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1.
Cell ; 160(3): 367-80, 2015 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-25619691

RESUMEN

The discovery that enhancers are regulated transcription units, encoding eRNAs, has raised new questions about the mechanisms of their activation. Here, we report an unexpected molecular mechanism that underlies ligand-dependent enhancer activation, based on DNA nicking to relieve torsional stress from eRNA synthesis. Using dihydrotestosterone (DHT)-induced binding of androgen receptor (AR) to prostate cancer cell enhancers as a model, we show rapid recruitment, within minutes, of DNA topoisomerase I (TOP1) to a large cohort of AR-regulated enhancers. Furthermore, we show that the DNA nicking activity of TOP1 is a prerequisite for robust eRNA synthesis and enhancer activation and is kinetically accompanied by the recruitment of ATR and the MRN complex, followed by additional components of DNA damage repair machinery to the AR-regulated enhancers. Together, our studies reveal a linkage between eRNA synthesis and ligand-dependent TOP1-mediated nicking-a strategy exerting quantitative effects on eRNA expression in regulating AR-bound enhancer-dependent transcriptional programs.


Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Receptores Androgénicos/metabolismo , Línea Celular Tumoral , Roturas del ADN de Cadena Simple , Reparación del ADN , ADN-Topoisomerasas de Tipo I/genética , Proteínas de Unión al ADN/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/metabolismo , Humanos , Proteína Homóloga de MRE11 , Factores de Transcripción/metabolismo , Transcripción Genética
2.
Cell ; 159(2): 358-73, 2014 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-25303530

RESUMEN

Enhancers provide critical information directing cell-type-specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. Here, we report that the most strongly activated estrogen (E2)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ERα at ERE-containing enhancers. We refer to enhancers recruiting these factors as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome.


Asunto(s)
Elementos de Facilitación Genéticos , Receptor alfa de Estrógeno/metabolismo , Factores de Transcripción/metabolismo , Estrógenos/metabolismo , Factor de Transcripción GATA3/metabolismo , Regulación de la Expresión Génica , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , Complejos Multiproteicos/metabolismo
3.
Nature ; 595(7867): 444-449, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34194047

RESUMEN

The size of the transcriptional program of long non-coding RNAs in the mammalian genome has engendered discussions about their biological roles1, particularly the promoter antisense (PAS) transcripts2,3. Here we report the development of an assay-referred to as chromatin isolation by RNA-Cas13a complex-to quantitatively detect the distribution of RNA in the genome. The assay revealed that PAS RNAs serve as a key gatekeeper of a broad transcriptional pause release program, based on decommissioning the 7SK small nuclear RNA-dependent inhibitory P-TEFb complex. Induction of PAS RNAs by liganded ERα led to a significant loss of H3K9me3 and the release of basally recruited HP1α and KAP1 on activated target gene promoters. This release was due to PAS RNA-dependent recruitment of H3K9me3 demethylases, which required interactions with a compact stem-loop structure in the PAS RNAs, an apparent feature of similarly regulated PAS RNAs. Activation of the ERα-bound MegaTrans enhancer, which is essential for robust pause release, required the recruitment of phosphorylated KAP1, with its transfer to the cognate promoters permitting 17ß-oestradiol-induced pause release and activation of the target gene. This study reveals a mechanism, based on RNA structure, that mediates the function of PAS RNAs in gene regulation.


Asunto(s)
Conformación de Ácido Nucleico , Regiones Promotoras Genéticas/genética , ARN sin Sentido/química , ARN sin Sentido/genética , Activación Transcripcional/genética , Línea Celular , Homólogo de la Proteína Chromobox 5/metabolismo , Proteína Sustrato Asociada a CrK , Receptor alfa de Estrógeno/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ligandos , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN Polimerasa II/metabolismo , Estabilidad del ARN , Proteína 28 que Contiene Motivos Tripartito/metabolismo
4.
Cell ; 147(4): 773-88, 2011 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-22078878

RESUMEN

Although eukaryotic nuclei contain distinct architectural structures associated with noncoding RNAs (ncRNAs), their potential relationship to regulated transcriptional programs remains poorly understood. Here, we report that methylation/demethylation of Polycomb 2 protein (Pc2) controls relocation of growth-control genes between Polycomb bodies (PcGs) and interchromatin granules (ICGs) in response to growth signals. This movement is the consequence of binding of methylated and unmethylated Pc2 to the ncRNAs TUG1 and MALAT1/NEAT2, located in PcGs and ICGs, respectively. These ncRNAs mediate assembly of multiple corepressors/coactivators and can serve to switch mark recognition by "readers" of the histone code. Additionally, binding of NEAT2 to unmethylated Pc2 promotes E2F1 SUMOylation, leading to activation of the growth-control gene program. These observations delineate a molecular pathway linking the actions of subnuclear structure-specific ncRNAs and nonhistone protein methylation to relocation of transcription units in the three-dimensional space of the nucleus, thus achieving coordinated gene expression programs.


Asunto(s)
Núcleo Celular/metabolismo , Regulación de la Expresión Génica , ARN no Traducido/metabolismo , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Línea Celular , Cromatina/metabolismo , Factor de Transcripción E2F1/metabolismo , Células HeLa , Humanos , Ligasas , Metilación , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Proteínas del Grupo Polycomb , ARN Largo no Codificante , Proteínas Represoras/química , Sumoilación , Transcripción Genética , Ubiquitina-Proteína Ligasas , Ubiquitinación
5.
Mol Cell ; 66(3): 321-331.e6, 2017 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-28475868

RESUMEN

The molecular mechanisms underlying the opposing functions of glucocorticoid receptors (GRs) and estrogen receptor α (ERα) in breast cancer development remain poorly understood. Here we report that, in breast cancer cells, liganded GR represses a large ERα-activated transcriptional program by binding, in trans, to ERα-occupied enhancers. This abolishes effective activation of these enhancers and their cognate target genes, and it leads to the inhibition of ERα-dependent binding of components of the MegaTrans complex. Consistent with the effects of SUMOylation on other classes of nuclear receptors, dexamethasone (Dex)-induced trans-repression of the estrogen E2 program appears to depend on GR SUMOylation, which leads to stable trans-recruitment of the GR-N-CoR/SMRT-HDAC3 corepressor complex on these enhancers. Together, these results uncover a mechanism by which competitive recruitment of DNA-binding nuclear receptors/transcription factors in trans to hot spot enhancers serves as an effective biological strategy for trans-repression, with clear implications for breast cancer and other diseases.


Asunto(s)
Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/metabolismo , Regulación Neoplásica de la Expresión Génica , Receptor Cross-Talk , Receptores de Glucocorticoides/metabolismo , Transcripción Genética , Sitios de Unión , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Dexametasona/farmacología , Regulación hacia Abajo , Elementos de Facilitación Genéticos , Estradiol/farmacología , Receptor alfa de Estrógeno/agonistas , Receptor alfa de Estrógeno/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Células MCF-7 , Complejos Multiproteicos , Mutación , Co-Represor 1 de Receptor Nuclear/genética , Co-Represor 1 de Receptor Nuclear/metabolismo , Co-Represor 2 de Receptor Nuclear/genética , Co-Represor 2 de Receptor Nuclear/metabolismo , Unión Proteica , Interferencia de ARN , Receptor Cross-Talk/efectos de los fármacos , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/genética , Transducción de Señal , Sumoilación , Transcripción Genética/efectos de los fármacos , Transcriptoma , Transfección
6.
Cell ; 132(6): 996-1010, 2008 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-18358812

RESUMEN

While the transcriptional machinery has been extensively dissected at the molecular level, little is known about regulation of chromosomal organization in the three-dimensional space of the nucleus to achieve integrated transcriptional responses to diverse signaling events. Here, we report that ligand induces rapid interchromosomal interactions among subsets of estrogen receptor alpha-bound transcription units, with a dramatic reorganization of nuclear territories requiring nuclear actin/myosin-I transport machinery, dynein light chain 1 (DLC1), and a specific subset of transcriptional coactivators and chromatin remodeling complexes. We establish a requirement for the histone lysine demethylase, LSD1, in directing specific interchromosomal interaction loci to distinct interchromatin granules, long thought to be "storage" sites for splicing machinery, and demonstrate that these three-dimensional motor-dependent interactions are required to achieve enhanced transcription of specific estrogen-receptor target genes. These findings reveal roles for the modulation of nuclear architecture in orchestrating regulated gene-expression programs in the mammalian nucleus.


Asunto(s)
Cromatina/metabolismo , Receptor alfa de Estrógeno/metabolismo , Redes Reguladoras de Genes , Proteínas Motoras Moleculares/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Actinas/metabolismo , Línea Celular Tumoral , Núcleo Celular , Células Cultivadas , Histona Demetilasas , Humanos , Cuerpos de Inclusión Intranucleares/metabolismo , Transcripción Genética
8.
Nature ; 516(7530): 267-71, 2014 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-25252977

RESUMEN

Post-translational histone modifications have a critical role in regulating transcription, the cell cycle, DNA replication and DNA damage repair. The identification of new histone modifications critical for transcriptional regulation at initiation, elongation or termination is of particular interest. Here we report a new layer of regulation in transcriptional elongation that is conserved from yeast to mammals. This regulation is based on the phosphorylation of a highly conserved tyrosine residue, Tyr 57, in histone H2A and is mediated by the unsuspected tyrosine kinase activity of casein kinase 2 (CK2). Mutation of Tyr 57 in H2A in yeast or inhibition of CK2 activity impairs transcriptional elongation in yeast as well as in mammalian cells. Genome-wide binding analysis reveals that CK2α, the catalytic subunit of CK2, binds across RNA-polymerase-II-transcribed coding genes and active enhancers. Mutation of Tyr 57 causes a loss of H2B mono-ubiquitination as well as H3K4me3 and H3K79me3, histone marks associated with active transcription. Mechanistically, both CK2 inhibition and the H2A(Y57F) mutation enhance H2B deubiquitination activity of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, suggesting a critical role of this phosphorylation in coordinating the activity of the SAGA complex during transcription. Together, these results identify a new component of regulation in transcriptional elongation based on CK2-dependent tyrosine phosphorylation of the globular domain of H2A.


Asunto(s)
Quinasa de la Caseína II/metabolismo , Histonas/química , Histonas/metabolismo , Elongación de la Transcripción Genética , Tirosina/metabolismo , Secuencia de Aminoácidos , Línea Celular , Secuencia Conservada , Histonas/genética , Humanos , Datos de Secuencia Molecular , Fosforilación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tirosina/química , Ubiquitinación/genética
9.
Nature ; 514(7521): 257-61, 2014 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-25119036

RESUMEN

Homeodomain proteins, described 30 years ago, exert essential roles in development as regulators of target gene expression; however, the molecular mechanisms underlying transcriptional activity of homeodomain factors remain poorly understood. Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program. Pit1 association with Satb1 (ref. 8) and ß-catenin is required for this tethering event. A naturally occurring, dominant negative, point mutation in human PIT1(R271W), causing combined pituitary hormone deficiency, results in loss of Pit1 association with ß-catenin and Satb1 and therefore the matrin-3-rich network, blocking Pit1-dependent enhancer/coding target gene activation. This defective activation can be rescued by artificial tethering of the mutant R271W Pit1 protein to the matrin-3 network, bypassing the pre-requisite association with ß-catenin and Satb1 otherwise required. The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation. These studies have thus revealed an unanticipated homeodomain factor/ß-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.


Asunto(s)
Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Animales , Células Cultivadas , Proteínas de Homeodominio/genética , Humanos , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Ratones , Hipófisis/embriología , Hipófisis/metabolismo , Unión Proteica , Factor de Transcripción Pit-1/genética , Factor de Transcripción Pit-1/metabolismo , Transcripción Genética/genética , beta Catenina/metabolismo
10.
Mol Cell ; 46(1): 91-104, 2012 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-22424771

RESUMEN

The association between hyperinflammatory states and numerous diseases is widely recognized, but our understanding of the molecular strategies that have evolved to prevent uncontrolled activation of inflammatory responses remains incomplete. Here, we report a critical, nontranscriptional role of GPS2 as a guardian against hyperstimulation of the TNF-α-induced gene program. GPS2 cytoplasmic actions are required to specifically modulate RIP1 ubiquitylation and JNK activation by inhibiting TRAF2/Ubc13 enzymatic activity. In vivo relevance of GPS2 anti-inflammatory role is confirmed by inhibition of TNF-α target genes in macrophages and by improved insulin signaling in the adipose tissue of aP2-GPS2 transgenic mice. As the nontranscriptional role is complemented by GPS2 functioning as positive and negative cofactor for nuclear receptors, in vivo overexpression also results in elevated circulating level of Resistin and development of hepatic steatosis. Together, these studies define GPS2 as a molecular guardian required for precise control of inflammatory responses involved in immunity and homeostasis.


Asunto(s)
Tejido Adiposo/metabolismo , Homeostasis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/metabolismo , Tejido Adiposo/inmunología , Animales , Línea Celular , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/inmunología , Proteínas Activadoras de GTPasa/metabolismo , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Insulina/genética , Insulina/inmunología , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/inmunología , MAP Quinasa Quinasa 4/metabolismo , Macrófagos/inmunología , Ratones , Ratones Transgénicos , Resistina/genética , Resistina/inmunología , Resistina/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/inmunología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/genética , Ubiquitinación/inmunología
11.
Nature ; 500(7464): 598-602, 2013 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-23945587

RESUMEN

Although recent studies have indicated roles of long non-coding RNAs (lncRNAs) in physiological aspects of cell-type determination and tissue homeostasis, their potential involvement in regulated gene transcription programs remains rather poorly understood. The androgen receptor regulates a large repertoire of genes central to the identity and behaviour of prostate cancer cells, and functions in a ligand-independent fashion in many prostate cancers when they become hormone refractory after initial androgen deprivation therapy. Here we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 (also known as PCAT8) and PCGEM1, bind successively to the androgen receptor and strongly enhance both ligand-dependent and ligand-independent androgen-receptor-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the carboxy-terminally acetylated androgen receptor on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM1, to the androgen receptor amino terminus that is methylated by DOT1L. Unexpectedly, recognition of specific protein marks by PCGEM1-recruited pygopus 2 PHD domain enhances selective looping of androgen-receptor-bound enhancers to target gene promoters in these cells. In 'resistant' prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for, the robust activation of both truncated and full-length androgen receptor, causing ligand-independent activation of the androgen receptor transcriptional program and cell proliferation. Conditionally expressed short hairpin RNA targeting these lncRNAs in castration-resistant prostate cancer cell lines strongly suppressed tumour xenograft growth in vivo. Together, these results indicate that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumours.


Asunto(s)
ARN Largo no Codificante/genética , Receptores Androgénicos/metabolismo , Activación Transcripcional/genética , Regulación hacia Arriba/genética , Animales , Castración , Línea Celular Tumoral , Proliferación Celular , Elementos de Facilitación Genéticos/genética , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Factores de Transcripción/metabolismo
13.
Proc Natl Acad Sci U S A ; 112(5): 1380-5, 2015 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-25605944

RESUMEN

Substantial evidence supports the hypothesis that enhancers are critical regulators of cell-type determination, orchestrating both positive and negative transcriptional programs; however, the basic mechanisms by which enhancers orchestrate interactions with cognate promoters during activation and repression events remain incompletely understood. Here we report the required actions of LIM domain-binding protein 1 (LDB1)/cofactor of LIM homeodomain protein 2/nuclear LIM interactor, interacting with the enhancer-binding protein achaete-scute complex homolog 1, to mediate looping to target gene promoters and target gene regulation in corticotrope cells. LDB1-mediated enhancer:promoter looping appears to be required for both activation and repression of these target genes. Although LDB1-dependent activated genes are regulated at the level of transcriptional initiation, the LDB1-dependent repressed transcription units appear to be regulated primarily at the level of promoter pausing, with LDB1 regulating recruitment of metastasis-associated 1 family, member 2, a component of the nucleosome remodeling deacetylase complex, on these negative enhancers, required for the repressive enhancer function. These results indicate that LDB1-dependent looping events can deliver repressive cargo to cognate promoters to mediate promoter pausing events in a pituitary cell type.


Asunto(s)
Corticotrofos/fisiología , Proteínas de Unión al ADN/fisiología , Elementos de Facilitación Genéticos , Proteínas con Dominio LIM/fisiología , Regiones Promotoras Genéticas , Animales , Línea Celular , Proteínas de Unión al ADN/metabolismo , Proteínas con Dominio LIM/metabolismo , Ratones , Ratones Noqueados
14.
Nature ; 474(7351): 390-4, 2011 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-21572438

RESUMEN

Mammalian genomes are populated with thousands of transcriptional enhancers that orchestrate cell-type-specific gene expression programs, but how those enhancers are exploited to institute alternative, signal-dependent transcriptional responses remains poorly understood. Here we present evidence that cell-lineage-specific factors, such as FoxA1, can simultaneously facilitate and restrict key regulated transcription factors, exemplified by the androgen receptor (AR), to act on structurally and functionally distinct classes of enhancer. Consequently, FoxA1 downregulation, an unfavourable prognostic sign in certain advanced prostate tumours, triggers dramatic reprogramming of the hormonal response by causing a massive switch in AR binding to a distinct cohort of pre-established enhancers. These enhancers are functional, as evidenced by the production of enhancer-templated non-coding RNA (eRNA) based on global nuclear run-on sequencing (GRO-seq) analysis, with a unique class apparently requiring no nucleosome remodelling to induce specific enhancer-promoter looping and gene activation. GRO-seq data also suggest that liganded AR induces both transcription initiation and elongation. Together, these findings reveal a large repository of active enhancers that can be dynamically tuned to elicit alternative gene expression programs, which may underlie many sequential gene expression events in development, cell differentiation and disease progression.


Asunto(s)
Elementos de Facilitación Genéticos/genética , Factor Nuclear 3-alfa del Hepatocito/metabolismo , ARN no Traducido/genética , Receptores Androgénicos/metabolismo , Transcripción Genética/genética , Secuencia de Bases , Línea Celular Tumoral , Linaje de la Célula , Dihidrotestosterona/farmacología , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genoma Humano/genética , Células HEK293 , Factor Nuclear 3-alfa del Hepatocito/deficiencia , Factor Nuclear 3-alfa del Hepatocito/genética , Histonas/metabolismo , Humanos , Calicreínas , Masculino , Antígeno Prostático Específico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo
15.
Proc Natl Acad Sci U S A ; 111(25): 9235-40, 2014 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-24928520

RESUMEN

Understanding the mechanisms by which compounds discovered using cell-based phenotypic screening strategies might exert their effects would be highly augmented by new approaches exploring their potential interactions with the genome. For example, altered androgen receptor (AR) transcriptional programs, including castration resistance and subsequent chromosomal translocations, play key roles in prostate cancer pathological progression, making the quest for identification of new therapeutic agents and an understanding of their actions a continued priority. Here we report an approach that has permitted us to uncover the sites and mechanisms of action of a drug, referred to as "SD70," initially identified by phenotypic screening for inhibitors of ligand and genotoxic stress-induced translocations in prostate cancer cells. Based on synthesis of a derivatized form of SD70 that permits its application for a ChIP-sequencing-like approach, referred to as "Chem-seq," we were next able to efficiently map the genome-wide binding locations of this small molecule, revealing that it largely colocalized with AR on regulatory enhancers. Based on these observations, we performed the appropriate global analyses to ascertain that SD70 inhibits the androgen-dependent AR program, and prostate cancer cell growth, acting, at least in part, by functionally inhibiting the Jumonji domain-containing demethylase, KDM4C. Global location of candidate drugs represents a powerful strategy for new drug development by mapping genome-wide location of small molecules, a powerful adjunct to contemporary drug development strategies.


Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Sistemas de Liberación de Medicamentos/métodos , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/genética , Receptores Androgénicos/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Antineoplásicos , Línea Celular Tumoral , Análisis Mutacional de ADN/métodos , Humanos , Masculino , Ratones , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Translocación Genética
16.
Nature ; 466(7305): 508-12, 2010 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-20622854

RESUMEN

While reversible histone modifications are linked to an ever-expanding range of biological functions, the demethylases for histone H4 lysine 20 and their potential regulatory roles remain unknown. Here we report that the PHD and Jumonji C (JmjC) domain-containing protein, PHF8, while using multiple substrates, including H3K9me1/2 and H3K27me2, also functions as an H4K20me1 demethylase. PHF8 is recruited to promoters by its PHD domain based on interaction with H3K4me2/3 and controls G1-S transition in conjunction with E2F1, HCF-1 (also known as HCFC1) and SET1A (also known as SETD1A), at least in part, by removing the repressive H4K20me1 mark from a subset of E2F1-regulated gene promoters. Phosphorylation-dependent PHF8 dismissal from chromatin in prophase is apparently required for the accumulation of H4K20me1 during early mitosis, which might represent a component of the condensin II loading process. Accordingly, the HEAT repeat clusters in two non-structural maintenance of chromosomes (SMC) condensin II subunits, N-CAPD3 and N-CAPG2 (also known as NCAPD3 and NCAPG2, respectively), are capable of recognizing H4K20me1, and ChIP-Seq analysis demonstrates a significant overlap of condensin II and H4K20me1 sites in mitotic HeLa cells. Thus, the identification and characterization of an H4K20me1 demethylase, PHF8, has revealed an intimate link between this enzyme and two distinct events in cell cycle progression.


Asunto(s)
Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Histona Demetilasas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Línea Celular , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Células HeLa , Histona Demetilasas/química , Histona Demetilasas/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Factor C1 de la Célula Huésped/genética , Factor C1 de la Célula Huésped/metabolismo , Humanos , Metilación , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Factores de Transcripción/química , Factores de Transcripción/deficiencia , Factores de Transcripción/genética
17.
Mol Cell ; 29(6): 755-66, 2008 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-18374649

RESUMEN

A key strategy to achieve regulated gene expression in higher eukaryotes is to prevent illegitimate signal-independent activation by imposing robust control on the dismissal of corepressors. Here, we report that many signaling pathways, including Notch, NF-kappaB, and nuclear receptor ligands, are subjected to a dual-repression "checkpoint" based on distinct corepressor complexes. Gene activation requires the release of both CtBP1/2- and NCoR/SMRT-dependent repression, through the coordinate action of two highly related exchange factors, the transducer beta-like proteins TBL1 and TBLR1, that license ubiquitylation and degradation of CtBP1/2 and NCoR/SMRT, respectively. Intriguingly, their function and differential specificity reside in only five specific Ser/Thr phosphorylation site differences, regulated by direct phosphorylation at the level of the promoter, as exemplified by the role of PKCdelta in TBLR1-dependent dismissal of NCoR. Thus, our data reveal a strategy of dual-factor repression checkpoints, in which dedicated exchange factors serve as sensors for signal-specific dismissal of distinct corepressors, with specificity imposed by upstream signaling pathways.


Asunto(s)
Oxidorreductasas de Alcohol/genética , Proteínas de Unión al ADN/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Transducina/metabolismo , Animales , Neoplasias de la Mama , Línea Celular , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Humanos , Co-Represor 2 de Receptor Nuclear , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Represoras/genética , Activación Transcripcional , Ubiquitina/metabolismo
18.
bioRxiv ; 2024 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-38798402

RESUMEN

Because most DNA-binding transcription factors (dbTFs), including the architectural regulator CTCF, bind RNA and exhibit di-/multimerization, a central conundrum is whether these distinct properties are regulated post-transcriptionally to modulate transcriptional programs. Here, investigating stress-dependent activation of SIRT1, encoding an evolutionarily-conserved protein deacetylase, we show that induced phosphorylation of CTCF acts as a rheostat to permit CTCF occupancy of low-affinity promoter DNA sites to precisely the levels necessary. This CTCF recruitment to the SIRT1 promoter is eliciting a cardioprotective cardiomyocyte transcriptional activation program and provides resilience against the stress of the beating heart in vivo . Mice harboring a mutation in the conserved low-affinity CTCF promoter binding site exhibit an altered, cardiomyocyte-specific transcriptional program and a systolic heart failure phenotype. This transcriptional role for CTCF reveals that a covalent dbTF modification regulating signal-dependent transcription serves as a previously unsuspected component of the oxidative stress response.

19.
Nature ; 446(7138): 882-7, 2007 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-17392792

RESUMEN

Precise control of transcriptional programmes underlying metazoan development is modulated by enzymatically active co-regulatory complexes, coupled with epigenetic strategies. One thing that remains unclear is how specific members of histone modification enzyme families, such as histone methyltransferases and demethylases, are used in vivo to simultaneously orchestrate distinct developmental gene activation and repression programmes. Here, we report that the histone lysine demethylase, LSD1--a component of the CoREST-CtBP co-repressor complex--is required for late cell-lineage determination and differentiation during pituitary organogenesis. LSD1 seems to act primarily on target gene activation programmes, as well as in gene repression programmes, on the basis of recruitment of distinct LSD1-containing co-activator or co-repressor complexes. LSD1-dependent gene repression programmes can be extended late in development with the induced expression of ZEB1, a Krüppel-like repressor that can act as a molecular beacon for recruitment of the LSD1-containing CoREST-CtBP co-repressor complex, causing repression of an additional cohort of genes, such as Gh, which previously required LSD1 for activation. These findings suggest that temporal patterns of expression of specific components of LSD1 complexes modulate gene regulatory programmes in many mammalian organs.


Asunto(s)
Regulación hacia Abajo/genética , Regulación del Desarrollo de la Expresión Génica , Oxidorreductasas N-Desmetilantes/metabolismo , Animales , Diferenciación Celular , Hormona del Crecimiento/genética , Histona Demetilasas , Proteínas de Homeodominio/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Lactotrofos/metabolismo , Ratones , Oxidorreductasas N-Desmetilantes/deficiencia , Oxidorreductasas N-Desmetilantes/genética , Hipófisis/citología , Hipófisis/metabolismo , Activación Transcripcional , Homeobox 1 de Unión a la E-Box con Dedos de Zinc
20.
Nature ; 434(7035): 921-6, 2005 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-15829968

RESUMEN

Defining the molecular strategies that integrate diverse signalling pathways in the expression of specific gene programmes that are critical in homeostasis and disease remains a central issue in biology. This is particularly pertinent in cancer biology because downregulation of tumour metastasis suppressor genes is a common occurrence, and the underlying molecular mechanisms are not well established. Here we report that the downregulation of a metastasis suppressor gene, KAI1, in prostate cancer cells involves the inhibitory actions of beta-catenin, along with a reptin chromatin remodelling complex. This inhibitory function of beta-catenin-reptin requires both increased beta-catenin expression and recruitment of histone deacetylase activity. The coordinated actions of beta-catenin-reptin components that mediate the repressive state serve to antagonize a Tip60 coactivator complex that is required for activation; the balance of these opposing complexes controls the expression of KAI1 and metastatic potential. The molecular mechanisms underlying the antagonistic regulation of beta-catenin-reptin and the Tip60 coactivator complexes for the metastasis suppressor gene, KAI1, are likely to be prototypic of a selective downregulation strategy for many genes, including a subset of NF-kappaB target genes.


Asunto(s)
Acetiltransferasas/metabolismo , Antígenos CD/genética , Proteínas del Citoesqueleto/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Glicoproteínas de Membrana/genética , Metástasis de la Neoplasia/genética , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/metabolismo , Transcripción Genética/genética , Acetiltransferasas/genética , Animales , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina , Colágeno , Regulación hacia Abajo/genética , Combinación de Medicamentos , Histona Acetiltransferasas , Humanos , Proteína Kangai-1 , Laminina , Lisina Acetiltransferasa 5 , Masculino , Ratones , FN-kappa B/metabolismo , Trasplante de Neoplasias , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/metabolismo , Proteoglicanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , beta Catenina
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