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1.
Cell Rep ; 27(2): 442-454.e5, 2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970248

RESUMO

Neural tube defects (NTDs) are common birth defects in humans and show an unexplained female bias. Female mice lacking the tumor suppressor p53 display NTDs with incomplete penetrance. We found that the combined loss of pro-apoptotic BIM and p53 caused 100% penetrant, female-exclusive NTDs, which allowed us to investigate the female-specific functions of p53. We report that female p53-/- embryonic neural tube samples show fewer cells with inactive X chromosome markers Xist and H3K27me3 and a concomitant increase in biallelic expression of the X-linked genes, Huwe1 and Usp9x. Decreased Xist and increased X-linked gene expression was confirmed by RNA sequencing. Moreover, we found that p53 directly bound response elements in the X chromosome inactivation center (XIC). Together, these findings suggest p53 directly activates XIC genes, without which there is stochastic failure in X chromosome inactivation, and that X chromosome inactivation failure may underlie the female bias in neural tube closure defects.

2.
Nucleic Acids Res ; 47(8): e46, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30793194

RESUMO

Systematic variation in the methylation of cytosines at CpG sites plays a critical role in early development of humans and other mammals. Of particular interest are regions of differential methylation between parental alleles, as these often dictate monoallelic gene expression, resulting in parent of origin specific control of the embryonic transcriptome and subsequent development, in a phenomenon known as genomic imprinting. Using long-read nanopore sequencing we show that, with an average genomic coverage of ∼10, it is possible to determine both the level of methylation of CpG sites and the haplotype from which each read arises. The long-read property is exploited to characterize, using novel methods, both methylation and haplotype for reads that have reduced basecalling precision compared to Sanger sequencing. We validate the analysis both through comparison of nanopore-derived methylation patterns with those from Reduced Representation Bisulfite Sequencing data and through comparison with previously reported data. Our analysis successfully identifies known imprinting control regions (ICRs) as well as some novel differentially methylated regions which, due to their proximity to hitherto unknown monoallelically expressed genes, may represent new ICRs.

3.
Nucleic Acids Res ; 47(6): 2822-2839, 2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30698748

RESUMO

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.

4.
Cell Rep ; 25(7): 1912-1923.e9, 2018 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-30428357

RESUMO

We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hox genes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist-HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xist or other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation.

5.
Nat Struct Mol Biol ; 25(9): 766-777, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30127357

RESUMO

The regulation of higher-order chromatin structure is complex and dynamic, and a full understanding of the suite of mechanisms governing this architecture is lacking. Here, we reveal the noncanonical SMC protein Smchd1 to be a novel regulator of long-range chromatin interactions in mice, and we add Smchd1 to the canon of epigenetic proteins required for Hox-gene regulation. The effect of losing Smchd1-dependent chromatin interactions has varying outcomes that depend on chromatin context. At autosomal targets transcriptionally sensitive to Smchd1 deletion, we found increased short-range interactions and ectopic enhancer activation. In contrast, the inactive X chromosome was transcriptionally refractive to Smchd1 ablation, despite chromosome-wide increases in short-range interactions. In the inactive X, we observed spreading of trimethylated histone H3 K27 (H3K27me3) domains into regions not normally decorated by this mark. Together, these data suggest that Smchd1 is able to insulate chromatin, thereby limiting access to other chromatin-modifying proteins.

6.
Blood ; 132(14): 1526-1534, 2018 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-30049810

RESUMO

The tendency of 5-methylcytosine (5mC) to undergo spontaneous deamination has had a major role in shaping the human genome, and this methylation damage remains the primary source of somatic mutations that accumulate with age. How 5mC deamination contributes to cancer risk in different tissues remains unclear. Genomic profiling of 3 early-onset acute myeloid leukemias (AMLs) identified germ line loss of MBD4 as an initiator of 5mC-dependent hypermutation. MBD4-deficient AMLs display a 33-fold higher mutation burden than AML generally, with >95% being C>T in the context of a CG dinucleotide. This distinctive signature was also observed in sporadic cancers that acquired biallelic mutations in MBD4 and in Mbd4 knockout mice. Sequential sampling of germ line cases demonstrated repeated expansion of blood cell progenitors with pathogenic mutations in DNMT3A, a key driver gene for both clonal hematopoiesis and AML. Our findings reveal genetic and epigenetic factors that shape the mutagenic influence of 5mC. Within blood cells, this links methylation damage to the driver landscape of clonal hematopoiesis and reveals a conserved path to leukemia. Germ line MBD4 deficiency enhances cancer susceptibility and predisposes to AML.

7.
J Biol Chem ; 293(25): 9841-9853, 2018 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-29748383

RESUMO

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.

8.
Biochem Soc Trans ; 46(3): 577-586, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29678955

RESUMO

Single-cell genomics is set to revolutionise our understanding of how epigenetic silencing works; by studying specific epigenetic marks or chromatin conformations in single cells, it is possible to ask whether they cause transcriptional silencing or are instead a consequence of the silent state. Here, we review what single-cell genomics has revealed about X chromosome inactivation, perhaps the best characterised mammalian epigenetic process, highlighting the novel findings and important differences between mouse and human X inactivation uncovered through these studies. We consider what fundamental questions these techniques are set to answer in coming years and propose that X chromosome inactivation is an ideal model to study gene silencing by single-cell genomics as technical limitations are minimised through the co-analysis of hundreds of genes.


Assuntos
Inativação do Cromossomo X , Animais , Compensação de Dosagem (Genética) , Epigênese Genética , Inativação Gênica , Humanos , Transcrição Genética
9.
Methods Mol Biol ; 1725: 177-184, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29322418

RESUMO

Chromatin Immunoprecipitation (ChIP) using antibodies specific for histone modifications is a powerful technique for assessing the epigenetic states of cell populations by either quantitative PCR (ChIP-PCR) or next generation sequencing analysis (ChIP-Seq). Here we describe the procedure for ChIP of histone marks in myeloid leukaemia cell lines and the subsequent purification of genomic DNA associated with repressive and activating histone modifications for further analysis. This procedure can be widely applied to a variety of histone marks to assess both activating and repressive modifications in the context of myeloid leukaemia.

10.
Hum Mol Genet ; 27(4): 716-731, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29281018

RESUMO

In humans, a copy of the DUX4 retrogene is located in each unit of the D4Z4 macrosatellite repeat that normally comprises 8-100 units. The D4Z4 repeat has heterochromatic features and does not express DUX4 in somatic cells. Individuals with facioscapulohumeral muscular dystrophy (FSHD) have a partial failure of somatic DUX4 repression resulting in the presence of DUX4 protein in sporadic muscle nuclei. Somatic DUX4 derepression is caused by contraction of the D4Z4 repeat to 1-10 units (FSHD1) or by heterozygous mutations in genes responsible for maintaining the D4Z4 chromatin structure in a repressive state (FSHD2). One of the FSHD2 genes is the structural maintenance of chromosomes hinge domain 1 (SMCHD1) gene. SMCHD1 mutations have also been identified in FSHD1; patients carrying a contracted D4Z4 repeat and a SMCHD1 mutation are more severely affected than relatives with only a contracted repeat or a SMCHD1 mutation. To evaluate the modifier role of SMCHD1, we crossbred mice carrying a contracted D4Z4 repeat (D4Z4-2.5 mice) with mice that are haploinsufficient for Smchd1 (Smchd1MommeD1 mice). D4Z4-2.5/Smchd1MommeD1 mice presented with a significantly reduced body weight and developed skin lesions. The same skin lesions, albeit in a milder form, were also observed in D4Z4-2.5 mice, suggesting that reduced Smchd1 levels aggravate disease in the D4Z4-2.5 mouse model. Our study emphasizes the evolutionary conservation of the SMCHD1-dependent epigenetic regulation of the D4Z4 repeat array and further suggests that the D4Z4-2.5/Smchd1MommeD1 mouse model may be used to unravel the function of DUX4 in non-muscle tissues like the skin.

11.
Nat Methods ; 14(11): 1055-1062, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28945704

RESUMO

Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference, we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore, our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs, underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs.


Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes/citologia , Diferenciação Celular , Fibroblastos/citologia , Instabilidade Genômica , Humanos
12.
Trends Genet ; 33(4): 233-243, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28222895

RESUMO

It has very recently become clear that the epigenetic modifier SMCHD1 has a role in two distinct disorders: facioscapulohumoral muscular dystrophy (FSHD) and Bosma arhinia and micropthalmia (BAMS). In the former there are heterozygous loss-of-function mutations, while both gain- and loss-of-function mutations have been proposed to underlie the latter. These findings have led to much interest in SMCHD1 and how it works at the molecular level. We summarise here current understanding of the mechanism of action of SMCHD1, its role in these diseases, and what has been learnt from study of mouse models null for Smchd1 in the decade since the discovery of SMCHD1.


Assuntos
Atresia das Cóanas/genética , Proteínas Cromossômicas não Histona/genética , Epigênese Genética , Microftalmia/genética , Distrofia Muscular Facioescapuloumeral/genética , Nariz/anormalidades , Animais , Metilação de DNA/genética , Heterozigoto , Humanos , Camundongos , Mutação
13.
Bioinformatics ; 33(13): 2050-2052, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28203714

RESUMO

Motivation: graphics for RNA-sequencing and microarray gene expression analyses may contain upwards of tens of thousands of points. Details about certain genes or samples of interest are easily obscured in such dense summary displays. Incorporating interactivity into summary plots would enable additional information to be displayed on demand and facilitate intuitive data exploration. Results: The open-source Glimma package creates interactive graphics for exploring gene expression analysis with a few simple R commands. It extends popular plots found in the limma package, such as multi-dimensional scaling plots and mean-difference plots, to allow individual data points to be queried and additional annotation information to be displayed upon hovering or selecting particular points. It also offers links between plots so that more information can be revealed on demand. Glimma is widely applicable, supporting data analyses from a number of well-established Bioconductor workflows ( limma , edgeR and DESeq2 ) and uses D3/JavaScript to produce HTML pages with interactive displays that enable more effective data exploration by end-users. Results from Glimma can be easily shared between bioinformaticians and biologists, enhancing reporting capabilities while maintaining reproducibility. Availability and Implementation: The Glimma R package is available from http://bioconductor.org/packages/Glimma/ . Contact: su.s@wehi.edu.au , law@wehi.edu.au or mritchie@wehi.edu.au.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Software , Animais , Camundongos
14.
Nat Genet ; 49(2): 249-255, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28067911

RESUMO

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.


Assuntos
Atresia das Cóanas/genética , Proteínas Cromossômicas não Histona/genética , Microftalmia/genética , Mutação de Sentido Incorreto/genética , Nariz/anormalidades , Animais , Linhagem Celular , Pré-Escolar , Epigênese Genética/genética , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distrofia Muscular Facioescapuloumeral/genética , Xenopus laevis/genética
15.
Genom Data ; 10: 97-100, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27766205

RESUMO

Reduced representation bisulfite sequencing (RRBS) provides an efficient method for measuring DNA methylation at single base resolution in regions of high CpG density. This technique has been extensively tested on the HiSeq2500, which uses a 4-colour detection method, however it is unclear if the method will also work on the NextSeq500 platform, which employs a 2-colour detection system. We created an RRBS library and sequenced it on both the HiSeq2500 and NextSeq500, and found no significant difference in the base composition of reads derived from either machine. Moreover, the methylation calls made from the data of each instrument were highly concordant, with methylation patterns across the genome appearing as expected. Therefore, RRBS can be sequenced on the Nextseq500 with comparable quality to that of the HiSeq2500. All sequencing data are deposited in the GEO database under accession number GSE87097.

16.
Nat Struct Mol Biol ; 23(7): 673-81, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27294782

RESUMO

Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis.


Assuntos
Regulação Leucêmica da Expressão Gênica , Histonas/genética , Leucemia Aguda Bifenotípica/genética , Metiltransferases/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Acetilação , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Proliferação de Células , Cromatina/química , Cromatina/metabolismo , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Feminino , Histonas/metabolismo , Humanos , Leucemia Aguda Bifenotípica/metabolismo , Leucemia Aguda Bifenotípica/patologia , Masculino , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Cultura Primária de Células , Ligação Proteica , Proteômica/métodos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Linfócitos T/patologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Transcrição Genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-27195021

RESUMO

BACKGROUND: The presence of histone 3 lysine 9 (H3K9) methylation on the mouse inactive X chromosome has been controversial over the last 15 years, and the functional role of H3K9 methylation in X chromosome inactivation in any species has remained largely unexplored. RESULTS: Here we report the first genomic analysis of H3K9 di- and tri-methylation on the inactive X: we find they are enriched at the intergenic, gene poor regions of the inactive X, interspersed between H3K27 tri-methylation domains found in the gene dense regions. Although H3K9 methylation is predominantly non-genic, we find that depletion of H3K9 methylation via depletion of H3K9 methyltransferase Set domain bifurcated 1 (Setdb1) during the establishment of X inactivation, results in failure of silencing for around 150 genes on the inactive X. By contrast, we find a very minor role for Setdb1-mediated H3K9 methylation once X inactivation is fully established. In addition to failed gene silencing, we observed a specific failure to silence X-linked long-terminal repeat class repetitive elements. CONCLUSIONS: Here we have shown that H3K9 methylation clearly marks the murine inactive X chromosome. The role of this mark is most apparent during the establishment phase of gene silencing, with a more muted effect on maintenance of the silent state. Based on our data, we hypothesise that Setdb1-mediated H3K9 methylation plays a role in epigenetic silencing of the inactive X via silencing of the repeats, which itself facilitates gene silencing through alterations to the conformation of the whole inactive X chromosome.

18.
Biochem J ; 473(12): 1733-44, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27059856

RESUMO

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic regulator that plays critical roles in gene regulation during development. Mutations in SMCHD1 were recently implicated in the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD), although the mechanistic basis remains of outstanding interest. We have previously shown that Smchd1 associates with chromatin via its homodimeric C-terminal hinge domain, yet little is known about the function of the putative GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPase domain at its N-terminus. To formally assess the structure and function of Smchd1's ATPase domain, we have generated recombinant proteins encompassing the predicted ATPase domain and the adjacent region. Here, we show that the Smchd1 N-terminal region exists as a monomer and adopts a conformation resembling that of monomeric full-length heat shock protein 90 (Hsp90) protein in solution, even though the two proteins share only ∼8% overall sequence identity. Despite being monomeric, the N-terminal region of Smchd1 exhibits ATPase activity, which can be antagonized by the reaction product, ADP, or the Hsp90 inhibitor, radicicol, at a nanomolar concentration. Interestingly, introduction of an analogous mutation to that identified in SMCHD1 of an FSHD patient compromised protein stability, suggesting a possible molecular basis for loss of protein function and pathogenesis. Together, these results reveal important structure-function characteristics of Smchd1 that may underpin its mechanistic action at the chromatin level.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Macrolídeos/farmacologia , Camundongos , Dados de Sequência Molecular , Domínios Proteicos/genética , Domínios Proteicos/fisiologia , Alinhamento de Sequência
19.
Genom Data ; 7: 144-7, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26981392

RESUMO

Smchd1 is an epigenetic repressor with important functions in healthy cellular processes and disease. To elucidate its role in transcriptional regulation, we performed two independent genome-wide RNA-sequencing studies comparing wild-type and Smchd1 null samples in neural stem cells and lymphoma cell lines. Using an R-based analysis pipeline that accommodates observational and sample-specific weights in the linear modeling, we identify key genes dysregulated by Smchd1 deletion such as clustered protocadherins in the neural stem cells and imprinted genes in both experiments. Here we provide a detailed description of this analysis, from quality control to read mapping and differential expression analysis. These data sets are publicly available from the Gene Expression Omnibus database (accession numbers GSE64099 and GSE65747).

20.
Biochem J ; 473(6): 733-42, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26733688

RESUMO

The structural maintenance of chromosomes (SMC) proteins are fundamental to chromosome organization. They share a characteristic domain structure, featuring a central SMC hinge domain that is critical for forming SMC dimers and interacting with nucleic acids. The structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is a non-canonical member of the SMC family. Although it has been well established that Smchd1 serves crucial roles in epigenetic silencing events implicated in development and disease, much less is known about the structure and function of the Smchd1 protein. Recently, we demonstrated that the C-terminal hinge domain of Smchd1 forms a nucleic acid-binding homodimer; however, it is unclear how the protomers are assembled within the hinge homodimer and how the full-length Smchd1 protein is organized with respect to the hinge region. In the present study, by employing SAXS we demonstrate that the hinge domain of Smchd1 probably adopts an unconventional homodimeric arrangement augmented by an intermolecular coiled coil formed between the two monomers. Such a dimeric structure differs markedly from that of archetypical SMC proteins, raising the possibility that Smchd1 binds chromatin in an unconventional manner.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Repressão Epigenética/fisiologia , Regulação da Expressão Gênica/fisiologia , Animais , Proteínas Cromossômicas não Histona/genética , Células HEK293 , Humanos , Imunoprecipitação , Camundongos , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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