RESUMO
Structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1) is an epigenetic regulator that mediates gene expression silencing at targeted sites across the genome. Our current understanding of SMCHD1's molecular mechanism, and how substitutions within SMCHD1 lead to the diseases, facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS), are only emerging. Recent structural studies of its two component domains - the N-terminal ATPase and C-terminal SMC hinge - suggest that dimerization of each domain plays a central role in SMCHD1 function. Here, using biophysical techniques, we demonstrate that the SMCHD1 ATPase undergoes dimerization in a process that is dependent on both the N-terminal UBL (Ubiquitin-like) domain and ATP binding. We show that neither the dimerization event, nor the presence of a C-terminal extension past the transducer domain, affect SMCHD1's in vitro catalytic activity as the rate of ATP turnover remains comparable to the monomeric protein. We further examined the functional importance of the N-terminal UBL domain in cells, revealing that its targeted deletion disrupts the localization of full-length SMCHD1 to chromatin. These findings implicate UBL-mediated SMCHD1 dimerization as a crucial step for chromatin interaction, and thereby for promoting SMCHD1-mediated gene silencing.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Multimerização Proteica , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Células HEK293 , Humanos , Immunoblotting , Microscopia de Fluorescência , Mutação , Ligação Proteica , Domínios Proteicos , Espalhamento a Baixo Ângulo , Especificidade por Substrato , Ubiquitina/química , Ubiquitina/metabolismo , Difração de Raios XRESUMO
The DNA methylation epigenetic signature is a key determinant during development. Rules governing its establishment and maintenance remain elusive especially at repetitive sequences, which account for the majority of methylated CGs. DNA methylation is altered in a number of diseases including those linked to mutations in factors that modify chromatin. Among them, SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain Containing 1) has been of major interest following identification of germline mutations in Facio-Scapulo-Humeral Dystrophy (FSHD) and in an unrelated developmental disorder, Bosma Arhinia Microphthalmia Syndrome (BAMS). By investigating why germline SMCHD1 mutations lead to these two different diseases, we uncovered a role for this factor in de novo methylation at the pluripotent stage. SMCHD1 is required for the dynamic methylation of the D4Z4 macrosatellite upon reprogramming but seems dispensable for methylation maintenance. We find that FSHD and BAMS patient's cells carrying SMCHD1 mutations are both permissive for DUX4 expression, a transcription factor whose regulation has been proposed as the main trigger for FSHD. These findings open new questions as to what is the true aetiology for FSHD, the epigenetic events associated with the disease thus calling the current model into question and opening new perspectives for understanding repetitive DNA sequences regulation.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Metilação de DNA , Proteínas de Homeodomínio/genética , Repetições de Microssatélites/genética , Células Cultivadas , Reprogramação Celular/genética , Atresia das Cóanas/genética , Atresia das Cóanas/metabolismo , Metilação de DNA/genética , Epigênese Genética/genética , Regulação da Expressão Gênica , Células HCT116 , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Microftalmia/genética , Microftalmia/metabolismo , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , Distrofia Muscular Facioescapuloumeral/patologia , Nariz/anormalidadesRESUMO
The structural maintenance of chromosomes hinge domain containing protein 1 (SMCHD1) is a large multidomain protein involved in epigenetic gene silencing. Variations in the SMCHD1 gene are associated with two debilitating human disorders, facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS). Failure of SMCHD1 to silence the D4Z4 macro-repeat array causes FSHD, yet the consequences on gene silencing of SMCHD1 variations associated with BAMS are currently unknown. Despite the interest due to these roles, our understanding of the SMCHD1 protein is in its infancy. Most knowledge of SMCHD1 function is based on its similarity to the structural maintenance of chromosomes (SMC) proteins, such as cohesin and condensin. SMC proteins and SMCHD1 share similar domain organisation and affect chromatin conformation. However, there are important differences between the domain architectures of SMC proteins and SMCHD1, which distinguish SMCHD1 as a non-canonical member of the family. In the last year, the crystal structures of the two key domains crucial to SMCHD1 function, the ATPase and hinge domains, have emerged. These structures reveal new insights into how SMCHD1 may bind and regulate chromatin structure, and address how amino acid variations in SMCHD1 may contribute to BAMS and FSHD. Here, we contrast SMCHD1 with canonical SMC proteins, and relate the ATPase and hinge domain structures to their roles in SMCHD1-mediated epigenetic silencing and disease.
Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Epigênese Genética , Adenosina Trifosfatases/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Inativação Gênica , Humanos , Polimorfismo de Nucleotídeo Único , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Structural maintenance of chromosomes flexible hinge domain-containing 1 (Smchd1) plays important roles in epigenetic silencing and normal mammalian development. Recently, heterozygous mutations in SMCHD1 have been reported in two disparate disorders: facioscapulohumeral muscular dystrophy type 2 (FSHD2) and Bosma arhinia microphthalmia syndrome (BAMS). FSHD2-associated mutations lead to loss of function; however, whether BAMS is associated with loss- or gain-of-function mutations in SMCHD1 is unclear. Here, we have assessed the effect of SMCHD1 missense mutations from FSHD2 and BAMS patients on ATP hydrolysis activity and protein conformation and the effect of BAMS mutations on craniofacial development in a Xenopus model. These data demonstrated that FSHD2 mutations only result in decreased ATP hydrolysis, whereas many BAMS mutations can result in elevated ATPase activity and decreased eye size in Xenopus Interestingly, a mutation reported in both an FSHD2 patient and a BAMS patient results in increased ATPase activity and a smaller Xenopus eye size. Mutations in the extended ATPase domain increased catalytic activity, suggesting critical regulatory intramolecular interactions and the possibility of targeting this region therapeutically to boost SMCHD1's activity to counter FSHD.
Assuntos
Trifosfato de Adenosina/metabolismo , Atresia das Cóanas/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Oftalmopatias/patologia , Microftalmia/genética , Distrofia Muscular Facioescapuloumeral/genética , Mutação de Sentido Incorreto , Nariz/anormalidades , Adenosina Trifosfatases , Sequência de Aminoácidos , Animais , Atresia das Cóanas/patologia , Proteínas Cromossômicas não Histona/genética , Cristalografia por Raios X , Oftalmopatias/genética , Oftalmopatias/metabolismo , Humanos , Camundongos , Microftalmia/patologia , Distrofia Muscular Facioescapuloumeral/patologia , Nariz/patologia , Conformação Proteica , Domínios Proteicos , Homologia de Sequência , Xenopus laevisRESUMO
Variants in the poorly characterised oncoprotein, MORC2, a chromatin remodelling ATPase, lead to defects in epigenetic regulation and DNA damage response. The C-terminal domain (CTD) of MORC2, frequently phosphorylated in DNA damage, promotes cancer progression, but its role in chromatin remodelling remains unclear. Here, we report a molecular characterisation of full-length, phosphorylated MORC2, demonstrating its preference for binding open chromatin and functioning as a DNA sliding clamp. We identified a phosphate interacting motif within the CTD that dictates ATP hydrolysis rate and cooperative DNA binding. The DNA binding impacts several structural domains within the ATPase region. We provide the first visual proof that MORC2 induces chromatin remodelling through ATP hydrolysis-dependent DNA compaction, regulated by its phosphorylation state. These findings highlight phosphorylation of MORC2 CTD as a key modulator of chromatin remodelling, presenting it as a potential therapeutic target.
RESUMO
The interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its silencing capacity at endogenous developmental targets. Moreover, it also results in enhanced silencing at the facioscapulohumeral muscular dystrophy associated macrosatellite-array, D4Z4, resulting in enhanced repression of DUX4 encoded by this repeat. Heightened SMCHD1 silencing perturbs developmental Hox gene activation, causing a homeotic transformation in mice. Paradoxically, the mutant SMCHD1 appears to enhance insulation against other epigenetic regulators, including PRC2 and CTCF, while depleting long range chromatin interactions akin to what is observed in the absence of SMCHD1. These data suggest that SMCHD1's role in long range chromatin interactions is not directly linked to gene silencing or insulating the chromatin, refining the model for how the different levels of SMCHD1-mediated chromatin regulation interact to bring about gene silencing in normal development and disease.
Assuntos
Cromatina , Proteínas Cromossômicas não Histona , Distrofia Muscular Facioescapuloumeral , Animais , Camundongos , Cromatina/genética , Epigenômica , Inativação Gênica , Genes Homeobox , Distrofia Muscular Facioescapuloumeral/genética , Proteínas Cromossômicas não Histona/genéticaRESUMO
Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) is an epigenetic regulator in which polymorphisms cause the human developmental disorder, Bosma arhinia micropthalmia syndrome, and the degenerative disease, facioscapulohumeral muscular dystrophy. SMCHD1 is considered a noncanonical SMC family member because its hinge domain is C-terminal, because it homodimerizes rather than heterodimerizes, and because SMCHD1 contains a GHKL-type, rather than an ABC-type ATPase domain at its N terminus. The hinge domain has been previously implicated in chromatin association; however, the underlying mechanism involved and the basis for SMCHD1 homodimerization are unclear. Here, we used x-ray crystallography to solve the three-dimensional structure of the Smchd1 hinge domain. Together with structure-guided mutagenesis, we defined structural features of the hinge domain that participated in homodimerization and nucleic acid binding, and we identified a functional hotspot required for chromatin localization in cells. This structure provides a template for interpreting the mechanism by which patient polymorphisms within the SMCHD1 hinge domain could compromise function and lead to facioscapulohumeral muscular dystrophy.
Assuntos
Proteínas Cromossômicas não Histona/química , Multimerização Proteica , Animais , Proteínas Cromossômicas não Histona/genética , Cristalografia por Raios X , Camundongos , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Domínios Proteicos , Estrutura Quaternária de Proteína , IrmãosRESUMO
Bosma arhinia microphthalmia syndrome (BAMS) is an extremely rare and striking condition characterized by complete absence of the nose with or without ocular defects. We report here that missense mutations in the epigenetic regulator SMCHD1 mapping to the extended ATPase domain of the encoded protein cause BAMS in all 14 cases studied. All mutations were de novo where parental DNA was available. Biochemical tests and in vivo assays in Xenopus laevis embryos suggest that these mutations may behave as gain-of-function alleles. This finding is in contrast to the loss-of-function mutations in SMCHD1 that have been associated with facioscapulohumeral muscular dystrophy (FSHD) type 2. Our results establish SMCHD1 as a key player in nasal development and provide biochemical insight into its enzymatic function that may be exploited for development of therapeutics for FSHD.