Background: SMCHD1 is a disease modifier and a causative gene for facioscapulohumeral muscular dystrophy (FSHD) type 1 and type 2, respectively. A large variety of different mutations in SMCHD1 have been identified as causing FSHD2. In many cases, it is unclear how these mutations disrupt the normal function of SMCHD1. Methods: We made and analyzed lenti-viral vectors that express Flag-tagged full-length or different mutant SMCHD1 proteins to better understand the functional domains of SMCHD1 in muscle cells. Results: We identified regions necessary for nuclear localization, dimerization, and cleavage sites. Moreover, we confirmed that some mutants increased DUX4 expression in FSHD1 myoblasts. Conclusions: These findings provide an additional basis for understanding the molecular consequences of SMCHD1 mutations. Keywords: Facioscapulohumeral muscular dystrophy, SMCHD1, Nuclear localization, Homo-dimerization, Protein cleavage, DUX4 Background single-nucleotide polymorphisms that create a polyadenyla- Facioscapulohumeral muscular dystrophy (FSHD) is char- tion signal for the DUX4 mRNA . In addition, SMCHD1 acterized by weakness initially of the facial, scapular, and is also a disease modifier of FSHD1 . upper arm muscles, but progresses to involve most of the The human SMCHD1 gene consists of 48 exons, and skeletal muscles of the body. DUX4 is normally not the protein has an ATPase domain in the amino-terminus expressed in the skeletal muscle, whereas it is and a hinge domain in the carboxy-terminus . The mis-expressed in the FSHD skeletal muscle . DUX4 is a ATPase domain hydrolyzes ATP and the hinge domain retrogene encoding a double-homeobox transcription fac- mediates SMCHD1 dimerization [7–11]. SMCHD1 is a tor and is present in each copy of the D4Z4 macrosatellite chromatin binding protein that has a role in the epigenetic repeat, a 3.3 kilobase unit in multicopy arrays in the subte- silencing of the D4Z4 region, the X chromosome, and lomeric regions of chromosomes 4 and 10. The most com- other regions of DNA repeats in the genome [4, 10, 12– mon form of FSHD is caused by a shortened D4Z4 array 16]. Although multiple different FSHD2-causing muta- with ten or fewer units on a permissive haplotype of tions in SMCHD1 have been reported, limited knowledge chromosome 4 (FSHD1) [2, 3]. A phenotypically identical of the functional regions of the SMCHD1 protein restrict form of FSHD2 is caused by mutations in SMCHD1, a our understanding of the consequences of each mutation. member of the condensin/cohesin family of chromatin fac- Here, to better understand the function of SMCHD1 as a tors required for silencing some repetitive regions . chromatin binding protein, we focused on the identifica- FSHD2 also requires the presence of a permissive haplotype tion of the region(s) necessary for nuclear localization and of chromosome 4, which is characterized by specific homo-dimerization. * Correspondence: email@example.com Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hiramuki and Tapscott Skeletal Muscle (2018) 8:24 Page 2 of 9 Fig. 1 Schematic of full-length and different mutant SMCHD1 constructs. The gene and protein SMCHD1 consists of 48 exons and 2005 amino acids, respectively, and the ATPase domain in the amino-terminus and the hinge domain in the carboxy-terminus are indicated in red and blue, respectively. A bar indicates recognition regions for anti-SMCHD1 antibody (HPA039441 and ab179456). Predicted molecular weight is shown in the right side. 3 × HA tag (green) and 3 × Flag tag (yellow) Methods length or different mutant SMCHD1 purified by PCR or Cell culture digested by restriction enzymes was inserted into the GFP Control (MB135) and FSHD1 (MB073) myoblasts were cul- site of pLenti CMV GFP Puro (Addgene plasmid no. 17448) tured in growth medium (F-10 including 10% fetal bovine . Lenti-viral construct that removed GFP from pLenti serum, 10 ng/ml human recombinant FGF, 1 μM dexa- CMV GFP Puro was used as “No insert” for RT-qPCR. Se- methasone, 100 U/ml penicillin, and 100 μg/ml strepto- quences of the primers are listed in Additional file 1. mycin) or differentiation medium (DMEM including 1% horse serum, 10 μg/ml insulin, 10 μg/ml transferrin, 100 U/ Western blot ml penicillin, and 100 μg/ml streptomycin). These cells were For preparation of sample, cells were lysed with lysis transduced with the lentivirus of interest using 8 μg/ml poly- buffer (50 mM Tris-HCl pH 6.8, 150 mM NaCl, brene. For RT-qPCR, these cells were selected using 2 μg/ml 0.2% Triton X-100) including protease inhibitors puromycin. For making lentivirus, 293T cells cultured in (Roche). Lysates were on ice for 15 min and spun DMEM including 10% fetal bovine serum, 100 U/ml penicil- down at 12000 rpm at 4 °C for 15 min. The super- lin, and 100 μg/ml streptomycin were transfected with a natant was transferred to a new tube, added with lenti-viral construct vector, psPAX2 (Addgene plasmid no. 2 × Laemmli sample buffer, and boiled for 5 min. For 12260), and pMD2.G (Addgene plasmid no. 12259) using li- immunoprecipitation (IP), lysates were pre-cleaned pofectamine 3000 following manufacture protocol. For hu- with protein A and protein G mixture (Millipore) at 4 °C man SMCHD1 cDNA lenti-viral constructs, cDNA for full for 1 h, immunoprecipitated with anti-Flag antibody (Sigma Hiramuki and Tapscott Skeletal Muscle (2018) 8:24 Page 3 of 9 Fig. 2 Identification of the nuclear localization signal. a Immunofluorescence for SMCHD1 in control myoblasts. b–d Immunofluorescence for Flag in control myoblasts transduced with Flag-tagged SMCHD1 lentivirus. DAPI was used for staining the nucleus. Scale bar is 20 μm. The regions necessary for nuclear localization were confirmed by repeat experiments F1804, 1:300) and protein A and protein G mixture at 4 °C ab179456, 1:3500), Flag (Sigma, F1804, 1:3500), HA overnight, washed with lysis buffer five times, added with (Abcam, ab18181, 1:3500), and α-Tubulin (Sigma, 2 × Laemmli sample buffer, and boiled for 5 min. The sam- T9026, 1:14000)) at 4 °C overnight, and secondary ple was resolved on 4–12% Bis-Tris gel (Invitrogen) and antibody (Jackson ImmunoResearch) at room transferred to PVDF membrane (Novex), and then, temperature for 1 h. Signals were detected using ECL the membrane was exposed to blocking buffer (5% Western Blotting Substrate (Thermo Scientific) or dry milk) for 1 h followed by primary antibody West Femto Maximum Sensitivity Substrate (Thermo (SMCHD1 (Sigma, HPA039441, 1:3500 or Abcam, Scientific) in X-Ray Film Processors (AFP Imaging). Hiramuki and Tapscott Skeletal Muscle (2018) 8:24 Page 4 of 9 Fig. 3 Single spot of SMCHD1 in female myotube. a Immunofluorescence for SMCHD1 and myosin heavy chain (MF20 antibody) in control female myotubes. b Immunofluorescence for SMCHD1 and H3K27me3 in control female myotubes. c Immunofluorescence for Flag in control female myotubes transduced with Flag-tagged SMCHD1 lentivirus. DAPI was used for staining the nucleus. Scale bar is 20 μm. Single spot of full length was confirmed by repeat experiments Immunofluorescence Statistical analysis Cells were fixed with 4% PFA for 12 min, permeabilized Statistical analysis was carried out using Dunnett’ stest. A with 0.5% Triton X-100 for 13 min, blocked with blocking value of P < 0.01 was considered statistically significant. buffer (0.1% goat serum, 0.1% BSA, 0.02% Tween20), and incubated with primary antibodies (Flag (Sigma, F1804, Results 1:500), SMCHD1 (Sigma, HPA039441, 1:500), MF20 Identification of the SMCHD1 nuclear localization signal (DSHB, MF 20 was deposited to the DSHB by Fischman, In order to characterize SMCHD1 proteins in muscle cells, D.A., 1:500), and H3K27me3 (active motif, 61017, 1:500)) at we made lenti-viral vectors that express full length or differ- 4 °C overnight and incubated with secondary antibodies ent mutant SMCHD1 protein (Fig. 1). SMCHD1 localized (Jackson ImmunoResearch) at room temperature for 1 h. to the nucleus in control (MB135) myoblasts under growth DAPI (Sigma) was used for staining nucleic acid. Fluores- medium (Fig. 2a). To identify the nuclear localization signal cence was obtained with a Zeiss Axiophot (AxioCam MRm (NLS) in SMCHD1, we used immunofluorescence to deter- camera and Axiovision 4.6 software). mine the localization of Flag-tagged full-length and mutant SMCHD1 proteins in control myoblasts. The mutant pro- teins from constructs Exon1-48-Flag, Exon1-9.41-48-Flag, RT-qPCR and Exon1-9.47M-48-Flag were mostly localized to Total RNA was isolated using NucleoSpin RNA (Macher- the nucleus, whereas proteins from construct ey-Nagel). After DNase I treatment, cDNA synthesis with Exon1-9.47A-48-Flag were localized to the cytoplasm oligo dT primers was performed using SuperScript III Re- (Fig. 2b). Therefore, the 32 amino acids (aa) between verse Transcriptase (Invitrogen). After RNase H treatment, residues 1961 and 1992 (MTPIRKCNDSLRHSPK- RT-qPCR was performed using iTaq Universal SYBR Green VETTDCPVPPKRMRRE) functioned as an NLS. This Supermix (Bio-Rad) on QuantStudio 7 Flex (Applied Bio- NLS was sufficient for SMCHD1 to localize to the nucleus systems). Sequences of the primers are listed in because Exon1-36-Flag was expressed in the cytoplasm and Additional file 2. Exon1-36.47M-48-Flag, which has NLS sequence, was Hiramuki and Tapscott Skeletal Muscle (2018) 8:24 Page 5 of 9 Fig. 4 Identification of regions necessary for SMCHD1 homo-dimerization. a, b IP of exogenous SMCHD1 in control myoblasts transduced with Flag-tagged SMCHD1 lentivirus followed by Western blot with an anti-SMCHD1 antibody (ab179456) detecting the carboxy-terminal region of SMCHD1. For confirming the expression of Exon1-36-Flag, anti-Flag antibody was used after stripping. α-Tubulin was used as a loading control. Red and blue arrows identify endogenous and exogenous SMCHD1, respectively. Blue double arrows identify smaller fragments of exogenous SMCHD1. IgG heavy chain (**) and light chain (*). IP (immunoprecipitation) expressed mostly in the nucleus (Fig. 2c). Similarly, cells were derived from a female, we tested whether the Exon37-48-Flag was expressed mostly in the nucleus, while single spot in myotube nuclei could be co-localized with Exon37-46.47A-48-Flag, which lacks the NLS, was the inactive X chromosome marker H3K27me3 (histone expressed in the cytoplasm (Fig. 2c). Moreover, we deleted H3 trimethyl lysine 27) [20, 21] and determined that the this NLS from full-length SMCHD1 (Exon1-46.47A-48-- spots for SMCHD1 and H3K27me3 were co-localized Flag) and confirmed that it was expressed in the cytoplasm (Fig. 3b). Similarly, the expression of Exon1-48-Flag also (Fig. 2d), which suggests that this NLS is necessary for localized to nuclear foci, although the overexpression SMCHD1 to localize to the nucleus. As these 32 amino showed some additional homogeneous nuclear staining. acids include the consensus sequence of K(K/R)X(K/R) In contrast, all of the mutants Exon1-36.47M-48-Flag, for a classical NLS (bold in the sequence above) , we Exon3748-Flag, and Exon1-9.41-48-Flag showed only dif- deleted these four amino acids (KRMR) from full-length fuse nuclear staining with no evident foci (Fig. 3c), sug- SMCHD1 (ΔKRMR) and confirmed that it was expressed gesting that the structural integrity of SMCHD1, rather in the cytoplasm (Fig. 2d). A FSHD2 causing mutation of than a single specific region, was necessary for the foci SMCHD1 introduces a stop codon between the hinge do- and presumably binding to the inactive X chromosome. main and the nuclear localization signal (R1868*) and expression of a tagged version of this mutation Identification of regions necessary for SMCHD1 homo- (R1868*-Flag) showed mostly cytoplasmic localization dimerization (Fig. 2d). Previous studies using IP showed that the hinge domain In control (MB135) myotubes in differentiation medium, was important for SMCHD1 homo-dimerization [8, 10]. SMCHD1 was mostlylocalized to onesinglespotinthe nu- We used IP to determine whether different SMCHD1 cleus (Fig. 3a). Taking into account that MB135 muscle mutants would dimerize with endogenous SMCHD1. Hiramuki and Tapscott Skeletal Muscle (2018) 8:24 Page 6 of 9 Fig. 5 DUX4 expression in FSHD1 myoblasts with mutant SMCHD1. RT-qPCR for DUX4, ZSCAN4, MBD3L2, and SMCHD1 in FSHD1 myoblasts transduced with different mutant SMCHD1 constructs. RPL27 was used as an internal control. (n = 5 in each group) Dunnett’ s test (*P < 0.01). These are representative data from three experiments Confirming the prior results, Exon37-48-Flag bound to SMCHD1 mutants in FSHD1 myoblasts and measured endogenous SMCHD1, whereas Exon37-41S.44R-48-Flag, the level of DUX4 expression. Exon1-9.41-48-Flag and which lacks the hinge domain, did not bind to endogenous Exon37-48-Flag increased DUX4 and DUX4 target genes SMCHD1 (Fig. 4a). Moreover, Exon1-36-Flag, which does (ZSCAN4 and MBD3L2) expression, whereas not have a hinge domain, also did not bind to endogenous Exon1-9.47M-48-Flag had less effect on DUX4 and its SMCHD1, whereas Exon37-46.47A-48-Flag, which lacks target genes expression (Fig. 5). the NLS but has the hinge domain, also bound to en- dogenous SMCHD1 (Fig. 4b). These results indicate that Identification of cleavage sites in the SMCHD1 protein the hinge domain can mediate dimerization between en- Unexpectedly, in addition to the band of predicted molecu- dogenous SMCHD1 and the mutant SMCHD1 proteins. lar size for the mutant SMCHD1 proteins, we also detected It is interesting to note that the expression of these mu- a smaller band(s) (see double arrows in Fig. 4b). To better tant SMCHD1 proteins did not alter the abundance of the understand how the smaller bands were produced, we fo- endogenous SMCHD1 protein, whether the mutant con- cused on the small band derived from Exon1-36-Flag. We tained the hinge domain or the NLS, or not (Fig. 4a, b), in- first determined whether the smaller fragment might repre- dicating that mutations in SMCHD1 might not alter the sent a cleavage product of SMCHD1 comparing immuno- abundance of the endogenous SMCHD1 in FSHD2 but reactivity to antibodies directed to either the amino- or might alter its function by forming inactive heterodimers. carboxy-terminus of the protein. Antibody HPA039441 de- To determine whether a mutant SMCHD1 capable of tected both the predicted full-length protein (173.9 kDa) forming a heterodimer with the wild-type might partly and a prominent smaller band (about 50 kDa), whereas the interfere with normal function, we overexpressed anti-Flag antibody detected the predicted full-length protein Hiramuki and Tapscott Skeletal Muscle (2018) 8:24 Page 7 of 9 Fig. 6 Identification of the cleavage sites. a, b, and d–f Western blot for SMCHD1 (HPA039441 and ab179456) and Flag in control myoblasts transduced with Flag-tagged SMCHD1. (c) Western blot for HA and Flag in control myoblasts transduced with HA-Exon1-14.47M-48-Flag. α- Tubulin was used as a loading control. Red and blue arrows indicate endogenous and exogenous SMCHD1, respectively. Red and blue double arrows indicate smaller fragments of endogenous and exogenous SMCHD1, respectively. 10A.11D (Exon1-10A.11D-14.47M-48). 11E.12D (Exon1- 11E.12D-14.47M-48). The smaller band from full length was confirmed by repeat experiments and a smaller band (about 125 kDa) (Fig. 6a). The total size HA-Exon1-14.47M-48-Flag, which has the HA tag in the of the two smaller bands, 50 and 125 kDa, would add to- amino-terminus and the Flag tag in the carboxy-terminus. gether to become a predicted size of the full-length protein. Similar to Exon1-14.47M-48-Flag, we detected smaller Similar to this, we confirmed the total size of the smaller bands using the anti-HA or Flag antibody (Fig. 6c). bands adds to the size of the full-length band in To further narrow down the site(s) of cleavage, we Exon1-36.47M-48-Flag (Fig. 6b). tested Exon1-10A.11D-14.47M-48-Flag (10A.11D), which To narrow down the location of a potential cleavage site, lacks the 44 aa between 409 and 452 aa in the area of we tested Exon1-14.47M-48-Flag. The HPA039441 de- cleavage, and Exon1-11E.12D-14.47M-48-Flag (11E.12D), tected the predicted band (82.4 kDa) and two smaller which lacks the 37 aa between 453 and 489 aa. The bands (approximately 50 and 60 kDa), whereas the anti-Flag antibody detected two smaller bands in anti-Flag antibody detected the full-length band and a Exon1-14.47M-48-Flag, whereas it detected only a single smaller band (about 30 kDa) (Fig. 6b). Similarly, the added smaller band (about 30 kDa) in 10A.11D and 11E.12D sizes of the smaller bands (50 and 30 kDa) match the size (Fig. 6d). These results indicate that there might be one of the full-length band. Moreover, we tested cleavage site between 409 and 452 aa and a second site Hiramuki and Tapscott Skeletal Muscle (2018) 8:24 Page 8 of 9 between 453 and 489 aa. Next, we tested to probe the functional significance of polymorphisms of Exon1-10A.12D-48-Flag, which lack these cleavage sites unknown significance in SMCHD1. from full length, and investigated whether there is any differ- As an additional finding, we identified cleavage sites in ence between Exon1-48-Flag and Exon1-10A.12D-48-Flag. the SMCHD1 protein between aa 409–452 and aa 453– We detected a smaller band from Exon1-48-Flag not 489.Wewereunable todetermine theproteasethatcleaves Exon1-10A.12D-48-Flag (Fig. 6e). Moreover, antibody at this site. In addition, since we detected additional smaller ab179456, which recognizes the carboxy-terminus, detected fragments from the endogenous SMCHD1 (see Fig. 6a, b), a smaller band in not only Exon1-48-Flag but also endogen- it is possible that SMCHD1 might have additional cleavage ous SMCHD1 (Fig. 6f). Together, these results suggest that sites. Further investigation is necessary to determine specific cleavage sites in the ectopic constructs could be also whether these cleavage sites could be involved in the bio- used in the endogenous SMCHD1. logical regulation of SMCHD1 degradation. Conclusions Discussion We identified regions of SMCHD1 necessary for nuclear The ATPase domain and the hinge domain make up localization, confirmed the region necessary for only 15% of the total protein, and it is important to pro- dimerization, and identified cleavage sites using lenti-viral gressively annotate additional functional domains to help vectors that express Flag-tagged full-length or different mu- identify functionally significant polymorphisms. In this tant SMCHD1 proteins. study, we identified the nuclear localization signal, con- firmed the dimerization domain, and identified cleavage sites Additional files in the SMCHD1 protein. The NLS of SMCHD1 was mapped from aa 1961 to 1992. The four residues (KRMR) Additional file 1: Sequences of the primers for constructions. (XLSX 23 kb) in the 32 amino acids is consistent with the consensus se- Additional file 2: Sequences of the primers for RT-qPCR. (XLSX 46 kb) quence of K(K/R)X(K/R) for a classical NLS . In addition to nuclear localization, the localization of SMCHD1 was ob- Abbreviations served as a bright intra-nuclear spot that co-localized with a 10A.11D: Exon1-10A.11D-14.47M-48-Flag; 11E.12D: Exon1-11E.12D-14.47M-48- marker for the inactive X chromosome [10, 13–15]. Flag; aa: Amino acid; FSHD: Facioscapulohumeral muscular dystrophy; IP: Immunoprecipitation; NLS: Nuclear localization signal Consistent with previous studies [8, 10], the hinge do- main was important for dimerization between endogen- Acknowledgements ous and exogenous SMCHD1. Since a variety of pLenti CMV GFP Puro (658-5) was a gift from Eric Campeau and Paul mutations in SMCHD1 coding region have been re- Kaufman. pMD2.G and psPAX2 were gifts from Didier Trono. ported that have the potential to produce different por- Funding tions of the SMCHD1 protein and might act as either This work was supported by the FSH Society and FSHD Canada Foundation haploinsufficient or possibly dominant negatives [4, 19, (YH). 22], understanding the domains that confer functional interactions, such as NLS and homo-dimerization, will Availability of data and materials All data generated or analyzed during this study are included in this be important to elucidate the molecular mechanisms for published article and additional files. the mutant SMCHD1 regulation of DUX4 expression. In this regard, it is interesting to note that based on our results Authors’ contributions YH and SJT designed the experiments and wrote the manuscript. YH with different SMCHD1 proteins, the amount of endogen- performed the experiments. Both authors read and approved the final ous SMCHD1 was not altered regardless of the presence or manuscript. absence of a hinge domain in the mutant; however, it is possible that the dimerization with a mutant partner might Ethics approval and consent to participate This study used pre-existing de-identified human cell lines and was deter- alter the function of the wild-type SMCHD1 because over- mined not to be Human Subjects Research by the Fred Hutchinson Cancer expression of mutants containing the hinge domain re- Research Center Institutional Review Board. sulted in increased expression of DUX4. A previous study showed that a mutation affecting the Consent for publication Not applicable. activity of the ATPase domain (E147A) or a deletion of the hinge domain failed to localize to the inactive X Competing interests −/− chromosome in Smchd1 female mouse embryonic fi- The authors declare that they have no competing interests. broblasts . Together with our finding that deletion of multiple different domains also results in a protein that Publisher’sNote does not localize to a nuclear focus suggests that Springer Nature remains neutral with regard to jurisdictional claims in lenti-viral expression of SMCHD1 might be an approach published maps and institutional affiliations. Hiramuki and Tapscott Skeletal Muscle (2018) 8:24 Page 9 of 9 Received: 29 March 2018 Accepted: 24 July 2018 21. Silva J, Mak W, Zvetkova I, Appanah R, Nesterova TB, Webster Z, et al. 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Published: Aug 2, 2018