RESUMEN
Histone post-translational modifications are extensively studied for their role in regulating gene transcription and cellular environmental adaptation. Research into these modifications has recently begun in the protozoan parasite Giardia lamblia, focusing on histone-modifying enzymes and specific post-translational changes. In the transformation from the trophozoite to the cyst form in the life cycle of this parasite, significant morphological and genetic alterations occur, culminating in the synthesis of cyst wall proteins responsible for forming the protective cyst wall. It has been previously demonstrated that histone deacetylation is required during encystation and that the enzyme lysine methyltransferase 1 is involved in the upregulation of encystation. Our study aims to extend the analysis to lysine methyltransferase 2 (GlKMT2) function. For this, two constructs were generated: one that downregulate the expression of GLKMT2 via antisense (glkmt2-as transgenic cells) and the other overexpressing GlKMT2 (glkmt2-ha transgenic cells). We found that the glktm2-as transgenic cells showed an arrest in progress at the late encystation stage. Consequently, the number of cysts produced was lower than that of the control cells. On the other hand, we found that the overexpression of GlKMT2 acts as a negative mutant of the enzyme. In this way, these glktm2-ha transgenic cells showed the same behavior during growth and encystation as glkmt2-as transgenic cells. This interplay between different enzymes acting during encystation reveals the complex process behind the differentiation of the parasite. Understanding how these enzymes play their role during the encystation of the parasite would allow the design of inhibitors to control the parasite.
Asunto(s)
Giardia lamblia , Enquistamiento de Parásito , Proteínas Protozoarias , Giardia lamblia/enzimología , Giardia lamblia/genética , Giardia lamblia/crecimiento & desarrollo , Giardia lamblia/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Enquistamiento de Parásito/fisiología , Enquistamiento de Parásito/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Procesamiento Proteico-PostraduccionalRESUMEN
NSD3 (nuclear receptor-binding SET domain protein 3) is a member of the NSD histone methyltransferase family of proteins. In recent years, it has been identified as a potential oncogene in certain types of cancer. The NSD3 gene encodes three isoforms, the long version (NSD3L), a short version (NSD3S) and the WHISTLE isoforms. Importantly, the NSD3S isoform corresponds to the N-terminal region of the full-length protein, lacking the methyltransferase domain. The chromosomal location of NSD3 is frequently amplified across cancer types, such as breast, lung, and colon, among others. Recently, this amplification has been correlated to a chromothripsis event, that could explain the different NSD3 alterations found in cancer. The fusion proteins containing NSD3 have also been reported in leukemia (NSD3-NUP98), and in NUT (nuclear protein of the testis) midline carcinoma (NSD3-NUT). Its role as an oncogene has been described by modulating different cancer pathways through its methyltransferase activity, or the short isoform of the protein, through protein interactions. Specifically, in this review we will focus on the functions that have been characterized as methyltransferase dependent, and those that have been correlated with the expression of the NSD3S isoform. There is evidence that both the NSD3L and NSD3S isoforms are relevant for cancer progression, establishing NSD3 as a therapeutic target. However, further functional studies are needed to differentiate NSD3 oncogenic activity as dependent or independent of the catalytic domain of the protein, as well as the contribution of each isoform and its clinical significance in cancer progression.
Asunto(s)
N-Metiltransferasa de Histona-Lisina , Neoplasias , Proteínas Nucleares , Humanos , Masculino , Carcinoma/enzimología , Leucemia/enzimología , Oncogenes , Isoformas de Proteínas/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias/enzimología , Neoplasias/patologíaRESUMEN
Psychostimulants regulate behavioral responses in zebrafish via epigenetic mechanisms. We have previously shown that DNA methylation and histone deacetylase (HDAC) inhibition abolish nicotine-induced conditioned place preference (CPP) but little is known about the role of histone methylation in addictive-like behaviors. To assess the influence of histone methylation on nicotine-CPP, zebrafish were treated with a histone (H3) lysine-9 (K9) dimethyltransferase G9a/GLP inhibitor, BIX-01294 (BIX), which was administered before conditioning sessions. We observed a dual effect of the inhibitor BIX: at high doses inhibited while at low doses potentiated nicotine reward. Transcriptional expression of α6 and α7 subunits of the nicotinic acetylcholine receptor and of G9a, DNA methyl transferase-3, and HDAC-1 was upregulated in zebrafish with positive scores for nicotine-CPP. Changes in relative levels of these mRNA molecules reflected the effects of BIX on nicotine reward. BIX treatment per sé did not affect transcriptional levels of epigenetic enzymes that regulate trimethylation or demethylation of H3. BIX reduced H3K9me2 protein levels in a dose-dependent manner in key structures of the reward pathway. Thus, our findings indicated that different doses of BIX differentially affect nicotine CPP via strong or weak inhibition of G9a/GLP activity. Additionally, we found that the lysine demethylase inhibitor daminozide abolished nicotine-CPP and drug seeking. Our data demonstrate that H3 methylation catalyzed by G9a/GLP is involved in nicotine-CPP induction. Dimethylation of K9 at H3 is an important epigenetic modification that should be considered as a potential therapeutic target to treat nicotine reward and perhaps other drug addictions.
Asunto(s)
N-Metiltransferasa de Histona-Lisina , Nicotina , Pez Cebra , Animales , Nicotina/farmacología , N-Metiltransferasa de Histona-Lisina/metabolismo , Azepinas/farmacología , Histonas/metabolismo , Recompensa , Quinazolinas/farmacología , Receptores Nicotínicos/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , MasculinoRESUMEN
During the tumorigenic process, cancer cells may become overly dependent on the activity of backup cellular pathways for their survival, representing vulnerabilities that could be exploited as therapeutic targets. Certain molecular vulnerabilities manifest as a synthetic lethality relationship, and the identification and characterization of new synthetic lethal interactions may pave the way for the development of new therapeutic approaches for human cancer. Our goal was to investigate a possible synthetic lethal interaction between a member of the Chromodomain Helicase DNA binding proteins family (CHD4) and a member of the histone methyltransferases family (SETDB1) in the molecular context of a cell line (Hs578T) representing the triple negative breast cancer (TNBC), a subtype of breast cancer lacking validated molecular targets for treatment. Therefore, we employed the CRISPR-Cas9 gene editing tool to individually or simultaneously introduce indels in the genomic loci corresponding to the catalytic domains of SETDB1 and CHD4 in the Hs578T cell line. Our main findings included: a) introduction of indels in exon 22 of SETDB1 sensitized Hs578T to the action of the genotoxic chemotherapy doxorubicin; b) by sequentially introducing indels in exon 22 of SETDB1 and exon 23 of CHD4 and tracking the percentage of the remaining wild-type sequences in the mixed cell populations generated, we obtained evidence of the existence of a synthetic lethality interaction between these genes. Considering the lack of molecular targets in TNBC, our findings provided valuable insights for development of new therapeutic approaches not only for TNBC but also for other cancer types.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Humanos , Histona Metiltransferasas/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Ensamble y Desensamble de Cromatina/genética , Mutaciones Letales Sintéticas/genética , Línea Celular , Factores de Transcripción/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismoRESUMEN
Changes in epigenetic programming have been proposed as being key events in the initiation and progression of childhood cancers. HMT euchromatic histone lysine methyltransferase 2 (G9a, EHMT2), which is encoded by the G9a (Ehmt2) gene, as well as its related protein GLP, which is encoded by the GLP/Ehmt1 gene, participate in epigenetic regulation by contributing to a transcriptionally repressed chromatin state. G9a/GLP activation has been reported in several cancer types. Herein, we evaluated the role of G9a in two solid pediatric tumors: neuroblastoma (NB) and Ewing sarcoma (ES). Our results show that G9a/Ehmt2 and GLP/Ehmt1 expression is higher in tumors with poorer prognosis, including St4 International Neuroblastoma Staging System (INSS) stage, MYCN amplified NB, and metastatic ES. Importantly, higher G9a and GLP levels were associated with shorter patient overall survival (OS) in both NB and ES. Moreover, pharmacological inhibition of G9a/GLP reduced cell viability in NB and ES cells. These findings suggest that G9a and GLP are associated with more aggressive NB and ES tumors and should be further investigated as being epigenetic targets in pediatric solid cancers.
Asunto(s)
Neuroblastoma , Sarcoma de Ewing , Niño , Humanos , Supervivencia Celular/genética , Epigénesis Genética , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , Histona Metiltransferasas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Neuroblastoma/genética , Sarcoma de Ewing/genéticaRESUMEN
MLL3, also known as KMT2C, is a lysine mono-methyltransferase in charge of the writing of an epigenetic mark on lysine 4 from histone 3. The catalytic site of MLL3 is composed of four tyrosines, namely, Y44, Y69, Y128, and Y130. Tyrosine residues are highly conserved among lysine methyltransferases' catalytic sites, although their complete function is still unclear. The exploration of how modifications on these residues from the enzymatic machinery impact the enzymatic activity of MLL3 could shed light transversally into the inner functioning of enzymes with similar characteristics. Through the use of QMMM calculations, we focus on the effect of the mutation of each tyrosine from the catalytic site on the enzymatic activity and the product specificity in the current study. While we found that the mutations of Y44 and Y128 by phenylalanine inactivated the enzyme, the mutation of Y128 by alanine reactivated the enzymatic activity of MLL3. Moreover, according to our models, the Y128A mutant was even found to be capable of di- and tri-methylate lysine 4 from histone 3, what would represent a gain of function mutation, and could be responsible for the development of diseases. Finally, we were able to establish the inactivation mechanism, which involved the use of Y130 as a water occlusion structure, whose conformation, once perturbed by its mutation or Y128 mutant, allows the access of water molecules that sequester the electron pair from lysine 4 avoiding its methylation process and, thus, increasing the barrier height.
Asunto(s)
N-Metiltransferasa de Histona-Lisina , Histonas , Alanina/genética , Sitios de Unión , Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Metilación , Fenilalanina/metabolismo , Tirosina/metabolismo , Agua/metabolismoRESUMEN
Oncogene-induced senescence (OIS) is a stable cell cycle arrest that occurs in normal cells upon oncogene activation. Cells undergoing OIS express a wide variety of secreted factors that affect the senescent microenvironment termed the senescence-associated secretory phenotype (SASP), which is beneficial or detrimental in a context-dependent manner. OIS cells are also characterized by marked epigenetic changes. We globally assessed histone modifications of OIS cells and discovered an increase in the active histone marks H3K79me2/3. The H3K79 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) was necessary and sufficient for increased H3K79me2/3 occupancy at the IL1A gene locus, but not other SASP genes, and was downstream of STING. Modulating DOT1L expression did not affect the cell cycle arrest. Together, our studies establish DOT1L as an epigenetic regulator of the SASP, whose expression is uncoupled from the senescence-associated cell cycle arrest, providing a potential strategy to inhibit the negative side effects of senescence while maintaining the beneficial inhibition of proliferation.
Asunto(s)
Senescencia Celular , Metilación de ADN , Epigénesis Genética , Fibroblastos/enzimología , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Interleucina-1alfa/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Puntos de Control del Ciclo Celular , Proliferación Celular , Femenino , Células HEK293 , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Humanos , Interleucina-1alfa/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Microscopía Fluorescente , Papiloma/inducido químicamente , Papiloma/genética , Papiloma/metabolismo , Papiloma/patología , Fenotipo , Vías Secretoras , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Acetato de TetradecanoilforbolRESUMEN
Inhibition of the H3K79 histone methyltransferase DOT1L has exhibited encouraging preclinical and early clinical activity in KMT2A (MLL)-rearranged leukemia, supporting the development of combinatorial therapies. Here, we investigated two novel combinations: dual inhibition of the histone methyltransferases DOT1L and EZH2, and the combination with a protein synthesis inhibitor. EZH2 is the catalytic subunit in the polycomb repressive complex 2 (PRC2), and inhibition of EZH2 has been reported to have preclinical activity in KMT2A-r leukemia. When combined with DOT1L inhibition, however, we observed both synergistic and antagonistic effects. Interestingly, antagonistic effects were not due to PRC2-mediated de-repression of HOXA9. HOXA cluster genes are key canonical targets of both KMT2A and the PRC2 complex. The independence of the HOXA cluster from PRC2 repression in KMT2A-r leukemia thus affords important insights into leukemia biology. Further studies revealed that EZH2 inhibition counteracted the effect of DOT1L inhibition on ribosomal gene expression. We thus identified a previously unrecognized role of DOT1L in regulating protein production. Decreased translation was one of the earliest effects measurable after DOT1L inhibition and specific to KMT2A-rearranged cell lines. H3K79me2 chromatin immunoprecipitation sequencing patterns over ribosomal genes were similar to those of the canonical KMT2A-fusion target genes in primary AML patient samples. The effects of DOT1L inhibition on ribosomal gene expression prompted us to evaluate the combination of EPZ5676 with a protein translation inhibitor. EPZ5676 was synergistic with the protein translation inhibitor homoharringtonine (omacetaxine), supporting further preclinical/clinical development of this combination. In summary, we discovered a novel epigenetic regulation of a metabolic process-protein synthesis-that plays a role in leukemogenesis and affords a combinatorial therapeutic opportunity.
Asunto(s)
Epigénesis Genética , Regulación Leucémica de la Expresión Génica , Reordenamiento Génico , Leucemia Mieloide Aguda/metabolismo , Biosíntesis de Proteínas , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteína de la Leucemia Mieloide-Linfoide/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/genética , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismoAsunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Proteínas de Unión al ADN/genética , Regulación Leucémica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Osteopontina/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Factores de Elongación Transcripcional/genética , Empalme Alternativo , Linfocitos B/metabolismo , Linfocitos B/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Niño , Preescolar , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Lactante , Masculino , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Osteopontina/antagonistas & inhibidores , Osteopontina/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Pronóstico , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factores de Riesgo , Factores de Elongación Transcripcional/metabolismoAsunto(s)
Metilación de ADN/inmunología , Epigénesis Genética/inmunología , Pénfigo/genética , Adulto , Estudios de Casos y Controles , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Haplotipos/inmunología , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteína del Locus del Complejo MDS1 y EV11/genética , Proteína del Locus del Complejo MDS1 y EV11/metabolismo , Masculino , Pénfigo/inmunología , Polimorfismo de Nucleótido Simple , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Approximately 5% of human T-cell leukemia virus type 1 (HTLV-1)-infected individuals will develop one of the HTLV-1-related diseases, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T-cell leukemia. However, the mechanisms responsible for the appearance of symptoms have not been fully clarified. It is believed that viral factors, host genetic and epigenetic mechanisms are implicated in this process. Studies have shown the involvement of histone methyltransferases in retrovirus infection, but no study observed their expression in HTLV-1-infected patients. Among them, euchromatic histone-lysine N-methyltransferase (EHMT)-1 and EHMT-2 were related to retroviral latency in HIV-1 infection. We investigated whether histone methyltransferases EHMT1 and EHMT2 exert any influence on HAM/TSP development by assessing their expression levels in CD4+ T-cells from HTLV-1-infected patients. CD4+ T-cells were immunomagnetically isolated from peripheral blood mononuclear cells of HTLV-1-infected or non-infected individuals and the expression levels of EHMT1 and EHMT2 were determined by RT-qPCR. We observed that EHMT2 was negatively regulated in HTLV-1 asymptomatic carriers compared to non-infected individuals. No difference was observed for EHMT1. These results suggest that EHMT2 downregulation in CD4+ T-cells may be linked to a protection mechanism against the development of HAM/TSP.
Asunto(s)
Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Virus Linfotrópico T Tipo 1 Humano , Paraparesia Espástica Tropical/genética , Paraparesia Espástica Tropical/virología , Adulto , Linfocitos T CD4-Positivos , Femenino , Regulación Enzimológica de la Expresión Génica , Predisposición Genética a la Enfermedad , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Masculino , Persona de Mediana EdadRESUMEN
In eukaryotes, histone lysine methylation is associated with either active or repressed chromatin states, depending on the status of methylation. Even when the amino-terminus of Giardia lamblia histones diverges from other organisms, these regions contain lysine residues that are potential targets for methylation. When we examined the role of the histone methyltransferase 1 (HMT1) in the regulation of the encystation process by giardial histone methyltransferase 1 (GlHMT1) overexpression or downregulation, we observed an increase or a decrease in cyst production, respectively, compared to wild-type trophozoites. A time-lapse analysis of encystation showed that overexpression of GlHMT1 induced an earlier and faster process than in wild-type cells together with an upregulation of mRNA expression of cyst wall proteins. Subcellular localization studies indicated that GlHMT1-hemaglutinin was mainly associated with the nuclear and perinuclear region in both growing and encysting parasites, in agreement with bioinformatics analyses showing that GlHMT-1 possesses nuclear localization signals in addition to the classical SU(var)3-9, Enhancer-of-Zeste, Trithorax (SET), and post-SET domains. Altogether, these findings suggest that the function of HMT1 is critical for the success and timing of the encystation process, and reinforce the idea that epigenetic marks are critical for cyst formation in G. lamblia.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Giardia lamblia/enzimología , Giardia lamblia/crecimiento & desarrollo , N-Metiltransferasa de Histona-Lisina/metabolismo , Modelos Moleculares , Enquistamiento de Parásito , Proteínas Protozoarias/metabolismo , Cristalografía por Rayos X , Minería de Datos , Bases de Datos de Ácidos Nucleicos , Bases de Datos de Proteínas , Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Lisina , Señales de Localización Nuclear , Filogenia , Conformación Proteica , Transporte de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
Herein, we used computer simulation to evaluate the free energy activation barriers of the first and second methyl transfer for native SET8 PKMT and its Y334F mutant. The results suggest that the origin of SET8 catalytic power is mainly due to electrostatic preorganization.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Catálisis , Dominio Catalítico , Codón , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Metilación , Modelos Moleculares , Mutación , Conformación Proteica , Electricidad Estática , Especificidad por SustratoRESUMEN
Some studies have linked age-related beneficial effects of exercise and epigenetic mechanisms. Although, the impact of treadmill exercise on histone acetylation, histone and DNA methylation marks in aged cortices yet remains poorly understood. Considering the role of frontal cortex on brain functions, we investigated the potential of different exercise protocols, single session and daily exercise, to modulate epigenetic marks, namely global H4 acetylation, histone methyltransferase activity (HMT H3K27) and levels of DNA methytransferase (DNMT1 and DNMT3b) in prefrontal cortices from 3 and 21-months aged Wistar rats. The animals were submitted to two treadmill exercise protocols, single session (20min) or daily moderate (20min/day during 14days). The daily exercise protocol induced an increased in histone H4 acetylation levels in prefrontal cortices of 21-months-old rats, without any effects in young adult group. DNMT3b levels were increased in aged cortices of animals submitted to single session of exercise. These results indicate that prefrontal cortex is susceptible to epigenetic changes in a protocol dependent-manner and that H4 acetylation levels and DNMT3b content changes might be linked at least in part to exercise-induced effects on brain functions.
Asunto(s)
Epigénesis Genética , Actividad Motora , Corteza Prefrontal/metabolismo , Acetilación , Animales , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Masculino , Corteza Prefrontal/enzimología , Ratas , Ratas Wistar , ADN Metiltransferasa 3BRESUMEN
With about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients.
Asunto(s)
Cromatina/genética , Virus de la Hepatitis B/fisiología , Hepatitis B/metabolismo , Histona Demetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Transactivadores/metabolismo , Acetilación , Línea Celular , Cromatina/química , Cromatina/metabolismo , Regulación Viral de la Expresión Génica , Células Hep G2 , Hepatitis B/virología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/metabolismo , Histonas/metabolismo , Humanos , Metilación , Regiones Promotoras Genéticas , Proteínas Reguladoras y Accesorias Virales , Replicación ViralRESUMEN
Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10(+) isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.
Asunto(s)
Empalme Alternativo , N-Metiltransferasa de Histona-Lisina/genética , Animales , Azepinas/farmacología , Encéfalo/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/metabolismo , Exones , Transferencia Resonante de Energía de Fluorescencia , Genes Reporteros , Células HeLa , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Metilación/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Neuronas/citología , Neuronas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Quinazolinas/farmacología , Interferencia de ARN , Precursores del ARN/metabolismo , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Tretinoina/farmacologíaRESUMEN
SET8, a member of the SET domain-containing methyl-transferase, has been implicated in various biological processes. In this study, SET8 was immunostained in 100 samples of gastric cancer tissues and semi-quantified using the HSCORE method to determine the predictive value of SET8 expression levels for gastric cancer outcome. The relationship between SET8 expression and the 5-year survival rate of gastric cancer patients was assessed. High expression of SET8 was associated with a shorter survival time in gastric cancer patients, and the level of SET8 expression was found to be an independent predictor of gastric cancer outcome (relative risk = 1.939; 95% confidence interval = 1.025-3.668; P = 0.042). Analysis of SET8 levels may help in the identification of patient subgroups that are at high risk for poor disease outcomes.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidad , Adulto , Anciano , Terapia Combinada , Femenino , Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Neoplasias Gástricas/patología , Neoplasias Gástricas/terapia , Carga TumoralRESUMEN
Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9), a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed.
Asunto(s)
Histonas/metabolismo , Lisina/metabolismo , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Células HeLa , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Humanos , Metilación , Proteína Metiltransferasas/metabolismo , Ribosomas/enzimologíaRESUMEN
In eukaryotic organisms, the replication of the DNA sequence and its organization into chromatin are critical to maintain genome integrity. Chromatin components, such as histone variants and histone post-translational modifications, along with the higher-order chromatin structure, impact several DNA metabolic processes, including replication, transcription, and repair. In this review we focus on lysine methylation and the relationships between this histone mark and chromatin replication. We first describe studies implicating lysine methylation in regulating early steps in the replication process. We then discuss chromatin reassembly following replication fork passage, where the incorporation of a combination of newly synthesized histones and parental histones can impact the inheritance of lysine methylation marks on the daughter strands. Finally, we elaborate on how the inheritance of lysine methylation can impact maintenance of the chromatin landscape, using heterochromatin as a model chromatin domain, and we discuss the potential mechanisms involved in this process.
Asunto(s)
Cromatina/metabolismo , Replicación del ADN , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Cromatina/genética , Humanos , MetilaciónRESUMEN
PURPOSE: To analyze the presence of various histone modifications in ejaculated human spermatozoa METHODS: In this prospective study, seminal ejaculates from 39 normozoospermic individuals were evaluated for semen analysis and the presence of histone modifications in isolated nuclei. RESULTS: We observed heterogeneous presence of histone methylation in normal mature human sperm. We observed that 12 to 30 % of the nuclei of normal sperm contain a heterogeneous distribution of the marks H3K4Me1, H3K9Me2, H3K4Me3, H3K79Me2, and H3K36Me3. Moreover, the presence of these marks is higher in the poor motile fraction of the ejaculate, which is associated with poor morphology and functional quality. In contrast, we did not observe histone acetylation (H3K4Ac and H4K5Ac) in normal or abnormal mature human sperm CONCLUSIONS: Defects in the process of spermatogenesis may alter the correct epigenetic programming in mature sperm. Further studies are required to evaluate the impact of these findings in human infertility.