Articles in PresS. Physiol Genomics (July 26, 2005). doi:10.1152/physiolgenomics.00060.2005
Alterations of nuclear envelope and chromatin organization in
mandibuloacral dysplasia, a rare form of laminopathy
Ilaria Filesi1, Francesca Gullotta2, Giovanna Lattanzi3, Maria Rosaria D’Apice2,
Cristina Capanni4, Anna Maria Nardone2, Marta Columbaro4, Gioacchino Scarano5
Elisabetta Mattioli4, Patrizia Sabatelli3, Nadir M. Maraldi3,4, Silvia Biocca1 and
Giuseppe Novelli2
1
Laboratory of Clinical Biochemistry and Department of Neuroscience, 2Laboratory of
Medical Genetics, University of Roma Tor Vergata, Italy; 3ITOI, CNR, Unit of Bologna,
c/o IOR, Bologna, Italy; 4Laboratory of Cell Biology, Istituti Ortopedici Rizzoli, Bologna,
Italy; 5 Medical Genetics Division, Hospital of Benevento, Italy
Correspondence to: Giuseppe Novelli
Department of Biopathology and Diagnostic Imaging,
University of Tor Vergata, Via Montpellier 1, 00133, Rome, Italy
(e-mail: [email protected])
Running Title: Chromatin organization in mandibuloacral dysplasia
Copyright © 2005 by the American Physiological Society.
2
The autosomal recessive mandibuloacral dysplasia (MADA; OMIM #248370) is caused by a
mutation in LMNA encoding lamin A/C. Here we show that this mutation causes accumulation
of the lamin A precursor protein, a marked alteration of the nuclear architecture and, hence,
chromatin disorganization. Heterochromatin domains are altered or completely lost in MADA
nuclei, consistent with the finding that heterochromatin-associated proteins HP1 and histone
H3 methylated at lysine 9 and their nuclear envelope partner protein lamin B receptor (LBR)
are delocalised and solubilised. Both accumulation of lamin A precursor and chromatin defects
become more severe in older patients. These results strongly suggest that altered chromatin
remodelling is a key event in the cascade of epigenetic events causing MADA and could be
related to the premature aging phenotype.
Keywords: mandibuloacral dysplasia; LMNA; heterochromatin; HP1 ; pre-lamin A
3
MANDIBULOACRAL DYSPLASIA type A (MADA; OMIM #248370) is a rare and complex
disease characterised by postnatal growth retardation, craniofacial anomalies, skeletal
malformations, mottled cutaneous pigmentation, partial lipodystrophy (type A pattern) and
insulin resistance (11, 17, 49). MADA patients seem to be genetically homogeneous since they
show the same mutation (R527H) in the LMNA gene that encodes A-type lamins, lamins A and
C (35, 42). In contrast, patients with generalized loss of subcutaneous fat involving the face,
trunk, and extremities (type B pattern) carry mutations in the ZMPSTE24 gene (MADB;
OMIM #608612) (1, 43).
Lamins are type V intermediate filament proteins that display a central rod domain, a Nterminal head domain and a C-terminal globular tail. Lamins A and C, together with B type
lamins, lamin B1 and B2, are the major components of the nuclear lamina, located between the
inner nuclear membrane and the chromatin. A growing number of proteins are known to
interact with lamins. Numerous experimental evidences suggest that nuclear lamins are
involved in many functions including nuclear positioning and shape, chromatin organization,
nuclear envelope assembly/disassembly, DNA replication, and regulation of gene
transcriptional activity (18). The multifunctional aspect of lamins inside cells may explain the
large phenotype spectrum observed in patients with a hereditary dysfunction in lamin A and C
gene (27, 31, 36).
MADA is a specific genetic entity belonging to a class of genetic disorders called
“laminopathies” including the autosomal dominant and autosomal recessive Emery-Dreifuss
muscular dystrophy (EDMD2; OMIM #181350; EDMD3; OMIM #604929), the limb-girdle
muscular dystrophy type 1B (LGMD1B; OMIM #159001), the Hutchinson-Gilford progeria
syndrome (HGPS; OMIM #176670), the “atypical” Werner syndrome, a dilated
cardiomyopathy with conduction defect (CMD1A OMIM #115200), the Charcot-Marie-Tooth
disorder type 2B1 (CMT2B1; OMIM #605588), the Dunnigan-type familial partial
lypodystrophy (FPLD2; OMIM #151660) and the restrictive dermopathy (RD; OMIM
#275210) (31). There is a considerable debate on how mutations in the LMNA gene promote a
large number and different phenotypes and why certain mutations can give rise to tissuespecific effects (for a review: 5, 22, 23, 24, 28). Several models have been proposed to
explicate this paradox: nuclear fragility, alteration of gene expression patterns, and
4
modification of the relationships between the nuclear membrane and the endoplasmic
reticulum (ER) (for a Review: 27, 36).
Here, we show that the R527H lamin A/C mutation which causes MADA, produces
accumulation of unprocessed pre-lamin A, altered distribution of the lamin B receptor (LBR)
and destabilization of two heterochromatin-associated proteins, histone H3 methylated at lysine
9 (Me9H3) and heterochromatin protein 1 (HP1 ).
5
MATERIALS AND METHODS
Patient Samples.
Human fibroblasts were isolated from skin biopsies (dorsal foream) obtained from three
MADA patients and from three control subjects. All biopsies were obtained under
institutionally approved protocols (Tor Vergata University, Rome, “Gaetano Rummo”
Hospital, Benevento, and Italian Dermatological Institute, Rome).
MADA patients (MADA-1, female, and MADA-2 and MADA-3, males) underwent skin
biopsy at 18 (MADA-1), 35 (MADA-2) and 50 (MADA-3) years of age. All MADA patients
were homozygous for the R527H mutation and they showed the same clinical phenotype
without important differences. The three control biopsies were age and sex matched. Fibroblast
cultures were established by mechanical and enzymatic methods and cultured in Dulbecco’s
modified Eagle’s-F12 medium (Cambrex) supplemented with 15% fetal bovine serum
(Cambrex) and antibiotics. The passage number of each cell type was recorded and cells were
analysed between passage 2 and 6.
Immunofluorescence staining
Human fibroblasts were grown on coverslips coated with poly-L-Lysine, rinsed in PBS and
fixed for 10 min with 4% (w/v) paraformaldehyde (in PBS). Cells were permeabilized for 5
min with 0.1% Triton X-100 in 100 mM Tris/HCl pH 7,5. Incubation with affinity purified rat
anti-HP1 IgG (Mac 353) (46) and rabbit anti-Me9H3 IgG (13) was carried out at room
temperature for 1h. For LBR, pre-lamin A and emerin detection, cells were fixed and
permeabilised with cold methanol at -20°C for 7 min, rinsed in PBS and incubated over-night
at 4°C with polyclonal rabbit anti-LBR IgG, or polyclonal goat anti pre-lamin A antibody
(Santa Cruz, sc-6214), or anti-emerin mouse monoclonal antibody (Novocastra). CyTM2conjugated*AffiniPure Donkey anti-rabbit IgG (Jackson), FITCconjugated anti-goat IgG
(DAKO), Cy3-conjugated anti-mouse IgG (Sigma) and Texas Red anti-mouse IgG
(Calbiochem) were used as secondary antibodies. Hoechst 33342 dye was used at 300ng/ml.
Samples were examined with a Leica fluorescence microscope equipped with a CCD camera.
Acquired images were deconvolved using Leica Qfluoro software and processed using Adobe
Photoshop.
6
Western blot analysis
Human fibroblasts were lysed in ice-cold 10 mM Tris-HCl buffer pH 7.4 containing 1% Triton
X-100, 0.25% sodium deoxycholate, 150mM NaCl, 1mM EGTA, 1mM PMFS, 10 µM
aprotinin, leupeptin, pepstatin. For pre-lamin A detection, 1% SDS was added to the extraction
buffer. Blots were probed with anti-lamin A/C (mouse monoclonal, Novocastra), anti-pre
lamin A (goat polyclonal, Santa Cruz, sc-6214), anti-emerin (mouse monoclonal, Novocastra),
anti-actin (goat polyclonal, Santa Cruz), anti-HP1 (rat monoclonal Mac 353) and anti-Me9H3
(rabbit polyclonal) (13). Immunoreactive bands were detected with horse-radish peroxidaseconjugated secondary antibodies (Pierce) and visualized by enhanced chemiluminescence
(Amersham, UK). Densitometry was performed using a Biorad GS-800 calibrated
densitometer. Data were reported as percentage of control fibroblast densitometry and means
of three different analyses were calculated.
Electron microscopy
Cell pellets from confluent control and MADA fibroblast cultures were fixed with 2.5%
glutaraldehyde-0.1 M phosphate buffer pH 7.6 for 1 hour at room temperature. After treatment
with 1% OsO4 in veronal buffer for 1 hour, pellets were dehydrated in an ethanol series and
embedded in Epon resin. Thin sections stained with uranyl acetate and lead citrate were
observed with a Philips EM 400 transmission electron microscope, operated at 100 kV. At least
200 nuclei per sample were observed. Statistical analysis was performed by counting nuclei
from three different preparations per each examined sample.
Preparation of nuclear and cytoplasmic fractions and Western Blot analysis
Human fibroblasts were washed twice in PBS, scraped and collected. The nuclear and
cytoplasmic fractions were prepared by suspending cells in 0,3 ml of hypotonic isolation buffer
(IB) (10 mM Tris-HCl pH 7.6, 10 mM NaCl, 1.5 mM MgCl2, protease inhibitor cocktail
[1:1000](Calbiochem) and 0.1 mM phenyl-methyl-sulfonyl-fluoride (PMSF). Cells were
passed through an ice-cold cylinder cell homogenizer and nuclei isolated by centrifuging at
4°C for 15 min at 290 g. Nuclear pellets were washed twice with 0.3 ml of IB, incubated for 30
min on ice in modified IB with 1% Triton X-100 and centrifuged at 12,000 g for 15 min to
separate the soluble and the insoluble nuclear fractions. The cytoplasmic supernatant, after two
subsequent centrifugations for clearing from cell debris, was detergent extracted by adding
7
0.5% NonidetP40 (Sigma) for 30 min on ice and centrifuged twice (12,000 g at 4°C for 15
min). The cytoplasmic pellet was washed twice in NP40 enriched IB buffer by subsequent
centrifugations at 12,000 g for 15 min. Equal amounts of soluble (S) and insoluble (I) proteins
from nuclei and cytoplasm were separated on SDS-PAGE in 12% acrylamide gels and blotted.
8
RESULTS
The cellular level of pre-lamin A is increased in cells of MADA patients.
Fibroblast cultures from an healthy patient, aged 35 and three MADA patients aged 18
(MADA-1), 35 (MADA-2) and 50 years (MADA-3), carrying the same R527H LMNA
mutation were analyzed. In particular, we examined, by Western blot, the LMNA products prelamin A, lamin A and lamin C. We observed accumulation of pre-lamin A in fibroblasts
derived from MADA patients, with a linear increase of protein amount in older patients (Fig.
1a and b). Lamin A level was unaffected in the younger subject and progressively reduced in
fibroblasts derived from the older patients (Fig. 1a and b). Lamin C level was slightly reduced
in MADA-3 fibroblasts (Fig. 1a). The expression level of the lamin A/C-binding protein
emerin was not altered in MADA fibroblasts (Fig. 1a).
We then analysed, by double immunofluorescence, the intracellular localisation of pre-lamin A
and emerin in control and MADA nuclei. Control cells (Fig. 2, panel a) expressed low amount
of pre-lamin A that appeared mostly distributed at the nuclear envelope. In marked contrast,
MADA nuclei showed accumulation of pre-lamin A in the nuclear envelope, associated with
formation of intra-nuclear pre-lamin labelled structures (Fig. 2, panels b, c and d). Emerin was
localized at the nuclear envelope of MADA-1 and MADA-2 nuclei (Fig. 2, panels f and g),
while it showed a honeycomb labelling pattern in MADA-3 cells (Fig. 2, panel h). Partial colocalization of pre-lamin A with emerin was observed in MADA-1 and MADA-2 nuclei, but
co-localization was lost in most MADA-3 nuclei (Fig. 2, panels j, k and l).
LBR staining of the nuclear envelope is lost in MADA nuclei
To further investigate whether the observed accumulation of pre-lamin A in MADA nuclei is
related with changes in the distribution of proteins of the nuclear envelope, we studied the
localization of lamin B receptor (LBR) in these cells (Fig. 3). In control fibroblasts, LBR was
localized at the nuclear rim (Fig. 3, panel a). A minor percentage of nuclei (7%) showed
intranuclear diffuse staining of LBR (Fig. 3, panel b). Interestingly, these cells presented also a
higher pre-lamin A level at the nuclear envelope (Fig. 3, panels f and g). In MADA cells, on
the contrary, LBR distribution was altered in a high percentage of cells. Thus, about 50% of
MADA-1 nuclei showed both nuclear rim and diffuse nucleoplasmic staining (Fig. 3, panels c
9
and h). Nucleoplasmic staining, cytoplasmic localization and a reduced nuclear envelope
labelling of LBR was typical of 60% of MADA-2 (Fig. 3, panels d and i) and 65% of MADA-3
cells (panels e and j). In some cells, LBR staining at the nuclear rim was almost completely lost
(Fig. 3, panel e and j). Both lamin A precursor accumulation at the nuclear envelope (Fig. 2,
panels b, c and d) and LBR nucleoplasmic localization (Fig 3, panels h, i and j) increased with
patient age. Nucleoporins and lamin B were correctly localized in all examined fibroblasts
(data not shown).
Heterochromatin organization is lost in MADA nuclei.
The ultrastructural morphology of MADA fibroblasts was examined by electron microscopy.
This analysis revealed striking nuclear alterations (Fig. 4). In particular, invaginations of the
nuclear envelope or thin papillary projections characterized 15-40% of nuclei (Fig. 4, panels b
and c). We observed nuclear dysmorphysm, irregular thickness of the nuclear lamina (Fig. 4,
panel d), focal absence of peripheral heterochromatin or complete heterochromatin loss (Fig. 4,
panel e). In some nuclei, projections, invaginations of the nuclear envelope and peripheral
heterochromatin loss were simultaneously observed. A minor percentage of nuclei showed
scarce density of interchromatin when compared with controls (Fig. 4, panel f). All these
alterations were absent in control nuclei (Fig. 4, panel a). The observed nuclear defects were
more represented in older MADA cells (Fig. 4, panel g). In particular, complete absence of
heterochromatin from the nuclear periphery ranged from 1-2% to 40% (out of 200 nuclei
examined) according to patient’s age (Fig. 4, panel g).
The distribution of HP1 and Me9H3 is affected in MADA nuclei.
To test whether nuclear envelope alterations affect the heterochromatin organization, two
major structural components of heterochromatin, the heterochromatin protein 1 (HP1 ), and
trimethylated histone H3 Lys9 (Me9H3) were studied by double immunofluorescence. In
control fibroblasts (Fig. 5, panels a, e, i and m) the nuclei were round or ovoid and the
distribution pattern of Me9H3 and HP1 appeared dispersed in multiple small foci filling the
whole nuclear area. A strong co-localisation of both proteins was evident in merged images
(Fig. 5, panel m). This pattern appeared partially modified in MADA cells in a way that
reflected the age of the patient. In particular, in fibroblasts derived from MADA-1 patient the
nuclear morphology was essentially preserved although a more distinctive localization of
10
Me9H3 and HP1
was detected in about 15% of cells (Fig. 5, panels b, f, j and n).
Interestingly, these cells displayed also a punctuate DNA distribution, visualized by Hoechst
staining (panel b) different from the typical more uniform DNA staining of control cells (panel
a). These fibroblasts accumulated a distinct chromatin structure enriched with heterochromatin
proteins, similar to the recently described senescence-associated heterochromatin foci (SAHF)
(33). In 5-10% of MADA-2 (Fig. 5, panels c, g, k and o) and in 30-40% of MADA-3 nuclei
(Fig. 5, panels d, h, l and p) a pronounced alteration of the nuclear morphology and a different
distribution pattern of the investigated heterochromatin proteins was observed. MADA-3
nuclei showed severe signs of degeneration with multiple invaginations and lobulations. In
most of the lobules the co-localization of the proteins was irremediably lost (Fig. 5, panel p).
While Me9H3 was detected inside the papillary extroflessions, no apparent HP1 was present
in these area.
To further analyse the biochemical features of the two heterochromatin markers we
fractionated fibroblasts derived from control and MADA patients. We first separated the
nuclear and the cytoplasmic fractions in the absence of detergent. Subsequently, the two
fractions were Triton-extracted and centrifuged to isolate the soluble and insoluble pools from
each fraction. After separation, we examined, by Western blot, the distribution of the HP1
and Me9H3 in these fractions. In control human fibroblasts, both HP1 and Me9H3 are mostly
found in the insoluble pool of the nuclear pellet (Fig. 6A, lanes 1 and 2). On the contrary, in
fibroblasts derived from MADA-1 and MADA-3 patients these proteins are differently
partitioned and found also in the nuclear soluble pool (Fig. 6A, lanes 3 and 5). The
densitometric analysis of HP1 and Me9H3 bands is shown in Fig. 6B. Interestingly, both
HP1 and Me9H3 accumulation in the soluble pool of the nucleus increased with patient age,
varying from 40-42% in MADA-1 to 48-50% in MADA-3 fibroblasts. It is worth noting that
no immunoreactive proteins were present in the cytoplasm neither in control fibroblasts nor in
MADA cells (data not shown). The fact that the expression level of HP1 and the methylation
state of H3 histone at lysine 9 was not altered, while the molecules were partially solubilised
by Triton X-100 treatment of MADA fibroblasts, is consistent with the finding that
heterochromatin in these cells is partly unstructured.
In order to investigate whether destabilization of HP1 and Me9H3 correlate with alteration of
the lamina, we analysed the distribution of pre-lamin A, lamin A/C and emerin between
11
supernatant and pellet fractions. In control human fibroblasts, lamin A/C and pre-lamin A
distribute in the nuclear pellet and emerin is also partly found in the nuclear soluble pool
(Figure 6C, lanes 1 and 2). These three molecules are not differently partitioned in MADA
cells (Figure 6C, lanes 3 and 4).
12
DISCUSSION
In this report we have documented the presence of nuclear envelope and chromatin alterations in
primary cultured fibroblasts from patients carrying a missense mutation in the LMNA gene
(R527H) resulting in MADA phenotype. We demonstrated accumulation of pre-lamin A, altered
stability of heterochromatin proteins HP1
and Me9H3 and a redistribution of the nuclear
envelope protein LBR in MADA cells. These cells showed evident alterations of the nuclear
periphery at the interface between peripheral heterochromatin and the nuclear envelope.
Interestingly, the degree of morphological alterations correlated with patient’s age. In fact,
fibroblasts derived from the oldest patient (MADA-3) revealed a more pronounced irregularity in
envelope organization and heterochromatin distribution. This finding further supports a key role of
lamins in chromatin organization and mechanical integrity of the nucleus, crucial to maintain cell
and tissue integrity during aging (29). In this contest, it has been recently provided a molecular
link between cellular senescence and heterochromatin structure (33). These authors showed that
senescent human fibroblasts accumulate a distinct chromatin structure enriched with
heterochromatin proteins, designated “senescence-associated heterochromatin foci” (SAHF) that
excludes active transcription and is characterized by the accumulation of Me9H3 and HP1
proteins. Interestingly, we observed Hoescht-positive foci highly resembling to SAHF in about
15% of MADA-1 nuclei with HP1 and methylated histone H3 at lysine 9 concentrated in these
foci, suggesting a process of accelerated cellular senescence in these cells. In agreement with this
observation, we found increase in senescence associated- -Gal staining in MADA cells, which
correlates with patient’s age (data not shown). Moreover, we demonstrated that HP1 and Me9H3
become partially solubilised by Triton X-100 treatment, consistent with the finding that
heterochromatin in these cells is partly unstructured. This was also confirmed by the fact that
Me9H3 looses its intracellular localisation in 30% of MADA-3’s nuclei. According with the
histone code hypothesis, Me9H3 is required to create high-affinity binding sites for HP1, crucial
to promote the formation of higher-ordered heterochromatin structures (4, 13, 47). This
observation may help explaining the dramatic loss of heterochromatin areas we observed in
MADA nuclei. In fact the ultrastructural microscopy shows a progressive alterations in nuclear
architecture in fibroblasts obtained from MADA patients bearing the common R527H mutation.
Focal loss and, in many cases, detachment of peripheral heterochromatin and alteration of nuclear
morphology (nuclear envelope invaginations and/or papillary extroflessions), similar to those
found in other laminopathies were observed (2, 8, 15, 19, 26, 32, 34, 40, 41, 44, 45). These
13
changes, together with the altered distribution of the two major heterochromatin components
Me9H3 and HP1 proteins, strongly support the hypothesis that R527H mutation may alter the
normal formation of heterochromatin-nuclear lamina proteins complex. Chromatin defects
observed in MADA nuclei are comparable with those observed in the nuclei from lamin A/C-/mouse fibroblasts (44) and with alterations shown in EDMD, FPLD and HGPS nuclei (9, 19, 37,
41). However, at least two features are exclusively found in MADA and in HGPS nuclei: the
complete absence of heterochromatin areas and the nuclear lamina thickening (present study and
19). Both these nuclear defects could be related to the accumulation of unprocessed lamin A
precursor, as observed in HGPS (14, 19), and MADA cells (present study and 10). Altered prelamin A processing and defective nuclear envelope organization has been also demonstrated in
Zmpste24-deficient mice (6, 38). Interestingly, Fong et al. (16) recently demonstrated that the
accumulation of pre-lamin A is responsible for many aspects of the disease-associated phenotype,
including the misshapen nuclei and that lowering pre-lamin A level may modify the evolution of
the disease (16). In this contest, notwithstanding the fact that heterozygous R527H cells exhibit
nuclear abnormalities (35), we didn’t observe any significant increase of pre-lamin A level with
respect to age-matched controls (data not shown).
We observed a marked redistribution of lamin B receptor in MADA cells. Mislocalization of LBR
was also reported in EDMD2 fibroblasts and in myoblasts, suggesting that lamin A/C mutations,
directly or indirectly, affect the localization of the nuclear envelope protein LBR (40). Moreover,
several studies have highlighted the importance of the association of LBR with components of the
heterochromatin such as HP1 proteins (25, 39, 48). Therefore, our study provides a link between
lamin A mutations and altered chromatin remodelling, supporting a common pathogenetic
mechanism. Recent findings suggest that the multisystem nature and the wide spectrum of
phenotype variation of several monogenic disorders is attributable to defects of chromatin
remodelling (3, 7, 12, 21). Laminopathies represent an excellent model to investigate the
molecular basis of this phenomena. In fact, it is not clear why mutations in LMNA, EMD, or LBR,
which are expressed in most cells, cause tissue-specific disorders. On the other hand, it is unclear
why different mutations in LMNA cause different diseases (27, 31, 36). Several hypotheses were
suggested
trying
to
explain
their
pathogenetic
mechanisms.
These
include
the
mechanical/structural model, and the gene expression model (20). Although these models are not
mutually exclusive, they do not explain the etiological link between an altered nuclear envelope
and transcriptional misregulation. Dramatic defects in nuclear envelope structure are evident in
14
cells from patients with EDMD, FLPD, or progerias and in mice carrying engineered mutations in
LMNA. In particular, the nuclei show frequent blebbing or ‘herniations with evident alterations in
nuclear shape, increased separation of the inner and outer nuclear membranes, clustering of
nuclear pores, loss of some inner nuclear membrane (INM) proteins from one pole of the nucleus
and disruption of the underlying electrondense heterochromatin (9, 19, 30, 37, 41, 44). Nuclear
envelopes from Lmna-/- mice exhibit increased fragility (2, 26, 34) and, in general, nuclei
containing defective lamins may be mechanically more fragile. Our study provides the first
evidence of an alteration of heterochromatin-associated protein distribution in laminopathies and
allows the first direct correlation between worsening of lamin A defect (precursor protein
accumulation) and increasing heterochromatin loss. Provided that HP1ß, LBR and Me9H3 belong
to the same functional complex and appear all affected in MADA cells, our results argue for a role
of lamin A in the correct assembly and/or stability of this chromatin-associated complex.
Moreover, mis-localization of emerin was also observed in MADA-3 cells, the cells obtained from
the oldest patient showing major nuclear defects. It is noteworthy that absence of interaction
between emerin and lamin A was previously found in FPLD fibroblasts (9), which also
accumulate pre-lamin A (10).
The phenotypic variations associated with mutations in LMNA gene reflect the functional
diversity, redundancy, and modulation of the lamin maturation process in different cellular types.
As a consequence, the spectrum of mutations that affect lamin-proteins interaction could give rise
to multiple phenotypes because each mutation could differentially affect this pathway.
Additionally, slight variations in the function of redundant and cooperative pathways could also
contribute to the phenotypic diversity. For example, because the regulation of gene expression
requires a fine compartmentalisation which is supported by chromatin architecture, mutations in
different lamin sites could generate alteration in gene transcription. Our results provide further
support to the hypothesis of a regulatory pathway connecting, in sequence, cellular
morphometry/nuclear architecture/chromatin structure/gene expression.
15
ACKNOWLEDGEMENTS
We are indebted to Dr. G. Zambruno for control fibroblasts, to Dr. P. Singh (Roslin Institute,
Edimburgh, UK) for anti-LBR, anti-HP1_ and anti-Me9H3 antibodies and to Dr. S. Squarzoni for
helpful discussions. This work was supported by grants from Italian Health Ministry, from Italian
Ministry for University and Research (FIRB Project n° RBNE01JJ45_005), and MIUR-Cofin
2004 to N.M.M., by a “Telethon” grant to G.N. and by a grant from “Fondazione Carisbo”, Italy,
by EU grant “Nuclear Envelope-linked Rare Human Diseases: From Molecular Pathophysiology
towards Clinical Applications” FP6-018690.
16
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Legends to figures
Figure 1 Western blot analysis of LMNA products in MADA fibroblasts.
(a) Whole cellular lysates from control and MADA fibroblasts (MADA-1, MADA-2, MADA-3)
were subjected to SDS-PAGE and Western blot using pre-lamin A, lamin A/C and emerin
antibodies. Actin staining shows equal loading amount. Molecular weight markers are reported in
kDa. (b) Data are reported as percentage of pre-lamin A (filled bars) or lamin A (empty bars)
densitometry measured in control samples. Means +/- SEM of three different experiments are
reported. Three different age matched controls were used in this experiment with identical results.
A control cell line derived from a 35 years patient is shown.
Figure 2 Double immunofluorescence staining of pre-lamin A and emerin in control and
MADA fibroblasts.
Pre-lamin A (panels a, b, c and d) and emerin (panels e, f, g and h) were labelled using specific
antibodies. Merged images are shown in panels i, j, k and l.
Figure 3 Double immunofluorescence staining of LBR and pre-lamin A in control and
MADA fibroblasts.
Control and MADA-1, MADA-2 and MADA-3 fibroblasts were double labelled with anti-LBR
(panels a, b, c, d and e) and anti-pre-lamin A antibodies (panels f, g, h, i and j). The DNA was
counterstained with Hoechst 33342 (panels h, l, m, n and o).
Figure 4 Electron microscopy analysis of MADA nuclei.
Cultured control (a) and MADA fibroblasts (b, c, d, e and f) were fixed with 2.5% glutaraldehyde,
included in epoxy resin and ultra-thin sections were examined by TEM. Ultrastructural alterations
observed in MADA nuclei are indicated by arrows. In (g) are reported the percentages of nuclear
morphological defects in control (c), MADA-1 (1), MADA-2 (2) or MADA-3 (3). Data are means
+/- SEM of three different observations. At least 200 nuclei per sample were counted.
22
Figure 5 Double immunofluorescence staining of Me9H3 and HP1 in control and MADA
fibroblasts.
Localization of trimethylated histone H3 at lysine 9 (green) and HP1 (red) was determined by
indirect immunofluorescence on control (C), MADA-1, MADA-2 and MADA-3 fibroblasts. The
DNA was counterstained with Hoechst 33342 (blue). Merged images are shown. Scale bar, 5 µm.
Figure 6 Western blot analysis of Me9H3 and HP1
in nuclear extracts of control and
MADA fibroblasts.
(A) Nuclear soluble (S) and insoluble (I) proteins from control (lanes 1 and 2), MADA-1 (lanes 3
and 4) and MADA-3 fibroblasts (lanes 5 and 6) were subjected to SDS-PAGE and Western blot
using anti-HP1 and anti-Me9H3 antibodies.
(B) Densitometric analysis of HP1 and Me9H3 immunoblotted bands was performed using a
Biorad GS-800 calibrated densitometer. Data are reported as percentage of HP1 (filled bars) or
Me9H3 (empty bars) densitometry measured in soluble and insoluble fractions. Means +/- SEM of
three different experiments are reported.
(C) Nuclear soluble (S) and insoluble (I) proteins from control (lanes 1 and 2), and MADA-3
fibroblasts (lanes 3 and 4) were subjected to SDS-PAGE and Western blot using anti-pre-lamin A,
anti-lamin A/C and anti-emerin antibodies.
23
Fig. 1
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Fig. 2
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Fig. 3
26
Fig. 4
27
Fig. 5
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Fig. 6