induction depends on MED1 and TR, and, consistent with a direct effect, that PRDM16 is recruited to the Ucp1 enhancer. Related studies have defined MED1 and PRDM16 interaction domains important for Ucp1 versus Ppargc1a induction by PRDM16. These results reveal novel mechanisms for PRDM16 function through the Mediator complex.


Asunto(s)
Adipocitos Marrones/citología , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Canales Iónicos/genética , Subunidad 1 del Complejo Mediador/metabolismo , Proteínas Mitocondriales/genética , Factores de Transcripción/metabolismo , Adipocitos Marrones/metabolismo , Animales , Línea Celular , Colforsina/farmacología , Elementos de Facilitación Genéticos/fisiología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Ratones , Unión Proteica , Estructura Terciaria de Proteína/genética , Proteína Desacopladora 1
11.
Proc Natl Acad Sci U S A ; 115(26): E6039-E6047, 2018 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-29891714

RESUMEN

Leptin expression decreases after fat loss and is increased when obesity develops, and its proper quantitative regulation is essential for the homeostatic control of fat mass. We previously reported that a distant leptin enhancer 1 (LE1), 16 kb upstream from the transcription start site (TSS), confers fat-specific expression in a bacterial artificial chromosome transgenic (BACTG) reporter mouse. However, this and the other elements that we identified do not account for the quantitative changes in leptin expression that accompany alterations of adipose mass. In this report, we used an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to identify a 17-bp noncanonical peroxisome proliferator-activated receptor gamma (PPARγ)/retinoid X receptor alpha (RXRα)-binding site, leptin regulatory element 1 (LepRE1), within LE1, and show that it is necessary for the fat-regulated quantitative control of reporter (luciferase) expression. While BACTG reporter mice with mutations in this sequence still show fat-specific expression, luciferase is no longer decreased after food restriction and weight loss. Similarly, the increased expression of leptin reporter associated with obesity in ob/ob mice is impaired. A functionally analogous LepRE1 site is also found in a second, redundant DNA regulatory element 13 kb downstream of the TSS. These data uncouple the mechanisms conferring qualitative and quantitative expression of the leptin gene and further suggest that factor(s) that bind to LepRE1 quantitatively control leptin expression and might be components of a lipid-sensing system in adipocytes.


Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Regulación de la Expresión Génica , Leptina , PPAR gamma , Elementos de Respuesta , Receptor alfa X Retinoide , Adipocitos/citología , Tejido Adiposo/citología , Animales , Línea Celular , Leptina/biosíntesis , Leptina/genética , Ratones , Ratones Obesos
12.
Genes Dev ; 27(14): 1596-609, 2013 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-23873942

RESUMEN

The multisubunit TFIID plays a direct role in transcription initiation by binding to core promoter elements and directing preinitiation complex assembly. Although TFIID may also function as a coactivator through direct interactions with promoter-bound activators, mechanistic aspects of this poorly defined function remain unclear. Here, biochemical studies show a direct TFIID-E-protein interaction that (1) is mediated through interaction of a novel E-protein activation domain (activation domain 3 [AD3]) with the TAF homology (TAFH) domain of TAF4, (2) is critical for activation of a natural target gene by an E protein, and (3) mechanistically acts by enhancing TFIID binding to the core promoter. Complementary assays establish a gene-specific role for the TAFH domain in TFIID recruitment and activation of a large subset of genes in vivo. These results firmly establish TAF4 as a bona fide E-protein coactivator as well as a mechanism involving facilitated TFIID binding through direct interaction with an E-protein activation domain.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Eliminación de Gen , Regulación de la Expresión Génica , Células HeLa , Humanos , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Terciaria de Proteína , Transcriptoma
13.
Nature ; 500(7460): 93-7, 2013 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-23812588

RESUMEN

Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression. AML1-ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia, is a transcription factor implicated in both gene repression and activation. AML1-ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis, making it important to identify co-regulatory factors that 'read' the NHR2 oligomerization and contribute to leukaemogenesis. Here we show that, in human leukaemic cells, AML1-ETO resides in and functions through a stable AML1-ETO-containing transcription factor complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, co-localize genome wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1-ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2-N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1-ETO-induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1-ETO, and provide a potential therapeutic target in t(8;21)-positive acute myeloid leukaemia.


Asunto(s)
Transformación Celular Neoplásica , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Complejos Multiproteicos/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , División Celular , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/química , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Proteínas de Fusión Oncogénica/química , Mutación Puntual , Unión Proteica , Multimerización de Proteína , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteína 1 Compañera de Translocación de RUNX1
15.
J Biol Chem ; 290(44): 26832-45, 2015 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-26381409

RESUMEN

The c-fos gene is rapidly induced to high levels by various extracellular stimuli. We used a defined in vitro transcription system that utilizes the c-fos promoter to purify a coactivator activity in an unbiased manner. We report here that NF45-NF90 and NF45-NF110, which possess archetypical double-stranded RNA binding motifs, have a direct function as transcriptional coactivators. The transcriptional activities of the nuclear factor (NF) complexes (NF45-NF90 and NF45-NF110) are mediated by both the upstream enhancer and core promoter regions of the c-fos gene and do not require their double-stranded RNA binding activities. The NF complexes cooperate with general coactivators, PC4 and Mediator, to elicit a high level of transcription and display multiple interactions with activators and the components of the general transcriptional machinery. Knockdown of the endogenous NF90/NF110 in mouse cells shows an important role for the NF complexes in inducing c-fos transcription. Chromatin immunoprecipitation assays demonstrate that the NF complexes occupy the c-fos enhancer/promoter region before and after serum induction and that their occupancies within the coding region of the c-fos gene increase in parallel to that of RNAPII upon serum induction. In light of their dynamic occupancy on the c-fos gene as well as direct functions in both transcription and posttranscriptional processes, the NF complexes appear to serve as multifunctional coactivators that coordinate different steps of gene expression to facilitate rapid response of inducible genes.


Asunto(s)
Genes fos , Proteína del Factor Nuclear 45/genética , Proteínas del Factor Nuclear 90/genética , ARN Polimerasa II/genética , Transcripción Genética , Animales , Baculoviridae/genética , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Cinética , Complejo Mediador/genética , Complejo Mediador/metabolismo , Ratones , Proteína del Factor Nuclear 45/metabolismo , Proteínas del Factor Nuclear 90/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Polimerasa II/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Transducción de Señal , Spodoptera , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
16.
Cancer Gene Ther ; 31(5): 736-745, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38429368

RESUMEN

Breast cancer is a heterogeneous disease, and breast cancer cell lines are invaluable for studying this heterogeneity. However, the epigenetic diversity across these cell lines remains poorly understood. In this study, we performed genome-wide chromatin accessibility analysis on 23 breast cancer cell lines, including 2 estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative (ER+/HER2-), 3 ER+/HER2+, 3 HER2+, and 15 triple-negative breast cancer (TNBC) lines. These cell lines were classified into three groups based on their chromatin accessibility: the receptor-positive group (Group-P), TNBC basal group (Group-B), and TNBC mesenchymal group (Group-M). Motif enrichment analysis revealed that only Group-P exhibited coenrichment of forkhead box A1 (FOXA1) and grainyhead-like 2 (GRHL2) motifs, whereas Group-B was characterized by the presence of the GRHL2 motif without FOXA1. Notably, Group-M did not show enrichment of either FOXA1 or GRHL2 motifs. Furthermore, gene ontology analysis suggested that group-specific accessible regions were associated with their unique lineage characteristics. To investigate the epigenetic landscape regulatory roles of FOXA1 and GRHL2, we performed knockdown experiments targeting FOXA1 and GRHL2, followed by assay for transposase-accessible chromatin sequencing analysis. The findings revealed that FOXA1 maintains Group-P-specific regions while suppressing Group-B-specific regions in Group-P cells. In contrast, GRHL2 preserves commonly accessible regions shared between Group-P and Group-B in Group-B cells, suggesting that FOXA1 and GRHL2 play a pivotal role in preserving distinct chromatin accessibility patterns for each group. Specifically, FOXA1 distinguishes between receptor-positive and TNBC cell lines, whereas GRHL2 distinguishes between basal-like and mesenchymal subtypes in TNBC lines.


Asunto(s)
Neoplasias de la Mama , Cromatina , Epigénesis Genética , Factor Nuclear 3-alfa del Hepatocito , Factores de Transcripción , Humanos , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Factor Nuclear 3-alfa del Hepatocito/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Femenino , Cromatina/metabolismo , Cromatina/genética , Línea Celular Tumoral , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Regulación Neoplásica de la Expresión Génica , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo
17.
Cell Res ; 33(2): 165-183, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36646760

RESUMEN

Estrogen-related receptors (ERRα/ß/γ) are orphan nuclear receptors that function in energy-demanding physiological processes, as well as in development and stem cell maintenance, but mechanisms underlying target gene activation by ERRs are largely unknown. Here, reconstituted biochemical assays that manifest ERR-dependent transcription have revealed two complementary mechanisms. On DNA templates, ERRs activate transcription with just the normal complement of general initiation factors through an interaction of the ERR DNA-binding domain with the p52 subunit of initiation factor TFIIH. On chromatin templates, activation by ERRs is dependent on AF2 domain interactions with the cell-specific coactivator PGC-1α, which in turn recruits the ubiquitous p300 and MED1/Mediator coactivators. This role of PGC-1α may also be fulfilled by other AF2-interacting coactivators like NCOA3, which is shown to recruit Mediator selectively to ERRß and ERRγ. Importantly, combined genetic and RNA-seq analyses establish that both the TFIIH and the AF2 interaction-dependent pathways are essential for ERRß/γ-selective gene expression and pluripotency maintenance in embryonic stem cells in which NCOA3 is a critical coactivator.


Asunto(s)
Furilfuramida , Receptores Nucleares Huérfanos , ADN , Regiones Promotoras Genéticas , Activación Transcripcional , Receptores de Estrógenos/metabolismo
18.
NPJ Breast Cancer ; 8(1): 70, 2022 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-35676392

RESUMEN

In breast cancer patients, tumor heterogeneity is associated with prognosis and therapeutic response; however, the epigenetic diversity that exists in primary tumors remains unknown. Using a single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq), we obtained the chromatin accessibility profiles of 12,452 cells from 16 breast cancer patients including 11 luminal, 1 luminal-HER2, 1 HER2+, and 3 triple-negative subtypes. Via this profiling process, tumors were classified into cancer cells and the tumor microenvironment, highlighting the heterogeneity of disease-related pathways including estrogen receptor (ER) signaling. Furthermore, the coexistence of cancer cell clusters with different ER binding motif enrichments was identified in a single ER+ tumor. In a cluster with reduced ER motif enrichment, we identified GRHL2, a transcription factor, as the most enriched motif, and it cooperated with FOXA1 to initiate endocrine resistance. Coaccessibility analysis revealed that GRHL2 binding elements potentially regulate genes associated with endocrine resistance, metastasis, and poor prognosis in patients that received hormonal therapy. Overall, our study suggests that epigenetic heterogeneity could lead to endocrine resistance and poor prognosis in breast cancer patients and it offers a large-scale resource for further cancer research.

19.
J Biol Chem ; 285(13): 9390-9401, 2010 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-20089855

RESUMEN

The rapid induction of the c-fos gene correlates with phosphorylations of histone H3 and HMGN1 by mitogen- and stress-activated protein kinases. We have used a cell-free system to dissect the mechanism by which MSK1 phosphorylates histone H3 within the c-fos chromatin. Here, we show that the reconstituted c-fos chromatin presents a strong barrier to histone H3 phosphorylation by MSK1; however, the activators (serum response factor, Elk-1, cAMP-response element-binding protein (CREB), and ATF1) bound on their cognate sites recruit MSK1 to phosphorylate histone H3 at Ser-10 within the chromatin. This activator-dependent phosphorylation of histone H3 is enhanced by HMGN1 and occurs preferentially near the promoter region. Among the four activators, CREB plays a predominant role in MSK1-mediated phosphorylation of histone H3, and the phosphorylation of Ser-133 in CREB is essential for this process. Mutational analyses of MSK1 show that its N-terminal inhibition domain is critical for the kinase to phosphorylate chromatin-embedded histone H3 in a CREB-dependent manner, indicating the presence of an intricate regulatory network for MSK1-mediated phosphorylation of histone H3.


Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Histonas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Cromatina/química , Análisis Mutacional de ADN , Redes Reguladoras de Genes , Células HeLa , Histonas/química , Humanos , Modelos Genéticos , Fosforilación , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Serina/química , Transcripción Genética
20.
Elife ; 102021 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-34747692

RESUMEN

The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.


Asunto(s)
Histonas/metabolismo , ARN Polimerasa II/genética , Sumoilación , Transcripción Genética , Extractos Celulares , Humanos , ARN Polimerasa II/metabolismo
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