Abstract
Myotonic dystrophy type 1 (DM1) is a multisystem genetic disorder caused by a triplet nucleotide repeat expansion in the 3′ untranslated region of the Dystrophia Myotonica-Protein Kinase (DMPK) gene. DMPK gene transcripts containing CUG expanded repeats accumulate in nuclear foci and ultimately cause altered splicing/gene expression of numerous secondary genes. The study of primary cell cultures derived from patients with DM1 has allowed the identification and further characterization of molecular mechanisms underlying the pathology in the natural context of the disease. In this study we show for the first time impaired nuclear structure in fibroblasts of DM1 patients. DM1-derived fibroblasts exhibited altered localization of the nuclear envelope (NE) proteins emerin and lamins A/C and B1 with concomitant increased size and altered shape of nuclei. Abnormal NE organization is more common in DM1 fibroblasts containing abundant nuclear foci, implying expression of the expanded RNA as determinant of nuclear defects. That transient expression of the DMPK 3′ UTR containing 960 CTG but not with the 3′ UTR lacking CTG repeats is sufficient to generate NE disruption in normal fibroblasts confirms the direct impact of mutant RNA on NE architecture. We also evidence nucleoli distortion in DM1 fibroblasts by immunostaining of the nucleolar protein fibrillarin, implying a broader effect of the mutant RNA on nuclear structure. In summary, these findings reveal that NE disruption, a hallmark of laminopathy disorders, is a novel characteristic of DM1.
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Introduction
Myotonic dystrophy type 1 (DM1) is an autosomal dominant inherited disease that represents the most common form of muscular dystrophy in adults, with a prevalence of 1 in 8,000 individuals worldwide [1]. DM1 is characterized mainly by myotonia (sustained muscle contraction), progressive muscle weakness and wasting as well as variable multisystemic features, including insulin resistance, cardiac conduction defects, gonadal atrophy, early posterior iridescent cataracts, and central nervous alterations [1, 2]. DM1 is caused by an expanded CTG repeat in the 3′-untranslated region (3′ UTR) of the DMPK gene in chromosome 19q [3–5]. Because of the location of CTG repeats in the 3′ UTR, it has been difficult to explain how a mutation in a non-coding region could cause the multisystemic features of DM1.
DM1 appears to be caused by different molecular mechanisms, including DMPK haploinsufficiency and a toxic gain-of-function by the expanded CUG repeat in mutant DMPK mRNA (reviewed in [6, 7]). Due to decreased levels of mRNA and protein of DMPK gene in adult DM1 tissue [8], the functional implications of a reduction in DMPK gene expression were genetically tested by generation of DMPK gene knockout mice [9]. However, Dmpk −/− mice exhibited only a mild late-onset, progressive skeletal myopathy with some cardiac- conduction abnormalities [10], implying that simply DMPK deficiency does not explain the multi systemic features of DM1. The latter hypothesis proposes that mutant DMPK mRNA containing expanded CUG repeats accumulates in the nucleus and folds into RNA hairpins, trapping alternative splicing factors, including muscleblind-like splicing regulator 1 (MBNL1) and hnRNP H, and activating the alternative splicing factor CUG triplet repeat RNA-binding protein 1 (CUG-BP1), also denominated CUGBP1/Elav-like family member 1 (CELF1), through hyperphosphorylation and stabilization in the cell nucleus [11–15], which in turns causes interference in developmentally regulated alternative splicing of defined pre-mRNAs [16–20]. In addition, mutant DMPK mRNA also binds and sequesters transcription factors, including Sp1 (specific protein 1) and STAT1 and 3 (members of the signal transducer and activator of transcription family), leading to reduce transcription of selected genes [19]. Furthermore, it has recently shown that CTG-repeat expansion induces repressive changes in chromatin dynamics [21].
In spite of the fact that expression of the CUG expanded repeats-containing mRNA interferes with different nuclear processes, and of the compelling evidence showing functional interrelationship between nuclear structure and function [22], the impact of this pathological transcript on nuclear architecture is largely unknown. In this study we show for the first time that expression of DM1-associated CUG repeats impairs the organization of different nuclear compartments, including the nuclear envelope (NE) and nucleoli, which results in nuclear morphology alterations in both DM1-derived fibroblasts and normal fibroblasts transiently expressing CUG repeats.
Materials and methods
Cell culture and transfection
Control [GM02673 (CTG5/19)] and DM1-derived skin fibroblasts [GM04033 (CTG1000) and GM03132 (CTG2000)] were purchased from Coriell Cell Repositories (Camden, NJ, USA). The length of the repeats in cell cultures was confirmed by Triplet repeat primed PCR as previously [23]. Fibroblast cultures were maintained in minimal essential medium (MEM; Invitrogen, Carlsbad, CA, USA) supplemented with 15 % fetal bovine serum and nonessential amino acids at 37 °C in a humidified 5 % CO2 atmosphere. For transfection, primary fibroblasts were seeded onto glass coverslips and grown overnight to ~50 % confluence. On the following day, fibroblasts were transfected with 2 µg of DNA pre-mixed with 4 µl of lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in 100 µl serum-free MEM medium for 20 min at room temperature. After 1 h, the cells were washed and changed to complete MEM medium for 48 h prior to analysis.
RNA extraction and quantitative polymerase chain reaction (RT-qPCR) analysis
Total RNA was isolated from fibroblast cultures using TRIzol Reagent (Invitrogen, Thermo Fisher Scientific Inc. MA. USA), quantified on a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc. MA. USA) and reverse transcribed using random hexanucleotides and the M-MLV reverse transcriptase (Invitrogen), according to manufacturer’s instructions. PCR was carried out in 25 µl, containing 12.5 μl Maxima SYBR Green/ROX qPCR Master Mix 2X, 1.5 μl cDNA, and 100 nM of each primer in a StepOnePlus™ Real-Time PCR System (Applied Biosystems). Expression of lamins A and C was quantified by the 2ΔΔct method normalized to GAPDH. Primer sequences were as follow: lamin A, forward 5′-CACTGGGGAAGAAGTGGCCA-3′ and reverse 5′-GAGCCGTGGTGGTGATGGAG-3′; lamin C forward 5′-CACTGGGGAAGAAGTGGCCA-3′ and reverse 5′ TACCACTCACGTGGTGGTGA-3′ and GAPDH, forward 5′-CGCTCTCTGCTCCTCCTGTT-3′ and reverse 5′-CCATGGTGTCTGAGCGATGT-3′.
Antibodies and vectors
The following primary antibodies were used: rabbit polyclonal antibodies anti-lamin A/C (H-110; 1:4,000 for WB), anti-emerin (FL-254; 1:25 for IF) and anti-lamin B1 (H90; 1:25 for IF), as well as a mouse polyclonal anti-GAPDH (6C5; 1:2,000 for WB) were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc., CA, USA). Rabbit polyclonal anti-fibrillarin (ab-5821 1:50 for IF) and mouse monoclonal anti-lamin A/C (1:250 dilution for IF) antibodies were acquired from Abcam (Abcam, Cambridge, MA, USA), while mouse monoclonal anti-actin antibody [24] (1:2,000 for WB) was kindly provided by Dr. Manuel Hernández from CINVESTAV.
Vectors pQBI960 and pQBIDM, expressing the DMPK 3′ UTR (exons 13–15) with 960 CTG or with no CTG repeats respectively, were generated by subcloning a BamHI–BamHI (0 CTG repeats) or BamHI–HindIII (960 CTG repeats) restriction fragment into pDGFP [25], using DT960 and DMPKS plasmids [26] as templates respectively.
Immunofluorescence and confocal microscopy analysis
Cells grown on coverslips were fixed with 4 % paraformaldehyde for 10 min and permeabilized by exposure to 0.2 % Triton X-100 in PBS for 10 min at room temperature. Cells were then incubated overnight at 4 °C with the appropriate primary antibody and the following day washed with PBS and incubated for 1 h at room temperature with a fluorescein-conjugated goat anti-mouse or -rabbit IgG (Zymed Laboratories, Inc. San Francisco, CA, USA). Nuclei were counterstained with 0.2 μg/μl DAPI (Sigma-Aldrich, St Louis, Missouri, USA) for 7 min at room temperature and washed with PBS, and then cell preparations were mounted on microscope slides with VectaShield (Vector Laboratories, Inc., Burlingame, CA, USA) prior to being examined on a confocal laser scanning microscope (TCP-SP5 or TCS-SP8 Leica, Heidelberg, Germany) employing a Plan Neo Fluor 63× (NA = 1.4) oil-immersion objective.
For nuclear envelope morphological analysis, 500 cells from each culture (control and DM1-derived fibroblasts) were analyzed. Fibroblast nuclei were scored as normal if showed the typical ring-like immunostaining pattern for lamin A/C, lamin B1 or emerin, and their appearance under DAPI-staining was spheroid or ellipsoid. On the other hand, nuclei were scored abnormal if exhibited intense nucleoplasmic staining, invaginations, lobules, and/or blebs under lamin A/C, lamin B1 or emerin immunolabeling analysis; and/or if they were at least twice larger than those in control cells under DAPI staining examination. Likewise, 500 cells from each culture (control and DM1-derived fibroblasts) were scored for nucleoli morphological analysis. Fibroblast nucleoli were scored as normal if 2–3 large rounded nucleoli were visible per cell after fibrillarin-immunostaining, and scored as distorted if cells contain a single elongated nucleolus or numerous dispersed small nucleoli after fibrillarin-immunostaining, as previously [27].
FISH (fluorescence in situ hybridization) and FISH-IF
The cells grown on coverslips were fixed with 4 % paraformaldehyde for 10 min and permeabilized by exposure to cold 2 % acetone in PBS for 5 min. Cell preparations were incubated in pre-hibrydation buffer (SSC 2×, 30 % formamide) for 10 min at room temperature and then incubated with hibrydation buffer [SSC 2×, 30 % formamide, 0.02 % BSA, 2 mM vanadyl ribonucleoside (Sigma-Aldrich Inc., MO, USA), 66 μg/ml yeast tRNA (Sigma-Aldrich Inc.), and 1 ng/μl Cy3-conjugated CAG6 probe (Sigma-Aldrich Inc.)] for 2 h in a humidified chamber at 37 °C. Preparations were washed as follows: twice in prehybridation buffer at 42 °C, twice in SCC 1× at room temperature, and once in PBS. For immunofluorescence after FISH, the slides were incubated in PBS with 0.5 % gelatin and 1.5 % BSA for 20 min at room temperature and then incubated overnight at 4 °C with primary anti-lamin A/C antibody. After washing three times with PBS cell preparations were incubated for 1 h at room temperature with a fluorescein-conjugated anti-mouse IgG (Zymed Laboratories, Inc., San Francisco, CA, USA). Nuclei were counterstained with DAPI (0.2 μg/μl, Sigma-Aldrich Inc.) for 7 min at room temperature, washed with PBS, and mounted on microscope slides with VectaShield (Vector Laboratories, Inc., Burlingame, CA, USA) for confocal microscopy analysis.
Western blotting
The cells were centrifuged and resuspended in 50 μl of lysis buffer (10 mM Tris-HCl pH 8.0, 2 mM MgCl2, 1 % Triton ×100, 1× Complete and 1 mM PMSF protease inhibitors cocktail). Homogenates were clarified by centrifugation at 15,000×g for 2 min at 4 °C, and protein concentrations were determined by the Bradford method. Protein extracts were electrophoresed on 10 % SDS–polyacrylamide gels and transferred onto nitrocellulose membranes (BIO-RAD, Germany). Membranes were blocked in TBST [100 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.5 % (v/v) Tween-20] with 10 % (w/v) low-fat dried milk and then incubated overnight at 4 °C with the appropriate primary antibody, and the following day washed three times with TBST and incubated for 1 h at room temperature with the appropriate secondary antibody and then washed three times with TBST. Specific proteins were visualized using the enhanced chemiluminescence (ECL™) western blotting detection system (Amersham Pharmacia, GE Healthcare) according to the manufacturer’s instructions.
Morphometric analysis
To analyze changes in nuclear shape, 500 randomly selected nuclei from each culture were measured as previously [28–30]. The nuclear contour ratio (4π × area/perimeter2) and the crossed diameter ratio (length/width) were calculated using the measure function in the NIS-Elements AR Version 4.13.01 (Nikon) Software. Objects were selected automatically using the smart threshold function and reviewed manually using the binary menu functions.
Statistical analysis
Statistical analyses were performed using the two-tailed Mann–Whitney test and the GraphPad Prism 6 software (La Jolla California USA, www.graphpad.com), with the exception of quantitative reverse transcription PCR (qRT-PCR) assays that were analyzed using one-way analysis of variance (one-way ANOVA).
Results
Altered nuclear morphology in DM1-derived fibroblasts
To examine whether DM1 gene mutation impacts nuclear morphology, fibroblasts derived from patients harboring mutant alleles with CTG1000 or CTG2000 repeats were immunolabeled for lamin A/C, and further analyzed by confocal microscopy, using fibroblast from age-matched healthy subject containing alleles with 5 and 19 CTG repeats (CTG5/19) as control. Confocal imaging revealed increased number of DM1-derived fibroblast nuclei showing irregular immunolabeling pattern for lamin A/C, characterized by increased immunostaining at both NE and nucleoplasm, invaginations of the NE, and an apparent augmented size of nuclei, compared with control fibroblasts (Fig. 1a). Quantitative analysis showed 30.8 and 23.7 % of nuclei with altered localization of lamin A/C in DM1-derived fibroblasts with CTG1000 and CTG2000 repeats respectively, compared to only about 12.1 % in control fibroblasts (Fig. 1b). Irregular NE immunostaining in DM1-derived fibroblasts were also evident using antibodies against lamin B1 and emerin, including increased immunolabeling and NE invaginations (Fig. 2a).
A quantitative analysis showed significantly higher percentage of cells with altered localization of both lamin B1 and emerin in CTG2000 DM1 than in control cultures (Fig. 2b) Morphometric analysis of 500 randomly chosen nuclei revealed a subtle but statistically significant differences in crossed diameter and nuclear contour ratios between control and DM1-derived fibroblasts (Table 1); however, selective analysis of 30 DM1 nuclei that exhibited altered localization of lamin A/C revealed greater alterations in crossed diameter (2.00 ± 0.03 and 2.46 ± 0.1 for control and CTG2000 DM1 fibroblasts respectively, p = < 0.0001) and nuclear contour (0.94 ± 0.005 and 0.86 ± 0.01 for control and CTG2000 DM1 fibroblasts respectively, p = < 0.0001) ratios. Increased crossed diameter ratio reflects an augment in size, while decreased contour ratio indicates nuclear deformation (i.e. blebbed or lobed nuclei) [28–30]. Overall these data imply that DM1 mutation affects global NE organization and ultimately nuclear morphology. In spite of the increased immunolabeling and mislocalization of lamin A/C found in DM1-derived fibroblasts, both qRT-PCR and western blotting assays showed similar lamin A/lamin C expression ratio between control and either CTG1000 and CTG2000 DM1 fibroblasts (Fig. 1c, d).
Nuclear foci containing CUG expanded repeats induce nuclear morphology defects
To ascertain whether nuclear shape abnormalities are related with expression of expanded CUG repeats, in situ hybridization assay using a fluorescent-labeled (CAG)6 probe (FISH), follow by immunofluorescence assay (FISH-IF) for lamin A/C was carried out in CTG2000 DMI fibroblasts. Interestingly, the presence of nuclear foci appears to be directly related with nuclear shape abnormalities (Fig. 3a); the higher the number of nuclear foci, the higher the percentage of misshapen nuclei (Fig. 3b). To test directly the hypothesis that expression of CUG expanded repeats induce nuclei morphology defects, control fibroblasts (CTG5/19) were transiently transfected with vector expressing the DMPK 3′ UTR containing 960 interrupted CTG repeats, and further subjected to FISH-IF. Transient expression of CTG-expanded repeats resulted in altered distribution of lamin A/C and abnormal nuclear shape, while typical ring-like distribution of lamin A/C in the NE and the presence of oval shaped nuclei were consistently observed in control fibroblasts transiently-transfected with vector expressing the DMPK 3′ UTR containing no CTG repeats (Fig. 4). A quantitative analysis showed 37.00 ± 2.7 and 79.27 ± 5.2 % of misshapen nuclei in control and DM1-derived fibroblasts respectively (right panel).
Nucleoli disruption in DM1-derived fibroblasts
To ascertain whether CTG-expanded repeats alter the organization of other subnuclear structures, the structure of nucleoli was analyzed by immunostaining nucleoli in control and CTG2000 DM1 fibroblasts using antibodies against the nucleolar protein fibrillarin. DM1-derived fibroblasts exhibited high percentage of distorted nucleoli, compared with control fibroblasts (Fig. 5; 36.0 ± 2.17 and 55.77 ± 2.08 % for control and DM1 fibroblasts respectively), which implies that expanded CTG repeats altered the spatial distribution of nucleoli.
Discussion
In this study we provide for the first time evidence showing both aberrant nuclear morphology and disorganization of nucleoli in DM1 cells. We found altered NE organization in a fraction of 30 % of fibroblasts derived from DM1 patients, evidenced by increased immunolabeling and mislocalization of the NE proteins, lamins A/C and B1 and emerin. Although metric measurements showed subtle changes in nuclear shape in DM1-derived cells, greater nuclear morphology alterations that resemble those reported in laminopathies [28–30] were found in the fraction of DM1 nuclei that displayed mislocalization of lamin A/C. Interestingly, the occurrence of misshapen nuclei was more frequent in DM1-fibroblasts containing numerous nuclear foci, which implicates the mutant DMPK mRNA in the development of nuclear morphology defects. A recent work showed that depletion of DPMK result in NE disruption, implicating the DM1 gene protein in NE stability [31]. Thus, it is possible that haploinsufficiency of DMPK in DM1-derived fibroblasts might alter NE organization. Nevertheless, that NE disorganization was induced in control fibroblasts through transient overexpression of the DMPK 3′ UTR containing 960 CTG but not with the 3′ UTR lacking CTG repeats implies that expression of the toxic expanded mRNA rather than reduction of DMPK is critical for NE impairing in our cell model.
Although molecular mechanisms that participate in defining nuclear shape have remained unclear, the important role of NE components in determining nuclear architecture is highlighted by the abnormal shaped nuclei observed in a variety of human disease, collectively called laminopathies, where genes encoding lamin or lamin-binding proteins are mutated [32, 33]. It is difficult at this point to hypothesize how the expression of CUG-containing mutant mRNA impacts nuclear morphology; however, in the light of the current knowledge of molecular bases underlying DM1 it could be speculated that aberrant RNA splicing caused by the expanded RNA [7] might affect the alternative splicing of LMNA gene, resulting in a shift in the balance of lamin A/C ratio, and ultimately in nuclear lamin disorganization. Nevertheless, qRT-PCR and immunoblotting analyses showing similar lamin A/lamin C expression ratio at mRNA and protein level between control and DM1-derived fibroblasts is inconsistent with this possibility. As lipins, a family of lipid phosphatases, have been implicated in the maintenance of nuclear shape [34–36], it should be noted that aberrant alternative splicing/gene expression of genes involved in lipid metabolism was found in a mouse model of DM1, including LPIN1, ATP9a, PICALM, and ACSL5 genes [37]. Clearly, the demonstration that the occurrence of mis-splicing events in genes involved in lipid metabolism in DM1 fibroblasts and its potential connection with nuclear membrane morphology requires further investigation.
An alternative likely mechanism to explain impaired nuclear integrity in DM1 fibroblasts is the effect exerted by the expanded RNA on chromatin dynamics, including altered nucleosome positioning [38–40] and chromatin remodeling [21]. Experimental evidence showing that conformational changes in chromatin structure are at least partially responsible for shaping the nucleus has recently been accumulated: downregulation of either BAF, an adaptor protein between nuclear lamin and chromatin, and p55, a component of histone modifying and chromatin assembly complexes, alters nuclear morphology in Drosophila cells [41]. Likewise, decreased expression of the chromatin remodeling ATPase BRG1 causes nuclear shaped changes in MCF-10A cells [42]. Furthermore, overexpression of Esc1p, an anchor for chromatin results in abnormal nuclear morphology in yeast cells [43]. Additional experiments focusing on setting out alterations in chromatin structure in DM1 fibroblasts and their potential connection with nuclear morphology defects will be undertaken in our laboratory.
Altered lamina A/C distribution in DM1 fibroblasts with a direct effect on nuclear structure implies that DM1 pathogenesis might share pathological mechanisms with muscular dystrophies caused by mutations in the LMNA gene, including different Emery-Dreifuss muscular dystrophies and limb-girdle muscular dystrophy type 1B. Besides some clinical features that are shared between DM1 and skeletal muscle laminopathies, including muscle weakness and wasting and cardiomyopathy [44, 45], it appears that altered myogenesis signaling pathway is a common molecular mechanism to the two types of muscular dystrophy. On the one hand, it has been shown that overexpression of the mutant DMPK 3′UTR mRNA disrupts myoblast fusion in both myoblast cell line C2C12 [46] and primary myoblasts from transgenic mice [47]. At molecular level, expression of the mutant DMPK 3′UTR mRNA results in downregulation of both the cell cycle regulator factor p21 [48] and the key myogenic transcription factor MyoD [49], which ultimately impair muscle differentiation. On the other hand, it is thought that mutation in the LMNA gene disrupts the interaction of lamin A/C and its partner LAP2α with different transcription factors and chromatin modifying complexes, leading to altered regulation of gene expression during muscle differentiation (reviewed in [50]). Supporting this idea, myoblasts generated from Lmna −/− mice exhibit decreased levels of proteins necessary for muscle cell differentiation, including MyoD, desmin, pRB and M-cadherin [51].
Fibroblasts derived from DM1 patients also exhibit nucleoli disorganization. The nucleolus is a multifunctional nuclear body with multiple roles, including ribosome biogenesis, cell proliferation, cell growth and survival, and the response to stress [52]. Importantly, disruption of nucleolar integrity is associated with aging and early response to cellular stress, and is underlying several human diseases, including neurodegenerative disorders and cancer [53]. Although the impact of the DMPK gene expanded RNA in specific functions associated with nucleoli remains to be elucidated, it is possible that mislocalization of lamin B1 observed in DM1 fibroblasts causes in turn altered nucleoli structure, because lamin B1 is involved in nucleoli organization [54], preserving its functional plasticity [27]. Interestingly, expanded mutant Huntingtin transcript interacts with the nucleolar protein nucleolin and this aberrant interaction leads to down-regulation of pre-45s rRNA transcription [55]. Thus, the possibility that the DMPK mutant mRNA sequesters components of nucleoli altering their architecture can not be ruled out.
In conclusion, we describe to the best of our knowledge for the first time alterations in the organization of the nuclear envelope and nucleoli in DM1 cells, and demonstrate that expression of the expanded DMPK gene RNA is underlying nuclear structure alterations. It is thought that impaired NE organization results in altered DNA repair, gene expression, and cell cycle, and ultimately causes apoptosis and aneuploidy [32, 33] Therefore, definition of the physiological relevance of altered NE disorganization in DM1 requires further investigation of NE-dependent processes in DM1 models.
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Acknowledgments
This work was supported by Science and Technology Institute of Mexico City Grant (PIFUTP08-164). We thank Dr. Thomas Cooper (Baylor College of Medicine) for donation of DMPKS and DT960 vectors.
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Rodríguez, R., Hernández-Hernández, O., Magaña, J. et al. Altered nuclear structure in myotonic dystrophy type 1-derived fibroblasts. Mol Biol Rep 42, 479–488 (2015). https://doi.org/10.1007/s11033-014-3791-4
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DOI: https://doi.org/10.1007/s11033-014-3791-4