RESUMEN
BACKGROUND: DNA methylation is an epigenetic modification that mainly repress expression of genes essential during embryogenesis and development. There are key ATPase-dependent enzymes that read or write DNA methylation to remodel chromatin and regulate gene expression. Structural maintenance of chromosome hinge domain containing 1 (SMCHD1) is an architectural protein that regulates expression of numerous genes, some of which are imprinted, that are sensitive to DNA methylation. In addition, SMCHD1 germline mutations lead to developmental diseases; facioscapulohumoral muscular dystrophy (FSHD), bosma arhinia and micropthalmia (BAMS). Current evidence suggests that SMCHD1 functions through maintenance or de novo DNA methylation required for chromatin compaction. However, it is unclear if DNA methylation is also essential for genomic recruitment of SMCHD1 and its role as an architectural protein. We previously isolated SMCHD1 using a methylated DNA region from mouse pituitary growth hormone (Gh1) promoter, suggesting that methylation is required for SMCHD1 DNA binding. The goal of this study was to further understand DNA methylation directed role of SMCHD1 in regulating gene expression. Therefore, we profiled SMCHD1 genome wide occupancy in human neuroblastoma SH-SY5Y cells and evaluated if DNA methylation is required for SMCHD1 genomic binding by treating cells with the DNA demethylating reagent, 5-azacytidine (5-azaC). RESULTS: Our data suggest that the majority of SMCHD1 binding occurs in intron and intergenic regions. Gene ontology analysis of genes associated with SMCHD1 genomic occupancy that is sensitive to 5-azaC treatment suggests SMCHD1 involvement in central nervous system development. The potassium voltage-gated channel subfamily Q member1 (KCNQ1) gene that associates with central nervous system is a known SMCHD1 target. We showed SMCHD1 binding to an intronic region of KCNQ1 that is lost following 5-azaC treatment suggesting DNA methylation facilitated binding of SMCHD1. Indeed, deletion of SMCHD1 by CRISPR- Cas9 increases KCNQ1 gene expression confirming its role in regulating KCNQ1 gene expression. CONCLUSION: These findings provide novel insights on DNA methylation directed function of SMCHD1 in regulating expression of genes associated with central nervous system development that impact future drug development strategies.
Asunto(s)
Azacitidina/farmacología , Sitios de Unión , Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN/efectos de los fármacos , Epigenómica , Epigénesis Genética/efectos de los fármacos , Epigenómica/métodos , Exones , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Intrones , Regiones Promotoras Genéticas , Unión Proteica , Sitio de Iniciación de la TranscripciónRESUMEN
The basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH-PAS) domain family of transcription factors mediates cellular responses to a variety of internal and external stimuli. As functional transcription factors, these proteins act as bHLH-PAS heterodimers and can be further sub-classified into sensory/activated subunits and regulatory or ARNT-like proteins. This class of proteins act as master regulators of the bHLH-PAS superfamily of transcription factors that mediate circadian rhythm gene programs, innate and adaptive immune responses, oxygen-sensing mechanisms and compensate for deleterious environmental exposures. Some contribute to the etiology of human pathologies including cancer because of their effects on cell growth and metabolism. We will review the canonical roles of ARNT and ARNT-like proteins with an emphasis on coactivator selectivity and recruitment. We will also discuss recent advances in our understanding of noncanonical DNA-binding independent or off-target roles of ARNT that are uncoupled from its classic heterodimeric bHLH-PAS binding partners. Understanding the DNA binding-independent functions of ARNT may identify novel therapeutic options for the treatment of a large spectrum of disease states.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Proteínas de Unión al ADN/genética , Secuencia de Aminoácidos , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Ritmo Circadiano/genética , Proteínas de Unión al ADN/metabolismo , Secuencias Hélice-Asa-Hélice , Humanos , Unión ProteicaRESUMEN
Steroid hormone receptors act to regulate specific gene transcription primarily as steroid-specific dimers bound to palindromic DNA response elements. DNA-dependent dimerization contacts mediated between the receptor DNA binding domains stabilize DNA binding. Additionally, some steroid receptors dimerize prior to their arrival on DNA through interactions mediated through the receptor ligand binding domain. In this report, we describe the steroid-induced homomeric interaction of the rat glucocorticoid receptor (GR) in solution in vivo. Our results demonstrate that GR interacts in solution at least as a dimer, and we have delimited this interaction to a novel interface within the hinge region of GR that appears to be both necessary and sufficient for direct binding. Strikingly, we also demonstrate an interaction between GR and the mineralocorticoid receptor in solution in vivo that is dependent on the ligand binding domain of GR alone and is separable from homodimerization of the glucocorticoid receptor. These results indicate that functional interactions between the glucocorticoid and mineralocorticoid receptors in activating specific gene transcription are probably more complex than has been previously appreciated.
Asunto(s)
Receptores de Glucocorticoides/química , Receptores de Mineralocorticoides/química , Animales , Sitios de Unión , Células COS , Línea Celular , Núcleo Celular/metabolismo , Citoplasma , Dimerización , Técnicas In Vitro , Estructura Cuaternaria de Proteína , Ratas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Soluciones , Técnicas del Sistema de Dos HíbridosRESUMEN
Steroid hormone receptors are distinguished from other members of the nuclear hormone receptor family through their association with heat shock proteins and immunophilins in the absence of ligands. Heat shock protein association represses steroid receptor DNA binding and protein-protein interactions with other transcription factors and facilitates hormone binding. In this study, we investigated the hormone-dependent interaction between the DNA binding domain (DBD) of the glucocorticoid receptor (GR) and the POU domains of octamer transcription factors 1 and 2 (Oct-1 and Oct-2, respectively). Our results indicate that the GR DBD binds directly, not only to the homeodomains of Oct-1 and Oct-2 but also to the homeodomains of several other homeodomain proteins. As these results suggest that the determinants for binding to the GR DBD are conserved within the homeodomain, we examined whether the ectopic expression of GR DBD peptides affected early embryonic development. The expression of GR DBD peptides in one-cell-stage zebra fish embryos severely affected their development, beginning with a delay in the epibolic movement during the blastula stage and followed by defects in convergence-extension movements during gastrulation, as revealed by the abnormal patterns of expression of several dorsal gene markers. In contrast, embryos injected with mRNA encoding a GR peptide with a point mutation that disrupted homeodomain binding or with mRNA encoding the DBD of the closely related mineralocorticoid receptor, which does not bind octamer factors, developed normally. Moreover, coinjection of mRNA encoding the homeodomain of Oct-2 completely rescued embryos from the effects of the GR DBD. These results highlight the potential of DNA-independent effects of GR in a whole-animal model and suggest that at least some of these effects may result from direct interactions with homeodomain proteins.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas Represoras , Animales , Sitios de Unión/genética , Tipificación del Cuerpo , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/aislamiento & purificación , Proteínas de Unión al ADN/genética , Proteína Goosecoide , Proteínas de Homeodominio/aislamiento & purificación , Factor C1 de la Célula Huésped , Leucina/genética , Mesodermo , Mutación , Factor 1 de Transcripción de Unión a Octámeros , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Prolina/genética , Unión Proteica/genética , Receptores de Glucocorticoides/genética , Distribución Tisular , Factores de Transcripción/metabolismo , Pez Cebra , Proteínas de Pez CebraRESUMEN
Transcriptional synergism between glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1 and Oct-2) in the induction of mouse mammary tumor virus (MMTV) transcription has been proposed to be mediated through directed recruitment of the octamer factors to their binding sites in the viral long terminal repeat. This recruitment correlates with direct binding between the GR DNA binding domain and the POU domain of the octamer factors. In present study, in vitro experiments identified several nuclear hormone receptors to have the potential to bind to the POU domains of Oct-1 and Oct-2 through their DNA binding domains, suggesting that POU domain binding may be a property shared by many nuclear hormone receptors. However, physiologically relevant binding to the POU domain appeared to be a property restricted to only a few nuclear receptors as only GR, progesterone receptor (PR), and androgen receptor (AR), were found to interact physically and functionally with Oct-1 and Oct-2 in transfected cells. Thus GR, PR, and AR efficiently promoted the recruitment of Oct-2 to adjacent octamer motifs in the cell, whereas mineralocorticoid receptor (MR), estrogen receptor alpha, and retinoid X receptor failed to facilitate octamer factor DNA binding. For MMTV, although GR and MR both induced transcription efficiently, mutation of the promoter proximal octamer motifs strongly decreased GR-induced transcription without affecting the total level of reporter gene activity in response to MR. These results suggest that the configuration of the hormone response element within the MMTV long terminal repeat may promote a dependence for the glucocorticoid response upon the recruitment of octamer transcription factors to their response elements within the viral promoter.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Virus del Tumor Mamario del Ratón/genética , Regiones Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Sitios de Unión , ADN Viral/metabolismo , Drosophila , Fibroblastos/metabolismo , Factor C1 de la Célula Huésped , Ratones , Factor 1 de Transcripción de Unión a Octámeros , Factor 2 de Transcripción de Unión a Octámeros , Pliegue de Proteína , Conejos , Receptores de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Activación TranscripcionalRESUMEN
Glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1/2) interact synergistically to activate the transcription of mouse mammary tumor virus and many cellular genes. Synergism correlates with cooperative DNA binding of the two factors in vitro. To examine the molecular basis for these cooperative interactions, we have studied the consequences of protein-protein binding between GR and Oct-1/2. We have determined that GR binds in solution to the octamer factor POU domain. Binding is mediated through an interface in the GR DNA binding domain that includes amino acids C500 and L501. In transfected mammalian cells, a transcriptionally inert wild-type but not an L501P GR peptide potentiated transcriptional activation by Oct-2 100-fold above the level that could be attained in the cell by expressing Oct-2 alone. Transcriptional activation correlated closely with a striking increase in the occupancy of octamer motifs adjacent to glucocorticoid response elements (GREs) on transiently transfected DNAs. Intriguingly, GR-Oct-1/2 binding was interrupted by the binding of GR to a GRE. We propose a model for transcriptional cooperativity in which GR-Oct-1/2 binding promotes an increase in the local concentration of octamer factors over glucocorticoid-responsive regulatory regions. These results reveal transcriptional cooperativity through a direct protein interaction between two sequence-specific transcription factors that is mediated in a way that is expected to restrict transcriptional effects to regulatory regions with DNA binding sites for both factors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Receptores de Glucocorticoides/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Factor C1 de la Célula Huésped , Virus del Tumor Mamario del Ratón/genética , Ratones , Conformación de Ácido Nucleico , Factor 1 de Transcripción de Unión a Octámeros , Factor 2 de Transcripción de Unión a Octámeros , Mutación Puntual , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Activación TranscripcionalRESUMEN
NRE1 is a DNA sequence element through which Ku antigen/DNA-dependent protein kinase (DNA-PK) catalytic subunit represses the induction of mouse mammary tumor virus transcription by glucocorticoids. Although Ku is an avid binder of DNA ends and has the ability to translocate along DNA, we report that direct sequence-specific Ku binding occurs with higher affinity (Kd = 0.84 +/- 0.24 nM) than DNA end binding. Comparison of Ku binding to several sequences over which Ku can accumulate revealed two classes of sequence. Sequences with similarity to NRE1 competed efficiently for NRE1 binding. Conversely, sequences lacking similarity to NRE1 competed poorly for Ku and were not recognized in the absence of DNA ends. Phosphorylation of glucocorticoid receptor (GR) fusion proteins by DNA-PK reflected Ku DNA-binding preferences and demonstrated that co-localization of GR with DNA-PK on DNA in cis was critical for efficient phosphorylation. Phosphorylation of the GR fusion protein by DNA-PK mapped to a single site, Ser-527. This site occurs adjacent the GR nuclear localization sequence between the DNA and ligand binding domains of GR, and thus its phosphorylation, if confirmed, has the potential to affect receptor function in vivo.
Asunto(s)
Antígenos Nucleares , ADN Helicasas , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Glucocorticoides/metabolismo , Factores de Transcripción/metabolismo , Animales , Unión Competitiva , Proteína Quinasa Activada por ADN , Autoantígeno Ku , Ratones , Mapeo Peptídico , Fosforilación , Ratas , Serina , Relación Estructura-ActividadRESUMEN
DNA-dependent protein kinase (DNA-PK) has been implicated in several nuclear processes including transcription, DNA replication, double-stranded DNA break repair, and V(D)J recombination. Linkage of kinase and substrate on DNA in cis is required for efficient phosphorylation. Recruitment of DNA-PK to DNA is by Ku autoantigen, a DNA-end-binding protein required for DNA-PK catalytic activity. Although Ku is known to translocate along naked DNA, how DNA-end binding by Ku might lead to DNA-PK-mediated phosphorylation of sequence-specific DNA-binding proteins in vivo has not been obvious. Here we report the identification of Ku as a transcription factor that recruits DNA-PK directly to specific DNA sequences. NRE1 (negative regulatory element 1) is a DNA sequence element (-394/ -381) in the long terminal repeat of mouse mammary tumour virus (MMTV) that is important for repressing inappropriate viral expression. We show that direct binding of Ku/DNA-PK to NRE1 represses glucocorticoid-induced MMTV transcription.