RESUMEN
Dynamic regulation of histone methylation represents a fundamental epigenetic mechanism underlying eukaryotic gene regulation, yet little is known about how the catalytic activities of histone demethylases are regulated. Here, we identify and characterize NPAC/GLYR1 as an LSD2/KDM1b-specific cofactor that stimulates H3K4me1 and H3K4me2 demethylation. We determine the crystal structures of LSD2 alone and LSD2 in complex with the NPAC linker region in the absence or presence of histone H3 peptide, at resolutions of 2.9, 2.0, and 2.25 Å, respectively. These crystal structures and further biochemical characterization define a dodecapeptide of NPAC (residues 214-225) as the minimal functional unit for its cofactor activity and provide structural determinants and a molecular mechanism underlying the intrinsic cofactor activity of NPAC in stimulating LSD2-catalyzed H3K4 demethylation. Thus, these findings establish a model for how a cofactor directly regulates histone demethylation and will have a significant impact on our understanding of catalytic-activity-based epigenetic regulation.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Coenzimas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Modelos Moleculares , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/metabolismo , Oxidorreductasas de Alcohol/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Estabilidad de Enzimas , Células HeLa , Histonas/química , Humanos , Metilación , Datos de Secuencia Molecular , Péptidos/química , Unión Proteica , Especificidad por SustratoRESUMEN
Dynamic histone H3K4 methylation is an important epigenetic component of transcriptional regulation. However, most of our current understanding of this histone mark is confined to the regulation of transcriptional initiation. We now show that human LSD2/KDM1b/AOF1, the human homolog of LSD1, is an H3K4me1/2 demethylase that specifically regulates histone H3K4 methylation within intragenic regions of its target genes. Genome-wide mapping reveals that LSD2 associates predominantly with the gene bodies of actively transcribed genes, but is markedly absent from promoters. Depletion of endogenous LSD2 results in an increase of H3K4me2 as well as a decrease of H3K9me2 at LSD2-binding sites and a consequent dysregulation of target gene transcription. Furthermore, characterization of the LSD2 complex reveals that LSD2 forms active complexes with euchromatic histone methyltransferases G9a and NSD3 as well as cellular factors involved in transcription elongation. These data provide a possible molecular mechanism linking LSD2 to transcriptional regulation after initiation.
Asunto(s)
Histona Demetilasas/metabolismo , Histonas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Transcripción Genética/fisiología , Sitios de Unión , Células HeLa , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , Histona Demetilasas/genética , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Humanos , Metilación , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
ALKBH7 is the mitochondrial AlkB family member that is required for alkylation- and oxidation-induced programmed necrosis. In contrast to the protective role of other AlkB family members after suffering alkylation-induced DNA damage, ALKBH7 triggers the collapse of mitochondrial membrane potential and promotes cell death. Moreover, genetic ablation of mouse Alkbh7 dramatically increases body weight and fat mass. Here, we present crystal structures of human ALKBH7 in complex with Mn(II) and α-ketoglutarate at 1.35 Å or N-oxalylglycine at 2.0 Å resolution. ALKBH7 possesses the conserved double-stranded ß-helix fold that coordinates a catalytically active iron by a conserved HX(D/E) Xn H motif. Self-hydroxylation of Leu-110 was observed, indicating that ALKBH7 has the potential to catalyze hydroxylation of its substrate. Unlike other AlkB family members whose substrates are DNA or RNA, ALKBH7 is devoid of the "nucleotide recognition lid" which is essential for binding nucleobases, and thus exhibits a solvent-exposed active site; two loops between ß-strands ß6 and ß7 and between ß9 and ß10 create a special outer wall of the minor ß-sheet of the double-stranded ß-helix and form a negatively charged groove. These distinct features suggest that ALKBH7 may act on protein substrate rather than nucleic acids. Taken together, our findings provide a structural basis for understanding the distinct function of ALKBH7 in the AlkB family and offer a foundation for drug design in treating cell death-related diseases and metabolic diseases.
Asunto(s)
Proteínas Mitocondriales/química , Enzimas AlkB , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Grasas/metabolismo , Humanos , Ácidos Cetoglutáricos , Manganeso/química , Manganeso/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Necrosis , Multimerización de Proteína , Estructura Secundaria de Proteína , Alineación de Secuencia , Difracción de Rayos XRESUMEN
Gene transcription is critically influenced by chromatin structure and the modification status of histone tails. Methylation of lysine residues in histone tails is dynamically regulated by the opposing activities of histone methyltransferases and histone demethylases. Here we show that JARID1C/SMCX, a JmjC-domain-containing protein implicated in X-linked mental retardation and epilepsy, possesses H3K4 tri-demethylase activity and functions as a transcriptional repressor. An SMCX complex isolated from HeLa cells contains additional chromatin modifiers (the histone deacetylases HDAC1 and HDAC2, and the histone H3K9 methyltransferase G9a) and the transcriptional repressor REST, suggesting a direct role for SMCX in chromatin dynamics and REST-mediated repression. Chromatin immunoprecipitation reveals that SMCX and REST co-occupy the neuron-restrictive silencing elements in the promoters of a subset of REST target genes. RNA-interference-mediated depletion of SMCX derepresses several of these targets and simultaneously increases H3K4 trimethylation at the sodium channel type 2A (SCN2A) and synapsin I (SYN1) promoters. We propose that loss of SMCX activity impairs REST-mediated neuronal gene regulation, thereby contributing to SMCX-associated X-linked mental retardation.
Asunto(s)
Regulación de la Expresión Génica/genética , Histonas/metabolismo , Discapacidad Intelectual Ligada al Cromosoma X/genética , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Células HeLa , Histona Demetilasas , Humanos , Discapacidad Intelectual Ligada al Cromosoma X/metabolismo , Metilación , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Neuronas/metabolismo , Oxidorreductasas N-Desmetilantes , Regiones Promotoras Genéticas/genética , Proteínas/genética , Proteínas/aislamiento & purificación , Interferencia de ARN , Spodoptera , Especificidad por SustratoRESUMEN
E2F1 promotes DNA damage-induced apoptosis and the post-translational modifications of E2F1 play an important role in the regulation of E2F1-mediated cell death. Here, we found that Set9 and LSD1 regulate E2F1-mediated apoptosis upon DNA damage. Set9 methylates E2F1 at lysine 185, a conserved residue in the DNA-binding domain of E2F family proteins. The methylation of E2F1 by Set9 leads to the stabilization of E2F1 and up-regulation of its proapoptotic target genes p73 and Bim, and thereby induces E2F1-mediated apoptosis in response to genotoxic agents. We also found that LSD1 demethylates E2F1 at lysine 185 and reduces E2F1-mediated cell death. The identification of the methylation/demethylation of E2F1 by Set9/LSD1 suggests that E2F1 is dynamically regulated by epigenetic enzymes in response to DNA damage.
Asunto(s)
Daño del ADN , Factor de Transcripción E2F1/metabolismo , Secuencia de Aminoácidos , Muerte Celular/efectos de los fármacos , Línea Celular , Factor de Transcripción E2F1/química , Factor de Transcripción E2F1/genética , Técnicas de Silenciamiento del Gen , Histona Demetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Lisina/metabolismo , Metilación/efectos de los fármacos , Datos de Secuencia Molecular , Estabilidad Proteica/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Protein O-mannose beta1,2-N-acetyglucosaminyltransferase 1 (POMGnT1) is an enzyme involved in the synthesis of O-mannosyl glycans. Mutations of POMGnT1 in humans result in the muscle-eye-brain (MEB) disease. In this study, we have characterized a null mutation generated by gene trapping with a retroviral vector inserted into the second exon of the mouse POMGnT1 locus. Expression of POMGnT1 mRNA was abolished in mutant mice. Glycosylation of alpha-dystroglycan was also reduced. POMGnT1 mutant mice were viable with multiple developmental defects in muscle, eye, and brain, similar to the phenotypes observed in human MEB disease. The present study provides the first genetic animal model to further dissect the roles of POMGnT1 in MEB disease.
Asunto(s)
Encefalopatías/patología , Modelos Animales de Enfermedad , Oftalmopatías/patología , Fertilidad , Enfermedades Musculares/patología , N-Acetilglucosaminiltransferasas/genética , Animales , Encéfalo/patología , Cerebelo/patología , Distroglicanos/genética , Glicosilación , Homocigoto , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/patología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patología , Prosencéfalo/patologíaRESUMEN
Slit3 along with Slit1 and Slit2 comprise the Slit family of proteins. The latter two proteins are known to be involved in axon guidance and cell migration during animal development. However, little is know about the functions of Slit3. We created a Slit3-deficient mouse model from an OmniBank ES cell line with a Slit3 allele trapped by insertional mutagenesis to analyze the in vivo functions of this protein. In this model, congenital diaphragmatic hernia is the most obvious phenotype. Herniation was found to be caused by a defective central tendon (CT) of the diaphragm that remained fused with the liver. Electron microscopic analyses of the defective CT revealed disorganized collagen fibrils that failed to form tight collagen bundles. The hearts of Slit3-deficient mice have an enlarged right ventricle. In addition, 20% of homozygous mice also showed a range of kidney defects that include unilateral or bilateral agenesis of the kidney and ureter, or varying degrees of renal hypoplasia. Thus, we concluded that Slit3 is involved in the development of multiple organ systems that include the diaphragm and the kidney. Slit3-deficient mice represent a genetic animal model for physiological and pathological studies of congenital diaphragmatic hernia.
Asunto(s)
Cardiopatías Congénitas/genética , Hernias Diafragmáticas Congénitas , Riñón/anomalías , Proteínas de la Membrana/deficiencia , Animales , Secuencia de Bases , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica , Hernia Diafragmática/genética , Hibridación in Situ , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Tendones/anomalías , Uréter/anomalíasRESUMEN
Flexibility is an intrinsic property of proteins and essential for their biological functions. However, because of structural flexibility, obtaining high-quality crystals of proteins with heterogeneous conformations remain challenging. Here, we show a novel approach to immobilize traditional precipitants onto molecularly imprinted polymers (MIPs) to facilitate protein crystallization, especially for flexible proteins. By applying this method, high-quality crystals of the flexible N-terminus of human fragile X mental retardation protein are obtained, whose absence causes the most common inherited mental retardation. A novel KH domain and an intermolecular disulfide bond are discovered, and several types of dimers are found in solution, thus providing insights into the function of this protein. Furthermore, the precipitant-immobilized MIPs (piMIPs) successfully facilitate flexible protein crystal formation for five model proteins with increased diffraction resolution. This highlights the potential of piMIPs for the crystallization of flexible proteins.
Asunto(s)
Cristalización , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/química , Polímeros , Humanos , Estructura Terciaria de ProteínaRESUMEN
The gene expression profile of developing human hippocampus is of particular interest and importance to neurobiologists devoted to development of the human brain and related diseases. To gain further molecular insight into the developmental and functional characteristics, we analyzed the expression profile of active genes in developing human hippocampus. Expressed sequence tags (ESTs) were selected by sequencing randomly selected clones from an original 3'-directed cDNA library of 150-day human fetal hippocampus, and a digital expression profile of 946 known genes that could be divided into 16 categories was generated. We also used for comparison 14 other expression profiles of related human neural cells/tissues, including human adult hippocampus. To yield more confidence regarding differential expression, a method was applied to attach normalized expression data to genes with a low false-positive rate (<0.05). Finally, hierarchical cluster analysis was used to exhibit related gene expression patterns. Our results are in accordance with anatomical and physiological observations made during the developmental process of the human hippocampus. Furthermore, some novel findings appeared to be unique to our results. The abundant expression of genes for cell surface components and disease-related genes drew our attention. Twenty-four genes are significantly different from adult, and 13 genes might be developing hippocampus-specific candidate genes, including wnt2b and some Alzheimer's disease-related genes. Our results could provide useful information on the ontogeny, development, and function of cells in the human hippocampus at the molecular level and underscore the utility of large-scale, parallel gene expression analyses in the study of complex biological phenomena.