Expression of the myodystrophic R453W mutation of lamin A in C2C12 myoblasts causes promoter-specific and global epigenetic defects

in C2C12 myoblasts epigenetic defects wan e Co partme way RS an ofXi, butmaintainsXistexpressionandcoatingofXi, indicating thatXi remains inactivated.Our E X P E R I M E N T A L C E L L R E S E A R C H 3 1 4 ( 2 0 0 8 ) 1 8 6 9 – 1 8 8 0 ava i l ab l e a t www.sc i enced i rec t . com com results argue that gene-specific and genome-wide chromatin rearrangementsmay constitute a molecular basis for laminopathies. © 2008 Elsevier Inc. All rights reserved. Introduction Laminopathies are diseases manifested by skeletal muscle, mutations in LMNB2 andmutations in genes encoding lamina- associated proteins have also been linked to disease [3–5]. LMNA encodes nuclear laminsA and C, intermediate filaments Available online 7 March 2008 regulation of the myogenin (Myog) gene and on global chromatin organization. Expression of R453W-, but not R482W-lamin A, impairs activation of Myog and maintains a repressive chromatin state on theMyog promoter upon induction of differentiation, marked by H3 lysine (K) 9 dimethylation and failure to hypertrimethylate H3K4. Cells expressing WT-LaA also fail to hypertrimethylate H3K4. No defect occurs at the level ofMyog promoter DNAmethylation in any of the clones. Expression of R453W-lamin A and to a lesser extent R482W-lamin A in undifferentiated C2C12 cells redistributes H3K9me3 frompericentric heterochromatin. R453W- laminAalsoelicits a redistributionofH3K27me3 frominactiveX (Xi) andpartial decondensation Keywords: Chromatin Differentiation Histone modification Myoblast Lamin A mutation 22 February 2008 Accepted 26 February 2008 cardiac muscle, adipose tissue, skeletal, n opmental defects.Most pathologies are cau the LMNA gene [1,2] although a duplication ⁎ Corresponding author. Fax: +47 22851058. E-mail address: philippe.collas@medisin. Abbreviations: DM, differentiation medium Hutchinson–Gilford progeria syndrome; ChIP RT-PCR, reverse transcription polymerase ch 0014-4827/$ – see front matter © 2008 Elsevi doi:10.1016/j.yexcr.2008.02.018 Weshowhere the influence of stable expression of the R453Wandof theDunnigan-type partial lipodystrophy R482W mutation of lamin A in C2C12 cells on transcription and epigenetic A R T I C L E I N F O R M A T I O N Article Chronology: Received 5 January 2008 Revised version received causes promoter-specific and global Delbarrea, Kristine G. Gaustada, llasa,⁎ nt of Biochemistry, Faculty of Medicine, University of Oslo, d Universités Paris 6 et 7, 2 Place Jussieu, 75251 Paris, France A B S T R A C T Autosomal dominant Emery–Dreifuss muscular dystrophy (EDMD) is characterized by muscle wasting and is caused bymutations in the LMNA gene encoding A-type lamins. Overexpression of the EDMD lamin A R453W mutation in C2C12 myoblasts impairs myogenic differentiation. Anne-Mari Håkeliena, Er Brigitte Buendiab, Philipp aInstitute of Basic Medical Sciences, De PO Box 1112 Blindern, 0317 Oslo, Nor bInstitut Jacques Monod, UMR7592, CN Expression of the myodystrophic R453W mutation of lamin A Research Article www.e l sev i e r. euronal and devel- sedbymutations in of the LMNB1 gene, uio.no (P. Collas). ; EDMD, Emery Dreifuss , chromatin immunoprec ain reaction; Rb, retinobla er Inc. All rights reserved / l oca te /yexc r of the nuclear lamina which extend into the nuclear interior [6]. The lamina provides structure to the nuclear envelope and is essential for nuclear processes such as DNA replication [7] muscular dystrophy; FPLD, familial partial lipodystrophy; HGPS, ipitation; GM, growth medium; Ig, immunoglobulin; LaA, lamin A; stoma protein; TSS, transcription start site . Detection System with IQ SYBR Green (BioRad). Primer se- A R and transcription [8]. The autosomal dominant formof Emery– Dreifuss muscular dystrophy (EDMD) is caused by primarily missense point mutations throughout the LMNA gene, whereas dominantmutations causing Dunnigan-type familial partial lipodystrophy (FPLD) are mainly restricted to the im- munoglobulin (Ig) fold in the carboxyl-terminal tail of A-type lamins [1]. Expression of the R453W-lamin A (LaA) mutation causing EDMD or of the R482W-LaA mutation causing FPLD in C2C12 myoblasts results in an abnormal nuclear phenotype analogous to that detected in cells fromEDMDor FPLDpatients [9]. In contrast to EDMD mutations, LMNA mutations respon- sible for FPLD, partial lipodystrophy with mandibuloacral dysplasia and progeroid syndromes such as Hutchinson– Gilfordprogeria syndrome (HGPS), causenuclear accumulation of uncleaved prelamin A in fibroblasts [10,11] with alterations in the architecture of the lamina [12]. PrelaminAaccumulation has been shown to promote sequestration of an adipogenic transcription factor which in turns impairs adipogenesis [10]. R453W-LaA overexpression also impairs expression of the myogenic transcription factor myogenin and inhibits differ- entiation of C2C12 cells into myotubes [13]. The molecular events behind the failure to upregulate myogenin expression in C2C12 cells expressing R453W-LaA remain undetermined. Interaction of the Ig fold of lamins A and C, which contains the arginine 453 residue [14,15], with DNA [16] and association of lamins with chromatin [17] raise the hypothesis of alterations in global and locus-specific chromatin topology. Epigenetic modifications of DNA and histones contribute to regulating gene expression [18]. Methylation of cytosine in CpG dinucleotides is a heritablemodification essential for long-term gene silencing in the context of development, imprinting and X chromosome inactivation [19]. In addition, covalentmodifica- tions of core histones, of which those of histones H3 and H4 are the best characterized [18,20,21], modulate DNA packaging around nucleosomes, and thereby transcription. Chromatin immunoprecipitation (ChIP) studies have shown that di- and trimethylation of H3K9 (H3K9me2, H3K9me3), H3K27me3 and H4K20me3, together with unacetylated H3K9, are generally associated with repressed genes or heterochromatin. In con- trast, di- and trimethylatedH3K4 (H3K4me2, H3K4me3),methy- latedH3K36 andH3K79, togetherwith acetylatedH3K9 (H3K9ac) generally mark a gene for transcription. Proper epigenetic control of expression of the transcription factor myogenin is essential for muscle differentiation. The myogenin (Myog) gene is transcriptionally silenced inmyoblasts, and its activation uponmyogenic differentiation correlateswith demethylation and acetylation of H3K9 on the promoter [22]. Manipulation of the level of Suv39h1, which catalyzes H3K9 trimethylation in pericentric heterochromatin [23,24], interferes with differentiation of C2C12 cells [25]. Thus, regulation of H3K9 methylation is important for initiationofmuscle differentiation. Activation of Myog also correlates with demethylation of a specific CpG in the promoter [26]. In addition, during myogenic differentiation in the mouse, pericentric heterochromatin ag- gregates to form large chromocenters, a process involving a global increase in DNAmethylation in these domains [27]. We examine here the influence of stable expression of the 1870 E X P E R I M E N T A L C E L L R E S E R453WandR482Wmutations of laminAon transcription, DNA methylation and histone modifications on theMyog promoter quences were for Myog 5′-CAATGCACTGGAGTTCGGTC-3′ and 5′-AGTTGGGCATGGTTTCGTCT-3′; forMyoD, 5′-AACGC- CATCCGCTACATC-3′ and 5′-ACACAGCCGCACTCTTCC-3′; for Myf5, 5′-GCCATCCGCTACATTGAGA-3′and 5′-GCTG- TTCTTTCGGGACCA-3′; and for G6pdx 5′-AGGTAGTGGT- CAATGCGGTAG-3′ and 5′-TGCCCGTAATTCCTATGTAGC-3′. SYBR Green PCR conditions were 95 °C for 4.5 min and 40 cycles and at the nucleus-wide level in C2C12 myoblasts. We show that R453W-LaA expression causes alterations in histone modifications locally on the Myog promoter, and at the global nucleus level. Our results suggest that locus-specific and genome-wide epigenetic rearrangementsmay provide amole- cular basis for laminopathies. Materials and methods Cells C2C12myoblasts were cultured in DMEM (Sigma-Aldrich) con- taining 15% fetal calf serum and 2 mM L-glutamine. Differ- entiation medium was DMEM containing 2% horse serum (Sigma-Aldrich) and 2 mM L-glutamine. C2C12 cell lines stably expressing green fluorescent protein (GFP)-tagged lamin A mutant (clones R453W G8 and R482W E1A), or wild-type lamin A (clone E2A) have been previously described [9,13] and were cultured with 850 µg/ml G-418. The relative expression level of GFP-lamin A in triplicate cultures of each clone is shown by Western blotting in Supplementary Figure 1A. 3T3-L1 pre- adipocytes were cultured and differentiated as described [28]. Mouse muscle biopsies were taken from the thigh and frozen in liquid nitrogen prior to DNA isolation (GenEluteMammalian Genomic DNA Isolation kit; Sigma-Aldrich). Bisulfite genomic sequencing DNA was purified from cultured cells by phenol-chloroform- isoamylalcohol extraction and bisulfite conversion was per- formed as described [29]. Converted DNA was amplified by PCR using two primer sets designed to cover nucleotides −451 to + 323 relative to the transcription start site (TSS) of Myog. Primer sequences were P1: 5′-TAAGTGGATTTTCAAGACCCCTTCC-3′, P2: 5′-GCCAGCAGGGAGGGTTTAAATGGCA-3′, P3: 5′-GGGA- AGGGGAATCACATGTAATCCACT-3′, and P4: 5′-GCAGGCTCAA- GAA AGTGAATGAGGC-3′. PCR conditions were 95 °C for 7 min and 40 cycles of 95 °C for 1 min, 54 °C for 2 min and 72 °C for 2 min, followed by 10 min at 72 °C. PCR products were cloned using the TOPO TA cloning kit (Invitrogen) and clones se- quenced. Sequences amplified by primer pairs P1–P2 and P3–P4 contained two overlapping CpGs (No. 10 and 11) of which only those included in the P1–P2 amplicon are shown. cDNA synthesis and quantitative (q)RT-PCR cDNA was synthesized from 500 ng total RNA using the Iscript cDNA synthesis kit (BioRad). qPCRs were performed in triplicates on cDNA templates using aMyiQ Real-time PCR C H 3 1 4 ( 2 0 0 8 ) 1 8 6 9 – 1 8 8 0 of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 smRNA levelswere calculated [30] using G6pdx as normalization control. (Sigma-Aldrich; 50 µg/ml) for 6 h, and DNA was purified by A R Immunofluorescence For myogenin detection, undifferentiated or differentiated cells were harvested from tissue culture flasks, sedimented onto poly-L-lysine-coated coverslips and processed as described [31]. This stepwasnecessary becauseC2C12cells donotdifferentiate well on glass. Myogenin was detected using anti-myogenin antibody F5D (Santa Cruz; sc-12732; diluted 1:100) and a Cy3- conjugated anti-mouse IgG (Jackson ImmunoResearch; 115-165- 044; 1:200 dilution). For histone detection, undifferentiated cells were plated onto poly-L-lysine-coated coverslips overnight and fixed with 3% paraformaldehyde for 15 min. Cells were per- meabilized with 0.5% Triton X-100 for 2 min followed by 0.1% Triton X-100 for 15 min. Primary antibodies were anti-H3K9ac (Upstate; 06–942), anti-H3K9me2 (Upstate; 07–441), anti- H3K9me3 (Upstate; 07–442) and anti-H3K27me3 (Upstate; 05– 851) all diluted 1:500. Secondary antibodies were Cy3-conju- gated IgGs (Jackson ImmunoResearch). Cells were observed on an Olympus IX71 microscope fitted with a piezo-driven 100× objective and an Olympus CellR Real-Time Imaging Station. For analysis of correlation between H3K9me3 and DAPI labeling patterns, the same illumination settings were used in each channel for all cells. For each acquisition, a 1 µm z-step stack of 13 planes was acquired in all channels and maximum fluo- rescence intensity z-projections were done based on DAPI staining. Treatment of images was done with the ImageJ 1.36b software (National Institutes of Health). For each cell, the same area of the nucleus was selected in the DAPI and H3K9me3 images, and the smooth continuousbackgroundwas subtracted using a rolling ball algorithm. The two resulting images were compared and the correlation coefficient (R) was calculated using Image CorrelationJ (http://www.gcsca.net). Intensities of GFP fluorescence (Igreen) were obtained by measuring mean fluorescence intensity over the whole nucleus in z-projection images. Western blotting Immunoblotting ofmyogeninwas done as described [13] using antibodies against myogenin (cat. no. sc-12732, Santa Cruz), MyoD1 (clone 5.8A, DAKO), Myf5 (C-20, Santa Cruz) and HP1β [32] and horseradish peroxidase-conjugated anti-rabbit or mouse IgGs. Chromatin immunoprecipitation (ChIP) ChIPs were performed as described [33] with modifications. Adherent C2C12 cells in flasks were fixed with 1% formalde- hyde for 8 min to cross-link DNA and proteins. Cross-linking was stopped with 125 mM glycine for 5 min. Cells were rinsed twice in phosphate buffered saline (PBS), harvested using a cell scraper and sedimented. Cells were resuspended in ice-cold lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.0) containing protease inhibitors and 5 mM Na-butyrate. Cells were sonicated on ice using a Labsonic M sonicator fitted with a 3-mm probe (Sartorius) for 6×30 s, 40% output and at 0.6 s intervals, to yield chromatin fragments of 300–500 bp. Chro- matin was cleared by centrifugation, concentration deter- E X P E R I M E N T A L C E L L R E S E mined by A260, and chromatin was diluted in RIPA buffer (0.1% SDS, 0.1% Na-deoxycholate, 1% Triton X-100, 1 mM EDTA, phenol-chloroform-isoamylalcohol extraction and DNA dis- solved in 100 µl H2O. Quantification of precipitated DNA was performed by qPCR using SYBR® green and Myog promoter-specific primers (5′-GAATCACATGTAATCCA- CTGGA-3′; 5′-ACGCCAACTGCTGGGTGCCA-3′). Relative enrichment of Myog promoter DNA in bound chromatin relative to a no-antibody control was calculated based on dif- ference in threshold (Ct) values (2[Ctantibody−Ctno antibody]). RNA-fluorescence in situ hybridization (FISH) RNA-FISH was performed as described [34]. Briefly, the probe was labeled from themouseXist genome clone p510 (a gift from E. Heard) using nick translation and SpectrumGreen dUTP. The probe was denatured in formamide for 7 min at 75 °C, and 1 volumeof 2×hybridization buffer (4× SSC, 20%dextran sulfate, 2 mg/ml BSA) was added. For hybridization, cells were cultured on glass coverslips, permeabilized in 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES (pH 6.8), 0.5% Triton X-100 and 2 mM vanadyl ribonucleoside complex, and fixed with 3% paraformaldehyde. Cellsweredehydrated in 80%, 95%and 100% ethanol, air dried and hybridized with the probe at 37 °C overnight.Cellswere rinsed in50% formamide/2×SSC then in2× SSC, counterstainedwith0.2μg/mlDAPIandmounted inCrystal mounting medium (Santa Cruz). Flow cytometry Undifferentiated cells were plated at 5000 cells/cm2, cultured in GM for 24 h and harvested for cell cycle analysis. Cells were fixed in 70% ethanol at −20 °C for 24 h, treated with 40 µg/ml RNase, stained with 20 µg/ml propidium iodide and analyzed by flow cytometry to determine DNA content. Results Expression of R453W-LaA inhibits myogenic differentiation C2C12 myoblasts form multinucleated myotubes when cul- tured in myogenic differentiation medium (DM) in flasks (C2C12 cells do not differentiate well on glass coverslips). Multinucleated cells were seen after 3 days of differentiation (data not shown) and myotube morphology was clearly evident after 6 days (Fig. 1A). Note that myotube morphology was lost when the differentiated cells were sedimented onto 0.5 mM EGTA, 140 mM NaCl, 10 mM Tris–HCl, pH 8.0) to 2 A260 units. Each immunoprecipitation was performed in 250 µl RIPA buffer with 5 µg antibody overnight at 4 °C. The following antibodies were used: anti-H3K9ac (Upstate; 06–942), anti- H3K9me2 (Upstate; 07–441), anti-H3K9me3 (Abcam; 8898), anti-H3K4me2 (Abcam; ab7766) and anti-H3K4me3 (Abcam; ab8580). Antibody-bound chromatin was precipitated using protein A/G PLUS-agarose beads (Santa Cruz) and washed 3 times in RIPA buffer, once in TE buffer (10mMTris–HCl, pH 8.0, 10 mM EDTA) and eluted in 1% SDS with 100 mM NaHCO3. Eluted chromatin was incubated at 65 °C with proteinase K 1871C H 3 1 4 ( 2 0 0 8 ) 1 8 6 9 – 1 8 8 0 coverslips for immunofluorescence analysis, such that rather, multinucleated cells were observed. A clone of C2C12 cells 1872 E X P E R I M E N T A L C E L L R E S E A R C H 3 1 4 ( 2 0 0 8 ) 1 8 6 9 – 1 8 8 0 stably expressing the myodystrophic R453W-LaA mutation was previously shown to exhibit severely impaired myogenic differentiation and myogenin expression [13]. We confirmed that this clone failed to form multinucleated cells, in contrast to clones expressing wild-type (WT)-lamin A or the lipody- strophic R482W-LaAmutation (Fig. 1A). Differentiation of cells Fig. 1 – Expression of R453W-LaA in C2C12 cells impairs myoge differentiated (day 6; D6) C2C12, R453W-LaA, WT-LaA and R482W (B) Immunofluorescence analysis of myogenin expression in C2C cultured in GM or DM for 1, 3 or 6 days and cytospun onto cover protein levels in the same clones as in (A) cultured in GM or DM bands correspond to phosphorylated and non-phosphorylated m loading control. (D) RT-qPCR analysis ofMyogmRNA expression days ofmyogenic stimulation relative to day 0 (GM) levels, and no each with triplicate RT-qPCRs). Expression levels were compared (ab, bc, bdp and expressed in undifferentiated C2C12 cells, was confirmed by immunoblotting in all clones after myogenic induction (Supplementary Fig. 1B) [13]. Thus, stable overexpression of R453W-LaA inhibits the upregulation of myogenin but does not affect expression of MyoD and Myf5. R453W-LaA inhibits upregulation of Myog upon induction of differentiation Failure toupregulatemyogeninupondifferentiationof R453W- LaA expressing cells was reflected at the transcript level. RT- 1873E X P E R I M E N T A L C E L L R E S E A R C H 3 1 4 ( 2 0 0 8 ) 1 8 6 9 – 1 8 8 0 qPCR data indicate that Myog transcripts were strongly up- regulated by day 3 of differentiation in untransfected C2C12 cells and in cells expressing WT-LaA or R482W-LaA (Fig. 1D). Nevertheless, WT-LaA and R482W-LaA overexpression im- paired upregulation of Myog relative to untransfected cells (p A R E X P E R I M E N T A L C E L L R E S E mutant lamin A did not affect the profile of CpG methylation in the Myog region examined. Failure to fully activate Myog in R453W-LaA expressing cells correlates with persistence of H3K9 methylation on the Myog promoter We next examined by ChIP-qPCR whether lowMyog expression in R453W-LaA expressing cells was related to defects in histone Fig. 4 – Expression of R453W-LaA redistributes H3K9me3 from p of H3K9me3 distribution in C2C12 and in R453W-LaA, R482W-LaA DAPI. Bar, 10 µm. (B) H3K9me3 and DAPI staining co-localization quantification of the overlap betweenDAPI andH3K9me3 labeling R453W-LaA expressing cell (bottom panels). Bars, 5 µm. Dual lab establish a correlation coefficient (R) as a function of H3K9me3 la fluorescence units). (C) Mean±SD correlation coefficient (R) of H3 correlation values were compared by Student t-tests (a,bp=4.6×1 Number of cells analyzed in each clone is indicated in Suppleme 1875C H 3 1 4 ( 2 0 0 8 ) 1 8 6 9 – 1 8 8 0 H3 modification on the Myog promoter (Fig. 3A). ChIPs were performed using antibodies to H3K4me2, H3K4me3, generally but not exclusively associated with active promoters [40,41], H3K9ac, associated with transcriptionally active promoters, and H3K9me2 and H3K9me3, markers of transcriptionally silent genes. To control for unspecific precipitation, ChIPs were performed with no antibody and a non-relevant antibody. No differenceweredetectedbetween the twonegativecontrols (data not shown), thus a no-antibody control was subsequently used. ericentric heterochromatin. (A) Immunofluorescence analysis andWT-LaA expressing cells. DNAwas counterstainedwith phenotypes and correlation coefficients. Examples of in an area in the nucleus of a C2C12 cell (top panels) and of an eling pattern in each pixel was plotted in each example to beling intensity and DAPI staining intensity (a.u., arbitrary K9me3-DAPI labeling overlap in each cell clone. Mean 0−19, a,cp=0.104, b,cp=3.4×10−15, c,dp=6.8×10−4, b,dp=0.0015). ntary Fig. 2 (n=31–52). Myogenic stimulation of C2C12 cells for 3 days elicited acetylation of H3K9 (p=0.040; unpaired t-test between undif- ferentiated and differentiated cells), and increased di- and tri methylation of H3K4 (p=0.001 and p=0.011, respectively; Fig. 3B) in agreement with activation of the gene. In con- cordance with the enrichment of activating modifications, H3K9me2 was demethylated (p=0.002). Induction of differen- tiation did not alter H3K9me3 levels on theMyog promoter. In R453W-LaA expressing cells, some H3K9 acetylation was detected aftermyogenic stimulation (p=0.051), reflectingMyog activation in a subpopulation of the cells (see Fig. 1). However, levels of H3K4me2, H3K4me3, H3K9me2 and H3K9me3 remained unchanged at day 3, indicative of improper chroma- tin remodeling at this site in response to differentiation stimuli (Fig. 3B). This illustrates the persistence of a suppressive chro- matin environment on theMyog promoter and is in agreement with the strong reduction inMyog transcript levels detected at day 3 of differentiation in R453W-LaA cells (Fig. 1D). In R482W-LaA expressing cells, theMyog promoter behaved as untransfected C2C12 cells, with a significant increase in H3K9ac (p=0.002), H3K4me2 (p=0.002) andH3K4me3 (p=0.002), a decrease in H3K9me2 (p=0.025) induced by differentiation, but no change in H3K9me3 (Fig. 3B). In WT-LaA cells, theMyog promoter responded to differentiation in an intermediate fa- shion compared to C2C12 and R482W-LaA cells with an in- crease of H3K9ac (p=0.004) and a decrease of H3K9me2 and H3K9me3 (p sing cells in different contexts. For example, the R482W A R exemplified by R453W-LaA expressing cells). Correlation analysis showed no difference in DAPI–H3K9me3 labeling overlap between C2C12 and WT-LaA expressing cells (R=0.61±0.09 and R=0.57±0.10, respectively; p=0.1, Student t-test; Fig. 4C). However, the correlation was dramatically lower in cells expressing R453W-LaA compared to WT-LaA (R=0.38±0.13; p K.L. Wilson, The nuclear lamina comes of age, Nat. Rev., Mol. Cell Biol. 6 (2005) 21–31. [5] H.J. Worman, G. Bonne, “Laminopathies”: A wide spectrum of A R domains (this paper). Whether the chromatin reorganization observed in our R453W-LaA expressing C2C12 cells is caused by direct interaction between themutant lamin andmyoblast specific proteins is unknown. Aggregation of pericentric heterochromatin into chromo- centers is a hallmark of myogenic differentiation [27], and stabilization of these chromocenters is enabled by maintaining histone methylation in these domains [54]. Our results indir- ectly implicate A-type lamins in this process. We found that expression of R453W-LaA quantitatively impairs the enrich- ment of H3K9me3 in pericentric heterochromatin in undiffer- entiated C2C12 cells. Insufficient histone methylation in pericentric heterochromatin may prevent the chromatin com- partmentalization required formyogenesis and the enrichment in methyl-binding proteins on chromocenters that takes place during myogenic differentiation [27]. The dynamics of facultative heterochromatin associated with H3K27me3 is also affected by overexpression of R453W- LaA, as shown by reduced H3K27me3 labeling on Xi. Tri- methylation of H3K27 on Xi is catalyzed by the polycomb repressor complex 2, however, the complex is dispensable for initiation and maintenance of X inactivation [55,56]. Thus, displacement of H3K27me3 from Xi would not be expected to affect X inactivation. Indeed, FISH analysis reveals clear Xist labeling, suggesting that regulation of X inactivation is intact in cells expressing mutant lamin A. However, as H3K27me3 is also associated with developmentally regulated genes on autosomes [57,58], defects in H3K27me3 regulation are ex- pected to severely affect differentiation. Effects of wild type lamin A on myogenic differentiation In addition to data linking mutated A-type lamins to defective cell function, increasing evidence also suggests that over- expression ofWT laminA affects differentiation. Interestingly, stable expression of WT lamin A was found to inhibit adipo- genic differentiation in 3T3-L1 preadipocytes [59]. Similarly, WT-laA overexpression reduces myofiber formation [13] (this paper) and slowsdownC2C12 cell differentiation [60].Whether these phenotypes are related to the lack of enrichment of H3K4me3 on the Myog promoter in WT-LaA expressing cells remains currently unclear. However, this is a possibility, in light of the reported enrichment of H3K4me3 near the TSS at promoters that are preferentially active [40]. The relatively high EGFP-laA level in WT clone relative to the others may explain why this clone behaves differently from the R482W-laA expressing clone and C2C12 cells in our ChIP experiments. Nevertheless, despite this, the WT clone displays myogenic differentiation, upregulation of Myog expression and normal global chromatin organization. This indicates that in our cellular model the negative effect of the R453W-laA mutation is stronger than the effect of EGFP-laA overexpression itself. At present, how lamin A overexpression affects differentia- tion is subject to speculation. A proportion of satellite cells from Lmna−/− or Lmna+/−mice show decreased myogenic differentiation potential, downregulation of MyoD and upre- gulation of Myf5 [47]. So, as MyoD regulates Myog expression 1878 E X P E R I M E N T A L C E L L R E S E [35], Myog is likely to be affected in these cells. We did not observe any transcriptional deregulation of MyoD or Myf5 in human diseases, Exp. Cell Res. 313 (2007) 2121–2133. [6] H.J. Worman, J.C. Courvalin, Nuclear envelope, nuclear lamina, and inherited disease, Int. Rev. Cytol. 246 (2005) 231–279. [7] R.D. Moir, T.P. Spann, H. Herrmann, R.D. Goldman, Disruption of nuclear lamin organization blocks the elongation phase of DNA replication, J. Cell Biol. 149 (2000) 1179–1192. [8] T.P. Spann, R.D. Moir, A.E. Goldman, R. Stick, R.D. Goldman, Disruption of nuclear lamin organization alters the distribution of replication factors and inhibits DNA synthesis, J. Cell Biol. 136 (1997) 1201–1212. [9] C. Favreau, E. Dubosclard, C. Ostlund, C. Vigouroux, J. Capeau, M. Wehnert, D. Higuet, H.J. Worman, J.C. Courvalin, B. Buendia, Expression of lamin A mutated in the carboxyl-terminal tail generates an aberrant nuclear phenotype similar to that observed in cells from patients with [3] Q.S. Padiath, K. Saigoh, R. Schiffmann, H. Asahara, T. Yamada, A. Koeppen, K. Hogan, L.J. Ptacek, Y.H. Fu, Lamin B1 duplications cause autosomal dominant leukodystrophy, Nat. Genet. 38 (2006) 1114–1123. [4] R.A. Hegele, H. Cao, D.M. Liu, G.A. Costain, V. Charlton-Menys, N.W. Rodger, P.N. Durrington, Sequencing of the reannotated LMNB2 gene reveals novel mutations in patients with acquired partial lipodystrophy, Am. J. Hum. Genet. 79 (2006) 383–389. WT-LaA expressing C2C12 cells, although upregulation of Myog transcript level, but not protein level, was impaired. Our observations suggest therefore that lamin A levels per se are critical for proper myogenin expression. Alternatively, mod- ified expression of the LMNA gene may create a shift in the ratio of A- to B-type lamins within the lamina, with a detri- mental effect on nuclear function. To support this view, the recent discovery of a laminopathy caused by a duplication of the LMNB1 gene [3] indicates that a defect in gene dosage resulting in an imbalance of A- to B-type lamins, and not necessarily a lamin mutation per se, can cause profound ef- fects on cell function. Acknowledgments We thank Dr. Soheil Naderi for assistance with FACS analysis and Dr. Edith Heard (Institut Curie, Paris) for the Xist clone. This work was supported by Association Française pour la Lutte Contre les Myopathies (BB, ED), the Norwegian Cancer Society (AMH, PC) and the Research Council of Norway (PC). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2008.02.018. R E F E R E N C E S [1] R. Ben Yaou, A. Muchir, T. Arimura, C. 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Introduction Materials and methods Cells Bisulfite genomic sequencing cDNA synthesis and quantitative (q)RT-PCR Immunofluorescence Western blotting Chromatin immunoprecipitation (ChIP) RNA-fluorescence in situ hybridization (FISH) Flow cytometry Results Expression of R453W-LaA inhibits myogenic differentiation R453W-LaA inhibits upregulation of Myog upon induction of differentiation Inhibition of Myog upregulation by R453W-LaA does not occur at the level of Myog promoter DNA m..... Failure to fully activate Myog in R453W-LaA expressing cells correlates with persistence of H3K..... Overexpression of R453W-LaA alters the nuclear distribution of H3K9me3 Cells expressing R453W-LaA harbor reduced H3K27me3 on the inactive X chromosome Discussion Functional implications of lamin A mutations on myogenesis Effects of wild type lamin A on myogenic differentiation Acknowledgments Supplementary data References