RESUMEN
Histone deacetylase 3 (HDAC3) is a crucial epigenetic modulator essential for various developmental and physiological functions. Although its dysfunction is increasingly recognized in abnormal phenotypes, to our knowledge, there have been no established reports of human diseases directly linked to HDAC3 dysfunction. Using trio exome sequencing and extensive phenotypic analysis, we correlated heterozygous de novo variants in HDAC3 with a neurodevelopmental disorder having variable clinical presentations, frequently associated with intellectual disability, developmental delay, epilepsy, and musculoskeletal abnormalities. In a cohort of six individuals, we identified missense variants in HDAC3 (c.277G>A [p.Asp93Asn], c.328G>A [p.Ala110Thr], c.601C>T [p.Pro201Ser], c. 797T>C [p.Leu266Ser], c.799G>A [p.Gly267Ser], and c.1075C>T [p.Arg359Cys]), all located in evolutionarily conserved sites and confirmed as de novo. Experimental studies identified defective deacetylation activity in the p.Asp93Asn, p.Pro201Ser, p.Leu266Ser, and p.Gly267Ser variants, positioned near the enzymatic pocket. In addition, proteomic analysis employing co-immunoprecipitation revealed that the disrupted interactions with molecules involved in the CoREST and NCoR complexes, particularly in the p.Ala110Thr variant, consist of a central pathogenic mechanism. Moreover, immunofluorescence analysis showed diminished nuclear to cytoplasmic fluorescence ratio in the p.Ala110Thr, p.Gly267Ser, and p.Arg359Cys variants, indicating impaired nuclear localization. Taken together, our study highlights that de novo missense variants in HDAC3 are associated with a broad spectrum of neurodevelopmental disorders, which emphasizes the complex role of HDAC3 in histone deacetylase activity, multi-protein complex interactions, and nuclear localization for proper physiological functions. These insights open new avenues for understanding the molecular mechanisms of HDAC3-related disorders and may inform future therapeutic strategies.
Asunto(s)
Epigénesis Genética , Histona Desacetilasas , Mutación Missense , Trastornos del Neurodesarrollo , Humanos , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Mutación Missense/genética , Trastornos del Neurodesarrollo/genética , Masculino , Femenino , Preescolar , Niño , Discapacidad Intelectual/genética , Secuenciación del Exoma , Adolescente , Discapacidades del Desarrollo/genética , Fenotipo , Lactante , Co-Represor 1 de Receptor Nuclear/genética , Co-Represor 1 de Receptor Nuclear/metabolismoRESUMEN
H1 linker histones are the most abundant chromatin-binding proteins1. In vitro studies indicate that their association with chromatin determines nucleosome spacing and enables arrays of nucleosomes to fold into more compact chromatin structures. However, the in vivo roles of H1 are poorly understood2. Here we show that the local density of H1 controls the balance of repressive and active chromatin domains by promoting genomic compaction. We generated a conditional triple-H1-knockout mouse strain and depleted H1 in haematopoietic cells. H1 depletion in T cells leads to de-repression of T cell activation genes, a process that mimics normal T cell activation. Comparison of chromatin structure in normal and H1-depleted CD8+ T cells reveals that H1-mediated chromatin compaction occurs primarily in regions of the genome containing higher than average levels of H1: the chromosome conformation capture (Hi-C) B compartment and regions of the Hi-C A compartment marked by PRC2. Reduction of H1 stoichiometry leads to decreased H3K27 methylation, increased H3K36 methylation, B-to-A-compartment shifting and an increase in interaction frequency between compartments. In vitro, H1 promotes PRC2-mediated H3K27 methylation and inhibits NSD2-mediated H3K36 methylation. Mechanistically, H1 mediates these opposite effects by promoting physical compaction of the chromatin substrate. Our results establish H1 as a critical regulator of gene silencing through localized control of chromatin compaction, 3D genome organization and the epigenetic landscape.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/genética , Epigénesis Genética , Histonas/metabolismo , Animales , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/genética , Cromatina/química , Cromatina/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Silenciador del Gen , Histonas/química , Activación de Linfocitos/genética , Masculino , Metilación , Ratones , Ratones NoqueadosRESUMEN
As the process that silences gene expression ensues during development, the stage is set for the activity of Polycomb-repressive complex 2 (PRC2) to maintain these repressed gene profiles. PRC2 catalyzes a specific histone posttranslational modification (hPTM) that fosters chromatin compaction. PRC2 also facilitates the inheritance of this hPTM through its self-contained "write and read" activities, key to preserving cellular identity during cell division. As these changes in gene expression occur without changes in DNA sequence and are inherited, the process is epigenetic in scope. Mutants of mammalian PRC2 or of its histone substrate contribute to the cancer process and other diseases, and research into these aberrant pathways is yielding viable candidates for therapeutic targeting. The effectiveness of PRC2 hinges on its being recruited to the proper chromatin sites; however, resolving the determinants to this process in the mammalian case was not straightforward and thus piqued the interest of many in the field. Here, we chronicle the latest advances toward exposing mammalian PRC2 and its high maintenance.
Asunto(s)
Epigénesis Genética , Regulación de la Expresión Génica , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Animales , Cromatina/metabolismo , Humanos , Mutación , Neoplasias/genética , Neoplasias/fisiopatología , Transporte de Proteínas , Investigación/tendenciasRESUMEN
The histone methyltransferase activity of PRC2 is central to the formation of H3K27me3-decorated facultative heterochromatin and gene silencing. In addition, PRC2 has been shown to automethylate its core subunits, EZH1/EZH2 and SUZ12. Here, we identify the lysine residues at which EZH1/EZH2 are automethylated with EZH2-K510 and EZH2-K514 being the major such sites in vivo. Automethylated EZH2/PRC2 exhibits a higher level of histone methyltransferase activity and is required for attaining proper cellular levels of H3K27me3. While occurring independently of PRC2 recruitment to chromatin, automethylation promotes PRC2 accessibility to the histone H3 tail. Intriguingly, EZH2 automethylation is significantly reduced in diffuse intrinsic pontine glioma (DIPG) cells that carry a lysine-to-methionine substitution in histone H3 (H3K27M), but not in cells that carry either EZH2 or EED mutants that abrogate PRC2 allosteric activation, indicating that H3K27M impairs the intrinsic activity of PRC2. Our study demonstrates a PRC2 self-regulatory mechanism through its EZH1/2-mediated automethylation activity.
Asunto(s)
Glioma/enzimología , Glioma/genética , Histonas/metabolismo , Niño , Activación Enzimática , Silenciador del Gen , Histonas/genética , Humanos , Lisina/metabolismo , Metilación , Complejo Represivo Polycomb 2/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
PRC2 is a therapeutic target for several types of cancers currently undergoing clinical trials. Its activity is regulated by a positive feedback loop whereby its terminal enzymatic product, H3K27me3, is specifically recognized and bound by an aromatic cage present in its EED subunit. The ensuing allosteric activation of the complex stimulates H3K27me3 deposition on chromatin. Here we report a stepwise feedback mechanism entailing key residues within distinctive interfacing motifs of EZH2 or EED that are found to be mutated in cancers and/or Weaver syndrome. PRC2 harboring these EZH2 or EED mutants manifested little activity in vivo but, unexpectedly, exhibited similar chromatin association as wild-type PRC2, indicating an uncoupling of PRC2 activity and recruitment. With genetic and chemical tools, we demonstrated that targeting allosteric activation overrode the gain-of-function effect of EZH2Y646X oncogenic mutations. These results revealed critical implications for the regulation and biology of PRC2 and a vulnerability in tackling PRC2-addicted cancers.
Asunto(s)
Regulación Alostérica/fisiología , Cromatina/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Anomalías Múltiples/metabolismo , Línea Celular Tumoral , Hipotiroidismo Congénito/metabolismo , Anomalías Craneofaciales/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Deformidades Congénitas de la Mano/metabolismo , Histonas/metabolismo , Humanos , Neoplasias/metabolismoRESUMEN
The maintenance of gene expression patterns during metazoan development is achieved, in part, by the actions of polycomb repressive complex 2 (PRC2). PRC2 catalyzes mono-, di-, and trimethylation of histone H3 at lysine 27 (H3K27), with H3K27me2/3 being strongly associated with silenced genes. We demonstrate that EZH1 and EZH2, the two mutually exclusive catalytic subunits of PRC2, are differentially activated by various mechanisms. Whereas both PRC2-EZH1 and PRC2-EZH2 are able to catalyze mono- and dimethylation, only PRC2-EZH2 is strongly activated by allosteric modulators and specific chromatin substrates to catalyze trimethylation of H3K27 in mouse embryonic stem cells (mESCs). However, we also show that a PRC2-associated protein, AEBP2, can stimulate the activity of both complexes through a mechanism independent of and additive to allosteric activation. These results have strong implications regarding the cellular requirements for and the accompanying adjustments in PRC2 activity, given the differential expression of EZH1 and EZH2 upon cellular differentiation.
Asunto(s)
Complejo Represivo Polycomb 2/metabolismo , Animales , Catálisis , Línea Celular , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación , RatonesRESUMEN
Polycomb repressive complex 2 (PRC2) maintains gene silencing by catalyzing methylation of histone H3 at lysine 27 (H3K27me2/3) within chromatin. By designing a system whereby PRC2-mediated repressive domains were collapsed and then reconstructed in an inducible fashion in vivo, a two-step mechanism of H3K27me2/3 domain formation became evident. First, PRC2 is stably recruited by the actions of JARID2 and MTF2 to a limited number of spatially interacting "nucleation sites," creating H3K27me3-forming Polycomb foci within the nucleus. Second, PRC2 is allosterically activated via its binding to H3K27me3 and rapidly spreads H3K27me2/3 both in cis and in far-cis via long-range contacts. As PRC2 proceeds further from the nucleation sites, its stability on chromatin decreases such that domains of H3K27me3 remain proximal, and those of H3K27me2 distal, to the nucleation sites. This study demonstrates the principles of de novo establishment of PRC2-mediated repressive domains across the genome.
Asunto(s)
Complejo Represivo Polycomb 2/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Animales , Cromatina/metabolismo , Silenciador del Gen , Código de Histonas , Histonas/metabolismo , Lisina/metabolismo , Metilación , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
BACKGROUND: We investigated the role of tumor cell-intrinsic PD-L1 signaling in the epithelial-mesenchymal transition (EMT) in non-small-cell lung cancer (NSCLC) and the role of EMT as a predictive biomarker for immune checkpoint inhibitor (ICI) therapy. METHODS: PD-L1-overexpressing or PD-L1-knockdown NSCLC cells underwent RNA-seq and EMT phenotype assessment. Mouse lung cancer LLC cells were injected into nude mice. Two cohorts of patients with NSCLC undergoing ICI therapy were analyzed. RESULTS: RNA-seq showed that EMT pathways were enriched in PD-L1-high NSCLC cells. EMT was enhanced by PD-L1 in NSCLC cells, which was mediated by transforming growth factor-ß (TGFß). PD-L1 promoted the activation of p38-MAPK by binding to and inhibiting the protein phosphatase PPM1B, thereby increasing the TGFß production. Tumor growth and metastasis increased in nude mice injected with PD-L1-overexpressing LLC cells. In the ICI cohort, EMT signature was higher in patients with progressive disease than in those with responses, and EMT was significantly associated with poor survival in PD-L1-high NSCLC. In PD-L1-high NSCLC, EMT was associated with increased M2-macrophage and regulatory T-cell infiltrations and decreased cytotoxic T-cell infiltration. CONCLUSIONS: Tumor cell-intrinsic PD-L1 function contributes to NSCLC progression by promoting EMT. EMT may predict an unfavorable outcome after ICI therapy in PD-L1-high NSCLC.
Asunto(s)
Antígeno B7-H1 , Carcinoma de Pulmón de Células no Pequeñas , Transición Epitelial-Mesenquimal , Inhibidores de Puntos de Control Inmunológico , Neoplasias Pulmonares , Ratones Desnudos , Transducción de Señal , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/inmunología , Animales , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Ratones , Línea Celular Tumoral , Factor de Crecimiento Transformador beta/metabolismo , FemeninoRESUMEN
PURPOSE: This study aims to evaluate the association between body image dissatisfaction and quality of life and depression among patients after hematopoietic stem cell transplantation (HSCT). METHODS: We conducted a cross-sectional survey at three university-based HSCT outpatient clinics and the Korea Blood Cancer Association. We assessed the body image using the body image scale; quality of life and depression were measured using the World Health Organization Quality of Life-BREF and the Patient Health Questionnaire 9, respectively. Univariate and multivariate linear regression models were used to find an association between body image, quality of life, and depression. RESULTS: Among 163 study participants, 71.8% were male, and the mean age of the participants was 48.3 (SD = 11.2). Over 70% of the participants reported that they felt less physically and sexually attractive due to HSCT, and 39.3% of the patients were dissatisfied with their body image. In fully adjusted models, patients with dissatisfied body image had significantly poorer quality of life (- 13.68, 95% confidence interval [CI] = - 18.16, - 9.21). Moreover, patients with body image dissatisfaction were 8.59 times (95% CI = 3.79, 19.48) more likely to have depressive symptoms than patients without it. CONCLUSION: The majority of HSCT patients experienced body image dissatisfaction, which was significantly associated with poor quality of life and depression. It would be essential to evaluate body image after HSCT and provide appropriate interventions for preventing further psychological consequences.
Asunto(s)
Insatisfacción Corporal/psicología , Depresión/psicología , Trasplante de Células Madre Hematopoyéticas/psicología , Calidad de Vida/psicología , Acondicionamiento Pretrasplante/psicología , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Satisfacción del Paciente , Encuestas y CuestionariosRESUMEN
Chromatin remodelers have been implicated in the regulation of histone-modifying complexes. However, the underlying mechanism remains poorly understood. The Rpd3S histone deacetylase complex is recruited by elongating RNA polymerase II to remove histone acetylation at coding regions in a manner that is dependent on methylation of lysine 36 on histone 3 (H3K36me), and Rpd3S prefers dinucleosomes. Here, we show that the binding of Rpd3S to dinucleosomes and its catalytic activity are sensitive to the length of nucleosomal linker in a nonlinear fashion. Intriguingly, we found that H3K36me on one nucleosome stimulates Rpd3S to deacetylate the neighboring nucleosomes when those two nucleosomes are within an optimal distance. Finally, we demonstrate that chromatin remodelers enhance Rpd3S activity by altering nucleosomal spacing, suggesting that chromatin remodelers prime chromatin configuration to fine-tune subsequent histone modification reactions. This mechanism is important for accurate temporal control of chromatin dynamics during the transcription elongation cycle.
Asunto(s)
Cromatina/metabolismo , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilación , Animales , Western Blotting , Línea Celular , Cromatina/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Células HeLa , Histona Desacetilasas/genética , Humanos , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Modelos Genéticos , Nucleosomas/genética , Unión Proteica , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
Recognition of histone post-translational modifications is pivotal for directing chromatin-modifying enzymes to specific genomic regions and regulating their activities. Emerging evidence suggests that other structural features of nucleosomes also contribute to precise targeting of downstream chromatin complexes, such as linker DNA, the histone globular domain, and nucleosome spacing. However, how chromatin complexes coordinate individual interactions to achieve high affinity and specificity remains unclear. The Rpd3S histone deacetylase utilizes the chromodomain-containing Eaf3 subunit and the PHD domain-containing Rco1 subunit to recognize nucleosomes that are methylated at lysine 36 of histone H3 (H3K36me). We showed previously that the binding of Eaf3 to H3K36me can be allosterically activated by Rco1. To investigate how this chromatin recognition module is regulated in the context of the Rpd3S complex, we first determined the subunit interaction network of Rpd3S. Interestingly, we found that Rpd3S contains two copies of the essential subunit Rco1, and both copies of Rco1 are required for full functionality of Rpd3S. Our functional dissection of Rco1 revealed that besides its known chromatin-recognition interfaces, other regions of Rco1 are also critical for Rpd3S to recognize its nucleosomal substrates and functionin vivo. This unexpected result uncovered an important and understudied aspect of chromatin recognition. It suggests that precisely reading modified chromatin may not only need the combined actions of reader domains but also require an internal signaling circuit that coordinates the individual actions in a productive way.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Histona Desacetilasas/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Cromosómicas no Histona/genética , Histona Desacetilasas/genética , Histonas/metabolismo , Metilación , Datos de Secuencia Molecular , Unión Proteica , Procesamiento Proteico-Postraduccional , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The Rpd3S histone deacetylase complex represses cryptic transcription initiation within coding regions by maintaining the hypo-acetylated state of transcribed chromatin. Rpd3S recognizes methylation of histone H3 at lysine 36 (H3K36me), which is required for its deacetylation activity. Rpd3S is able to function over a wide range of H3K36me levels, making this a unique system to examine how chromatin regulators tolerate the reduction of their recognition signal. Here, we demonstrated that Rpd3S makes histone modification-independent contacts with nucleosomes, and that Rpd3S prefers di-nucleosome templates since two binding surfaces can be readily accessed simultaneously. Importantly, this multivalent mode of interaction across two linked nucleosomes allows Rpd3S to tolerate a two-fold intramolecular reduction of H3K36me. Our data suggest that chromatin regulators utilize an intrinsic di-nucleosome-recognition mechanism to prevent compromised function when their primary recognition modifications are diluted.
Asunto(s)
Histona Desacetilasas/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Animales , Metilación , XenopusRESUMEN
The correct removal of 5'-flap structures by Rad27 and Dna2 during Okazaki fragment maturation is crucial for the stable maintenance of genetic materials and cell viability. In this study, we identified RAD52, a key recombination protein, as a multicopy suppressor of dna2-K1080E, a lethal helicase-negative mutant allele of DNA2 in yeasts. In contrast, the overexpression of Rad51, which works conjointly with Rad52 in canonical homologous recombination, failed to suppress the growth defect of the dna2-K1080E mutation, indicating that Rad52 plays a unique and distinct role in Okazaki fragment metabolism. We found that the recombination-defective Rad52-QDDD/AAAA mutant did not rescue dna2-K1080E, suggesting that Rad52-mediated recombination is important for suppression. The Rad52-mediated enzymatic stimulation of Dna2 or Rad27 is not a direct cause of suppression observed in vivo, as both Rad52 and Rad52-QDDD/AAAA proteins stimulated the endonuclease activities of both Dna2 and Rad27 to a similar extent. The recombination mediator activity of Rad52 was dispensable for the suppression, whereas both the DNA annealing activity and its ability to interact with Rad59 were essential. In addition, we found that several cohesion establishment factors, including Rsc2 and Elg1, were required for the Rad52-dependent suppression of dna2-K1080E. Our findings suggest a novel Rad52/Rad59-dependent, but Rad51-independent recombination pathway that could ultimately lead to the removal of faulty flaps in conjunction with cohesion establishment factors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Recombinación Homóloga , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ADN/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/genética , Immunoblotting , Mutación , Proteína Recombinante y Reparadora de ADN Rad52/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The removal of initiating primers from the 5'-ends of each Okazaki fragment, required for the generation of contiguous daughter strands, can be catalyzed by the combined action of DNA polymerase δ and Fen1. When the flaps generated by displacement of DNA synthesis activity of polymerase δ become long enough to bind replication protein A or form hairpin structures, the helicase/endonuclease enzyme, Dna2, becomes critical because of its ability to remove replication protein A-coated or secondary structure flaps. In this study, we show that the N-terminal 45-kDa domain of Dna2 binds hairpin structures, allowing the enzyme to target secondary structure flap DNA. We found that this activity was essential for the efficient removal of hairpin flaps by the endonuclease activity of Dna2 with the aid of its helicase activity. Thus, the efficient removal of hairpin structure flaps requires the coordinated action of all three functional domains of Dna2. We also found that deletion of the N-terminal 45-kDa domain of Dna2 led to a partial loss of the intra-S-phase checkpoint function and an increased rate of homologous recombination in yeast. We discuss the potential roles of the N-terminal domain of Dna2 in the maintenance of genomic stability.
Asunto(s)
ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Roturas del ADN de Doble Cadena , ADN Helicasas/química , Desoxirribonucleasas/química , Marcadores Genéticos , Genoma , Conformación de Ácido Nucleico , Oligonucleótidos/genética , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Recombinación Genética , Fase SRESUMEN
Saccharomyces cerevisiae Mph1 is a 3-5' DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3' to 5' directionality, Mph1 displaced 3'-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5'-flap strand more efficiently than the 3' flap strand from double-flap substrates, which is not expected for a 3-5' DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5' single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , ADN/química , ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicación del ADN , ADN de Cadena Simple/metabolismo , Conformación de Ácido NucleicoRESUMEN
Dna2 and Rad27 (yeast Fen1) are the two endonucleases critical for Okazaki fragment processing during lagging strand DNA synthesis that have been shown to interact genetically and physically. In this study, we addressed the functional consequences of these interactions by examining whether purified Rad27 of Saccharomyces cerevisiae affects the enzymatic activity of Dna2 and vice versa. For this purpose, we constructed Rad27DA (catalytically defective enzyme with an Asp to Ala substitution at amino acid 179) and found that it significantly stimulated the endonuclease activity of wild type Dna2, but failed to do so with Dna2Δ405N that lacks the N-terminal 405 amino acids. This was an unexpected finding because dna2Δ405N cells were still partially suppressed by overexpression of rad27DA in vivo. Further analyses revealed that Rad27 is a trans-autostimulatory enzyme, providing an explanation why overexpression of Rad27, regardless of its catalytic activity, suppressed dna2 mutants as long as an endogenous wild type Rad27 is available. We found that the C-terminal 16-amino acid fragment of Rad27, a highly polybasic region due to the presence of multiple positively charged lysine and arginine residues, was sufficient and necessary for the stimulation of both Rad27 and Dna2. Our findings provide further insight into how Dna2 and Rad27 jointly affect the processing of Okazaki fragments in eukaryotes.
Asunto(s)
ADN Helicasas/metabolismo , ADN/genética , Regulación hacia Abajo , Endonucleasas de ADN Solapado/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , ADN Helicasas/genética , Replicación del ADN , Endonucleasas de ADN Solapado/genética , Regulación Fúngica de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Unión Proteica , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
DNA replication is a primary mechanism for maintaining genome integrity, but it serves this purpose best by cooperating with other proteins involved in DNA repair and recombination. Unlike leading strand synthesis, lagging strand synthesis has a greater risk of faulty replication for several reasons: First, a significant part of DNA is synthesized by polymerase alpha, which lacks a proofreading function. Second, a great number of Okazaki fragments are synthesized, processed and ligated per cell division. Third, the principal mechanism of Okazaki fragment processing is via generation of flaps, which have the potential to form a variety of structures in their sequence context. Finally, many proteins for the lagging strand interact with factors involved in repair and recombination. Thus, lagging strand DNA synthesis could be the best example of a converging place of both replication and repair proteins. To achieve the risky task with extraordinary fidelity, Okazaki fragment processing may depend on multiple layers of redundant, but connected pathways. An essential Dna2 endonuclease/helicase plays a pivotal role in processing common structural intermediates that occur during diverse DNA metabolisms (e.g. lagging strand synthesis and telomere maintenance). Many roles of Dna2 suggest that the preemptive removal of long or structured flaps ultimately contributes to genome maintenance in eukaryotes. In this review, we describe the function of Dna2 in Okazaki fragment processing, and discuss its role in the maintenance of genome integrity with an emphasis on its functional interactions with other factors required for genome maintenance.
Asunto(s)
ADN Helicasas/fisiología , ADN/metabolismo , Células Eucariotas/fisiología , Inestabilidad Genómica , Animales , ADN/genética , Reparación del ADN , Replicación del ADN , HumanosRESUMEN
Genetic variants in chromatin regulators are frequently found in neurodevelopmental disorders, but their effect in disease etiology is rarely determined. Here, we uncover and functionally define pathogenic variants in the chromatin modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 methyltransferases of the PRC2 complex. Unlike the other PRC2 subunits, which are involved in cancers and developmental syndromes, the implication of EZH1 in human development and disease is largely unknown. Using cellular and biochemical studies, we demonstrate that recessive variants impair EZH1 expression causing loss of function effects, while dominant variants are missense mutations that affect evolutionarily conserved aminoacids, likely impacting EZH1 structure or function. Accordingly, we found increased methyltransferase activity leading to gain of function of two EZH1 missense variants. Furthermore, we show that EZH1 is necessary and sufficient for differentiation of neural progenitor cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell-derived neural cultures and forebrain organoids, we demonstrate that EZH1 variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorders.
Asunto(s)
Trastornos del Neurodesarrollo , Neurogénesis , Complejo Represivo Polycomb 2 , Animales , Embrión de Pollo , Humanos , Diferenciación Celular/genética , Núcleo Celular , Cromatina/genética , Metiltransferasas , Trastornos del Neurodesarrollo/genética , Neurogénesis/genética , Complejo Represivo Polycomb 2/genéticaRESUMEN
The two endonucleases, Rad27 (yeast Fen1) and Dna2, jointly participate in the processing of Okazaki fragments in yeasts. Mus81-Mms4 is a structure-specific endonuclease that can resolve stalled replication forks as well as toxic recombination intermediates. In this study, we show that Mus81-Mms4 can suppress dna2 mutational defects by virtue of its functional and physical interaction with Rad27. Mus81-Mms4 stimulated Rad27 activity significantly, accounting for its ability to restore the growth defects caused by the dna2 mutation. Interestingly, Rad27 stimulated the rate of Mus81-Mms4 catalyzed cleavage of various substrates, including regressed replication fork substrates. The ability of Rad27 to stimulate Mus81-Mms4 did not depend on the catalytic activity of Rad27, but required the C-terminal 64 amino acid fragment of Rad27. This indicates that the stimulation was mediated by a specific protein-protein interaction between the two proteins. Our in vitro data indicate that Mus81-Mms4 and Rad27 act together during DNA replication and resolve various structures that can impede normal DNA replication. This conclusion was further strengthened by the fact that rad27 mus81 or rad27 mms4 double mutants were synergistically lethal. We discuss the significance of the interactions between Rad27, Dna2 and Mus81-Mms4 in context of DNA replication.