A Functional Role for 4qA/B in the Structural
Rearrangement of the 4q35 Region and in the
Regulation of FRG1 and ANT1 in Facioscapulohumeral
Iryna Pirozhkova1, Andrei Petrov1, Petr Dmitriev1, Dalila Laoudj2, Marc Lipinski1, Yegor Vassetzky1*
1Universite ´ Paris-Sud 11, CNRS UMR 8126, Interactions mole ´culaires et cancer, Institut de Cance ´rologie Gustave-Roussy, Villejuif, France, 2INSERM, ERI25, F-34000,
Montpellier, France, Universite ´ Montpellier 1, Montpellier, France
The number of D4Z4 repeats in the subtelomeric region of chromosome 4q is strongly reduced in patients with Facio-
Scapulo-Humeral Dystrophy (FSHD). We performed chromosome conformation capture (3C) analysis to document the
interactions taking place among different 4q35 markers. We found that the reduced number of D4Z4 repeats in FSHD
myoblasts was associated with a global alteration of the three-dimensional structure of the 4q35 region. Indeed, differently
from normal myoblasts, the 4qA/B marker interacted directly with the promoters of the FRG1 and ANT1 genes in FSHD cells.
Along with the presence of a newly identified transcriptional enhancer within the 4qA allele, our demonstration of an
interaction occurring between chromosomal segments located megabases away on the same chromosome 4q allows to
revisit the possible mechanisms leading to FSHD.
Citation: Pirozhkova I, Petrov A, Dmitriev P, Laoudj D, Lipinski M, et al. (2008) A Functional Role for 4qA/B in the Structural Rearrangement of the 4q35 Region
and in the Regulation of FRG1 and ANT1 in Facioscapulohumeral Dystrophy. PLoS ONE 3(10): e3389. doi:10.1371/journal.pone.0003389
Editor: Peter Fraser, The Babraham Institute, United Kingdom
Received May 26, 2008; Accepted September 17, 2008; Published October 13, 2008
Copyright: ? 2008 Pirozhkova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants to YSV and DL from the Association Franc ¸aise contre les Myopathies (AFM). IP was a recipient of an AFM fellowship,
and AP of a fellowship from the Fondation pour la Recherche Me ´dicale.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Facio-scapulo-humeral muscular dystrophy (FSHD) is an auto-
somal dominant neuromuscular disease characterized by weakness
and atrophy of muscles of the face, upper arms and shoulder girdle.
In patients with FSHD, a deletion in a polymorphic locus of
chromosome 4qreducesthe numberofD4Z4 repeatstolessthan 10
vs up to 200 in normal individuals . Each 3.3 kbp D4Z4 element
harbors DUX4, a gene which encodes a double homeodomain
protein [2–4]. Three other genes FRG1 (FSHD Region Gene 1)
[5,6], FRG2 (FSHD Region Gene 2) [5,7] and ANT1 (Adenine
Nucleotide Translocator 1)  are located within the 4q35
chromosomal region and have been reported to be upregulated in
FSHD patients. Aberrant expression of FRG1, which is thought to
encode a splicing regulator [6,9], could explain the simultaneous
changes in expression of many genes. Nevertheless, the evidence of
their involvement in FSHD pathogenesis is missing. Some studies
even argue against the upregulation of FRG1 and FRG2 in FSHD
muscles [10,11]. Indeed, to date, the many proteomics and
transcriptome approaches have provided a wealth of data
suggesting that the contraction of the D4Z4 repeat array is not
sufficient to cause the disease and that FSHD is likely to be a
multifactorial disorder (reviewed in ).
Several years ago a transcriptional repressor was identified
within the D4Z4 repeat array . However, we have recently
demonstrated that overall, each D4Z4 repeat has an enhancer
activity due to the presence of a very strong enhancer .
Moreover, we have shown that a nuclear matrix attachment site
(S/MAR), which is positioned in the immediate vicinity of the
D4Z4 repeat array , may function as an insulator and block
the D4Z4 enhancer in normal, but not FSHD, cells . In fact,
this S/MAR is prominent in normal myoblasts and non-muscular
human cells, and much weaker in muscle cells derived from FSHD
patients . From this observation, we inferred that, in normal
human myoblasts, the D4Z4 repeat array and neighboring genes
are located in two distinct loops, whereas, in myoblasts from
FSHD patients, they are in a single one. This suggests that a
looping mechanism could lead to a direct contact between the
D4Z4 array and genes that are positioned in cis on the
chromosome but are too far away to be subjected to transcrip-
tional regulation through classical molecular mechanisms .
Intriguingly, FSHD occurs only in individuals bearing the 4qA
allele. 4qA/B is a 10 kb-long polymorphic segment directly
adjacent to the D4Z4 repeat array. It exists in two allelic forms,
4qA and 4qB, which are 92% identical and equally common in the
general population [15,16]. The main difference between the two
alleles resides in a tract of b-satellite repeats present in 4qA but not
4qB . This dissimilarity may bear consequences either in the
predisposition to deletions occurring within the D4Z4 repeat array
or in the structural consequences of the deletion.
Here, we have further investigated the three-dimensional
structure of the 4q subtelomeric region using the recently
described 3C technique. We now report significant differences
existing between FSHD and normal muscle cells.
PLoS ONE | www.plosone.org1 October 2008 | Volume 3 | Issue 10 | e3389
Role of 4qA/B Marker in FSHD
PLoS ONE | www.plosone.org2 October 2008 | Volume 3 | Issue 10 | e3389
3C analysis of DNA-DNA interactions at 4q35 in normal
The 3C technique evaluates the spatial proximity of two
genomic fragments based upon their relative propensity to get
crosslinked in vivo [17–19]. The method uses a restriction enzyme
to digest previously crosslinked chromatin. After ligation of very
dilute DNA to favor intramolecular rather than intermolecular
ligation of crosslinked DNA, the ligated fragments are amplified by
PCR using specifically designed primers. In the present study, we
have used the BglII enzyme whose recognition sequence is present
on average every 3,50061,500 bp within the studied region. Such
a DNA length is appropriate for the 3C assay.
We selected several genes and landmarks (Figure 1A) within the
5 Mb-long subtelomeric region of chromosome 4q to study their
propensity to get crosslinked in vivo. These included 4qA/B, a
distal segment adjacent to the polymorphic 4qA/4qB marker
[15,16]; D4Z4, a 3.3 kb fragment containing the D4Z4 repeat
array itself; FR-MAR, the fragment containing the S/MAR whose
function is weakened in FSHD muscle cells ; 5’NT, a non-
transcribed fragment located between the D4Z4 array and the
FRG2 gene; FRG2, the promoter region of FRG2 ; DUX4c, a
DNA fragment in the vicinity of the unique D4Z4 copy located
between the FRG2 and FRG1 genes ; two fragments, FRG1-1
and FRG1-2, that correspond to the distal and proximal part of
the FRG1 gene promoter, respectively ; and ANT1, the
promoter region of the ANT1 gene (Figure 1A). We then
designed specific PCR primers for each BglII restriction fragment
as detailed in Materials and Methods.
We carried out preliminary experiments (Figure 1B–E) to define
the optimal conditions for the 3C analysis. For PCR amplification
we chose a number of cycles that fell into the linear range of
amplification (Figure 1B, right panel). However, the 4q35 locus
contains repetitive sequences and copies of the FRG1 and FRG2
genes also exist elsewhere in the genome . We thus had to verify
that the primer pairs used in this study specifically amplified
genomic DNA from chromosome 4. To this aim we used genomic
DNA extracted from the GM1015 human/rodent hybrid cell line
in which chromosome 4 is the only human chromosome. Indeed,
all six amplification products obtained using DNA from this cell
line migrated identically to the control PCR products obtained
from total human DNA (Figure 1C). We then verified the
specificity of the primer pairs for DUX4c, a fragment with
considerable homology to D4Z4 using the pGEM42 construct
which contains two D4Z4 repeats and 59 and 39 flanking
sequences, but no DUX4c sequence . With this template we
obtained an amplification product with the D4Z4 but not with the
DUX4c specific primers (Figure 1D). Finally, we confirmed the
sequence specificity of the DUX4c and DUX4 products by
sequencing (data not shown), and verified that all primer pairs
used produced specific fragments from total DNA of normal and
FSHD myoblasts (Figure 1E).
We next used the 3C assay to evaluate the spatial proximity of
the selected 4q35 landmarks in normal human myoblasts
(Figure 2A). We did not detect any interaction between ANT1
and the other landmarks (Figure 2, upper left panel). This indicates
a lack of proximity between the ANT1 gene and all other
landmarks tested. This result was confirmed when the other
landmarks were tested for proximity with ANT1 (see the ANT1
point, first on the left on the x-axis in all the other panels of
Figure 2A). In contrast, we consistently detected an interaction
between FRG1-1 and FRG2 and DUX4c. Specifically, DUX4c
strongly interacted with the distal part of the promoter of FRG1
(FRG1-1) and, to a lower extent, with the promoter of FRG2, and
also with the subtelomeric region proximal to the 4qA/4qB
marker. FR-MAR and 5’NT did not interact with other
landmarks, whereas D4Z4 interacted only with the region
proximal to DUX4c. Thus, in normal myoblasts, we have found
that the D4Z4 repeat array does not directly interact with any
3C analysis of DNA-DNA interactions within 4q35 in FSHD
We next performed the same 3C analysis using myoblasts
derived from an FSHD patient. Differently from what observed in
normal muscle cells, we could not detect any interaction between
FRG1-1 and FRG2 or FGR1-2, whereas we consistently identified
a novel interaction between FRG1-1 and 4qA/4qB (Figure 2B).
Indeed, in FSHD myoblasts, the 4qA/B landmark strongly
interacted not only with DUX4c (as in control cells), but also
with FRG1-1, FRG1-2 and the promoter of the ANT1 gene. This
indicates that despite being located 5 Mb proximally on the 4q
chromosome, the ANT1 gene directly interacts with 4qA/B in the
nuclear space of FSHD cells. This interaction was indeed specific
as ANT1 did not crosslink with any other sequence but 4qA/B.
Additional differences also exist between normal and FSHD cells
regarding 4qA/B whose interactions with FRG1-1 and FRG1-2
were also FSHD-specific. As in control cells, we did not observe
any interaction between FR-MAR or 59NT and the other
landmarks, whereas the D4Z4 repeat directly interacted only with
DUX4c, but not with any of the gene promoters.
The major differences in the 3D organization of the 4q35 locus
between normal and FSHD myoblasts are summarized in Table 1
and Figure 2C.
The majority of the interactions detected in the 3C assay
occur in cis within 4q35
The data obtained with the 3C assay evidence the spatial
proximity of sequences along the subtelomeric region of
chromosome 4q. However, approximately 60 kbp of sequences
within this region are also present on chromosome 10q which
Figure 1. 3C analysis of nine landmarks in the 4q35 region. A. Map and genomic coordinates (in bp) of primer pairs used for the 3C analysis.
Genes are represented by unique arrows, promoters by ovals. The D4Z4 array is shown as green block arrows. B. Control digestion on crosslinked
templates. Genomic DNA was digested with BglII and amplified using the primer pairs that allow only the amplification of non-digested DNA. No PCR
products were observed in the absence of the ligation step. C. The PCR amplification linear range was obtained by titration of the template
concentration and number of amplification cycles. Finally, 10 ng of crosslinked template and 100 ng of control template in 15 ml of reaction mixture
were used in our experiments. The PCR cycling conditions were as follows: 94uC for 3 min; 94uC for 45 sec and 58uC for 30 sec, 72uC for 50 sec,
followed by a final extension at 72uC for 10 min using Taq DNA Polymerase (Invitrogen). D. The DNA GM10115A human/rodent hybrid cell line
containing a single chromosome 4 was digested with BglII, ligated and then amplified using specific primer pairs to verify the accuracy of the primer
pairs for the chromosome 4 sequences. E. The D4Z4 repeat cloned into the pGEM42 plasmid was amplified using one primer pair specific for D4Z4
and two different primer pairs specific for DUX4c (DUX4c1 and DUX4c2). Two different template concentrations, 100 ng and 200 ng were used for
Role of 4qA/B Marker in FSHD
PLoS ONE | www.plosone.org3 October 2008 | Volume 3 | Issue 10 | e3389
Role of 4qA/B Marker in FSHD
PLoS ONE | www.plosone.org4 October 2008 | Volume 3 | Issue 10 | e3389
contains a region homologous to a 4q35 segment . Thus, the
interactions detected by the 3C assays could have occurred in trans
between chromosomes 4q and 10q rather than in cis within 4q. To
investigate this possibility, we measured the proximity of the
homologous 4q and 10q regions. To this aim, we used the FISH
technology to localize the long arms of chromosome 4 and 10 in
interphase nuclei (Figure 3A). Some hybridization signals were in
direct contact with each other. In this case, we assumed that
somatic pairing did take place. From the analysis of 200 nuclei, the
level of somatic pairing ranged between 9 and 10.5% of all signals
in both control and FSHD myoblasts (Table S2). This was
consistent with the low level of pairing (4.5%) reported between
chromosomes 4 and 10 in a previous study . The higher
pairing level observed here corresponds to the fact that, in addition
to the 4q–10q interactions, we have also revealed contacts between
homologous chromosomes (4q-4q and 10q-10q). From these
results we can conclude that, although the existence of interactions
in trans cannot be completely excluded, these do not occur in more
than the 10% of the nuclei, whereas in 90% of the nuclei, the loci
of interest are too far away from each other to interact. Therefore,
the interactions detected by our 3C experiments mainly reflect
interactions occurring in cis within 4q35.
The 4qA allele contains a transcriptional enhancer
We then asked whether the 4qA/B marker, which in FSHD
myoblasts interacts directly with the promoters of FRG1 and
ANT1, could have a role in the transcriptional regulation of these
two genes. Since all FSHD patients carry the 4qA phenotype on
the deleted 4q chromosome , we tested whether the 4qA allele
could directly regulate gene transcription. To this aim we cloned
the 4qA marker in both orientations in the pGL3-promoter
plasmid, a luciferase reporter vector. We transfected constructs
and control plasmids in HeLa cells, and then measured reporter
gene expression 48 hours after transfection. The presence of the
SV40 enhancer in the positive control (pGL3con, Figure 3B)
resulted in a five-fold increase of the transcription levels in
comparison to the enhancer-less control plasmid (pGL3Pro). The
4qA fragment cloned into the enhancer-less pGL3–promoter
plasmid stimulated luciferase synthesis with 60% efficiency as
compared to the SV40 enhancer positive control. Thus, the 4qA
allele exhibited properties of a transcriptional enhancer. This
enhancer was also active in a cell line derived from a human
rhabdomyosarcoma, a tumor of muscular origin (data not shown).
Despite many studies performed in the last twenty years, the
mechanism leading to the emergence of FSHD remains poorly
understood. The 3C data reported here provide the first
experimental evidence that, in this genetic disease, molecular
events occur that involve chromosomal segments located at a very
large linear distance on the partially deleted chromosome 4q.
Specifically, we have observed that in FSHD myoblasts, the
subtelomeric 4qA/B marker strongly interacts with the promoter
of the FRG1 gene which is located dozens of kbp proximally on the
chromosome, depending on the number of remaining D4Z4
repeats. Even more strikingly, we documented a direct interaction
of 4qA/B with the promoter of the ANT1 gene which lies at a
linear distance greater than 5 Mbp on the centromeric side. This
interaction is FSHD-specific as, in control myoblast cells, the 4qA/
B marker did not interact with the FRG1, or the ANT1 promoters.
4qA/B is a 10 kb-long polymorphic segment directly adjacent to
the D4Z4 repeat array. It exists in two allelic forms, 4qA and 4qB,
which are 92% identical and equally common in the general
population. FSHD, however, has been reported to occur only in
individuals with the 4qA allele [15,16]. The main difference
between the two alleles resides ina tract of b-satellite repeats present
Table 1. Frequencies of cis-interactions within 4q35 in normal and FSHD myoblasts.
ANT1FRG1-1FRG1-2DUX4c FRG25’NT FR-MARD4Z4 4qa/b
Horizontal and vertical dashes indicate interactions detected by the 3C technique in normal and FSHD myoblasts, respectively.
Figure 2. 3C Analysis of the 4q35 locus. A–B. Representation of the spatial proximity in normal (A) and FSHD (B) myoblasts. The fragment tested
for crosslinking is indicated in each panel. An arbitrary score of 10 corresponds to the PCR amplification obtained using primers located on either side
of the restriction site separating two adjacent fragments within the corresponding genomic segment. The Y axis indicates relative levels of interaction
with the other landmarks tested which are represented along the X axis according to their localization along chromosome 4q. The data represent the
average results of three independent experiments. The panels below the charts show the 3C ligation products detected by PCR amplification using
specific primers. One experiment out of three independent ones is represented in the Figure. C. The differences in 3C interactions between the
normal (top) and FSHD myoblasts. Only interactions which are different between the normal and FSHD myoblasts are shown.
Role of 4qA/B Marker in FSHD
PLoS ONE | www.plosone.org5 October 2008 | Volume 3 | Issue 10 | e3389
in 4qA but not 4qB . This difference may bear consequences
either in the predisposition to deletions occurring within the D4Z4
repeat array or in the pathological consequences thereof.
Another surprising observation was that, in both normal and
FSHD cells, the D4Z4 marker interacted only with its related
sequence DUX4c among the various segments tested. No
interactions were detected with the promoter regions of ANT1,
FRG1 or FRG2. In accordance, the hypothesis of a transcriptional
regulation through a direct contact of the D4Z4 array with the
promoters of these three genes [5,7,13,14] appears unlikely. DUX4
and DUX4c are two genes that have been shown to be transcribed
within the D4Z4 repeats [2,3,25]. Thus, our results suggest that the
D4Z4 enhancer, within the D4Z4 repeat array, may directly
regulate the transcription of the DUX4 and DUX4c genes.
We then found that DUX4c crosslinked with the FRG1 and
FRG2 promoter regions in both normal and FSHD myoblasts
(Figure 2). We therefore postulate that DUX4c plays a key role in
the three-dimensional organization of the locus. Sequence
alignment analysis (data not shown) suggests that DUX4c contains
a transcriptional enhancer. Moreover, DUX4 interacts with
DUX4c which, in turn, makes contact with FRG1 and FRG2.
This may provide a molecular basis for the transcriptional
regulation of neighbor genes by DUX4/DUX4c.
We then detected a new enhancer element in the 4qA allele that
may regulate the expression of the FRG1 and ANT1 genes
specifically in FSHD cells through a direct interaction with the
respective gene promoters. Indeed, both ANT1 and FRG1 are
activated in FSHD patients [5,6,8]. It is noteworthy that the (1.5 to
3 fold) up-regulation of these two genes seen in FSHD patients is
consistent with the relatively weak effect of the 4qA enhancer in
the luciferase assay.
Recently, we have reported that in FSHD myoblasts, the
nuclear matrix attachment site FR-MAR was specifically delocal-
ized from the nuclear matrix . In normal cells, this S/MAR
may constrain the flexibility of the region by anchoring it to the
nuclear matrix, thus restricting interactions of adjacent sequences
in the three dimensional nuclear space. This could particularly
affect the 4qA/B marker which is separated from neighbor genes
by the S/MAR. In FSHD cells, the delocalization of FR-MAR
would thus result in an increased flexibility of the corresponding
chromosomal segment and additional possibilities of interaction
for the 4qA/B marker. This may provide an explanation for the
FSHD-specific, direct interaction of 4qA/B with the ANT1 and
FRG1 gene promoters we observed in FSHD myoblasts. In the
present 3C experiments, no interactions were detected that
involved FR-MAR. This should not be surprising since previous
3C studies have already stressed that S/MARs appear to interact
only with other SMARs .
The experimental approach used here provides new ways to
systematically explore the higher-order chromatin structure of any
chromosomal region. In this study, we have found that the binding
of DUX4c to the FRG1 and FRG2 gene promoters appears to play
a key role in structuring the 4q35 region in normal cells. Other
interactions take place in FSHD cells and this is the likely result of
a global reorganization of the locus in relation with the contraction
of the number of D4Z4 tandem repeats. This reorganization is
schematized in the three dimensional model shown in Figure 4. In
FSHD cells (Figure 4B), the deletion of D4Z4 repeats and the
delocalization of the proximal S/MAR would result in the
formation of a giant loop where the subtelomeric 4qA/B sequence
is now brought in close proximity not only to DUX4C and FRG1
but also to the proximal ANT1 gene promoter which lies 5 Mbp
away on the centromeric side of the region. This major structural
rearrangement, as compared to the normal situation (Figure 4A),
would make gene promoters accessible to the DUX4c and 4qA
enhancers specifically in FSHD myoblasts. One hypothesis to
explain how such long-range changes in higher order chromatin
structure can occur relates to differences in the methylation status
of the corresponding regions [27,28]. Further studies are clearly
needed to explore this and other hypotheses.
Materials and Methods
The HeLa cell line was purchased from the ATCC collection.
The GM10115A hybrid murine cell line containing the human
chromosome 4 was a kind gift of Dr. Rosella Tupler. Primary
muscle fibroblasts from two different healthy individuals and two
FSHD patients with 5.5 D4Z4 repeats and 7 repeats [14,29] in the
Figure 3. A. FISH analysis on primary human myoblasts. Nuclei
from normal (left panels) and FSHD (right panel) primary myoblasts
were hybridized to a FR-MAR probe (arrowed dots) and counterstained
with DAPI. B. The 4qA allele contains a transcriptional enhancer.
The transcriptional effect of the 4qA allele was tested 48 hrs after
transfection in HeLa cells. The enhancer strength was quantified relative
to the luciferase activity generated by the pGL3 plasmid with the SV40
enhancer (pGL3Con). Equal amounts of the plasmids were transfected.
Luciferase signals were normalized to the total protein content in the
extracts. pGL3Pro, enhancer-less, empty pGL3 plasmid; 4qA1 and 4qA2,
4qA allele cloned in the enhancer-less pGL3 plasmid.
Role of 4qA/B Marker in FSHD
PLoS ONE | www.plosone.org6 October 2008 | Volume 3 | Issue 10 | e3389
4q35 array, respectively, were cultured on a collagen-coated
support in DMEM supplemented with 20% bovine fetal serum.
The 3C assay was performed as described elsewhere  with
some specific adaptations for myoblast cells. Nuclei were prepared
using 2 volumes of ice-cold MES lysis buffer  for 1 volume of
packed cells; a protease inhibitors cocktail (Roche, Complete Mini)
was added immediately prior to use. The lysis of nuclei was checked
under a microscope. Formaldehyde (Sigma) was added to diluted
nuclei (final concentration of 161027/ml) to perform the cross-
linking. Nuclei were then diluted tenfold and digested overnight at
Figure 4. A–B. Models schematizing the proximity between 4q subtelomeric fragments in control (A) and FSHD (B) nuclei.
Role of 4qA/B Marker in FSHD
PLoS ONE | www.plosone.org7 October 2008 | Volume 3 | Issue 10 | e3389
37uC with BglII (New England BioLabs). The BglII restriction sites
occur with an average frequency 3500 bp61500 bp within the
4q35 locus, which is appropriate for the 3C assay.
Digestion mix was inactivated by adding SDS and digested
DNA ligated overnight at a low concentration with T4 DNA ligase
(Fermentas). Ligation products were detected by PCR amplifica-
tion using fragment-specific primers. PCR products were separat-
ed on 2% agarose gels; images acquired using a Bio-DOC
apparatus (Vilbour-Lourmat, France) and quantified using the
Image Gauge 4.0 software (Fuji, Japan).
Three independent controls were carried out using genomic
DNA from FSHD myoblasts, normal myoblasts and from the
murine hybrid cell line containing the human chromosome 4 as
the only human material. The DNA fragments spanning the BglII
restriction sites were mixed in equimolar amounts as described
elsewhere  and added to the appropriate non-crosslinked
Relative crosslinking frequencies for combinatorial interactions
were calculated as the ratio of the amount of product detected with
crosslinked DNA template to the amount of product obtained with
non-crosslinked, control DNA templates [17,30]. The experiments
were carried out in triplicate and were averaged. Data from two
independent experiments are presented.
3C primer design
The primers spanning the BglII sites were designed using
OLIGO Primer Analysis Software 6.71 at positions shown in
Figure 1A. Primer sequences are shown in Table S1.
The p13E11 probe was derived from the pGEM42 plasmid 
and labeled with biotin-14-dCTP. Hybridization on slides was
performed as described earlier  using anti-biotin mouse
antibodies conjugated with AlexaFluor 488 (Invitrogen, USA).
Nuclei were counterstained with 0,5 mg/ml 4,6-diamindo-2-
phenylindole (DAPI) and mounted using Vectashield antifade
mounting medium (Vector Laboratories, USA). Slides were
examined under an Olimpus Provis fluorescence microscope with
a 1006oil immersion objective and the appropriate filters. Images
were captured with a CCD camera (Photometrics, USA), using the
RSImage software (Scanalytics, USA).
Vectors and cloning
A series of pGL3 vectors (Promega, USA) was used for transient
transfection studies. The pGL3-Promoter vector contains an SV40
promoter upstream of the luciferase gene. The pGEM42 plasmid
containing the fragment of chromosome 4 corresponding to the
allelic variant 4qA  (a kind gift of Dr. A.Belayew) was digested
by BamHI and EcoRI (Fermentas, Lithuania). The 598 bp
fragment was blunt-ended by Klenow (Fermentas, Lithuania)
and cloned in two orientations upstream of the promoter region of
the reporter plasmid pGL3-Pro (Promega, USA) digested by SmaI
resulting in the plasmids pGL3-4qA1 and pGL3-4qA2.
The pGL3-Control vector contains the SV40 promoter and
enhancer sequences, resulting in strong expression of the reporter
gene in many types of mammalian cells. Therefore, it was used as
a positive control in the experiments on the identification of a
putative enhancer within the D4Z4.
HeLa cells were plated in 24 well/plates 24 hours before
transfection at the density of 50.000 cells per well. The plasmids
used for transfection were purified with the Nucleobond midiprep
kit (Macherey Nagel, Gremany) and 1 mg of each was transfected
using JetPEI (Polyplus Transfections Inc., USA). 48 hours after
transfection luciferase activity was measured with the Luciferase
Assay System (Promega, USA) using a Microlourmat LB96P
luminometer. The protein content of cell extracts was determined
with the QuantiPro BCA assay kit (Sigma, USA). Each
transfection was repeated at least 3 times.
Found at: doi:10.1371/journal.pone.0003389.s001 (0.04 MB
Primers used for the 3C assay.
of normal and FSHD myoblasts.
Found at: doi:10.1371/journal.pone.0003389.s002 (0.03 MB
Frequency of pairing between the 4q and 10q in nuclei
We thank Drs. Elvira Eivazova and Tom Sexton for helpful discussions;
Dr. A. Belayew for the kind gift of the pGEM42 plasmid, and Dr. R.
Tupler for the GM10115A strain.
Conceived and designed the experiments: IVP AVP PD YSV. Performed
the experiments: IVP AVP PD. Analyzed the data: IVP AVP PD ML YSV.
Contributed reagents/materials/analysis tools: DL. Wrote the paper: IVP
AVP ML YSV.
1. van Deutekom JC, Wijmenga C, van Tienhoven EA, Gruter AM, Hewitt JE, et
al. (1993) FSHD associated DNA rearrangements are due to deletions of integral
copies of a 3.2 kb tandemly repeated unit. Hum Mol Genet 2: 2037–2042.
2. van Geel M, Heather LJ, Lyle R, Hewitt JE, Frants RR, et al. (1999) The FSHD
region on human chromosome 4q35 contains potential coding regions among
pseudogenes and a high density of repeat elements. Genomics 61: 55–65.
3. Dixit M, Ansseau E, Tassin A, Winokur S, Shi R, et al. (2007) DUX4, a
candidate gene of facioscapulohumeral muscular dystrophy, encodes a
transcriptional activator of PITX1. Proc Natl Acad Sci U S A 104:
4. Dmitriev P, Lipinski M, Vassetzky YS (2008) Pearls in junk: dissecting the
molecular pathogenesis of facioscapulohumeral muscular dystrophy. Neuromus-
cular Disorders in press: DOI: 10.1016/j.nmd.2008.1009.1004.
5. Gabellini D, Green MR, Tupler R (2002) Inappropriate gene activation in
FSHD: a repressor complex binds a chromosomal repeat deleted in dystrophic
muscle. Cell 110: 339–348.
6. Gabellini D, D’Antona G, Moggio M, Prelle A, Zecca C, et al. (2005)
Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1.
7. Rijkers T, Deidda G, van Koningsbruggen S, van Geel M, Lemmers RJ, et al.
(2004) FRG2, an FSHD candidate gene, is transcriptionally upregulated in
differentiating primary myoblast cultures of FSHD patients. J Med Genet 41:
8. Laoudj-Chenivesse D, Carnac G, Bisbal C, Hugon G, Bouillot S, et al. (2005)
Increased levels of adenine nucleotide translocator 1 protein and response to
oxidative stress are early events in facioscapulohumeral muscular dystrophy
muscle. J Mol Med 83: 216–224.
9. van Koningsbruggen S, Straasheijm KR, Sterrenburg E, de Graaf N,
Dauwerse HG, et al. (2007) FRG1P-mediated aggregation of proteins involved
in pre-mRNA processing. Chromosoma 116: 53–64.
10. Winokur ST, Chen YW, Masny PS, Martin JH, Ehmsen JT, et al. (2003)
Expression profiling of FSHD muscle supports a defect in specific stages of
myogenic differentiation. Hum Mol Genet 12: 2895–2907.
11. Osborne RJ, Welle S, Venance SL, Thornton CA, Tawil R (2007) Expression
profile of FSHD supports a link between retinal vasculopathy and muscular
dystrophy. Neurology 68: 569–577.
12. van der Maarel SM, Frants RR, Padberg GW (2007) Facioscapulohumeral
muscular dystrophy. Biochim Biophys Acta 1772: 186–194.
Role of 4qA/B Marker in FSHD
PLoS ONE | www.plosone.org8 October 2008 | Volume 3 | Issue 10 | e3389
13. Petrov AV, Allinne J, Pirozhkova IV, Laoudj D, Lipinski M, et al. (2008) A
nuclear matrix attachment site in the 4q35 locus has an enhancer-blocking
activity in vivo: implications for the facio-scapulo-humeral dystrophy. Genome
Res 18: 39–45.
14. Petrov A, Pirozhkova I, Laoudj D, Carnac G, Lipinski M, et al. (2006)
Chromatin loop domain organization within the 4q35 locus in facioscapulohu-
meral dystrophy patients versus normal human myoblasts. Proc Natl Acad Sci
USA 103: 6982–6987.
15. Lemmers RJ, de Kievit P, Sandkuijl L, Padberg GW, van Ommen GJ, et al.
(2002) Facioscapulohumeral muscular dystrophy is uniquely associated with one
of the two variants of the 4q subtelomere. Nat Genet 32: 235–236.
16. Lemmers RJ, Wohlgemuth M, Frants RR, Padberg GW, Morava E, et al. (2004)
Contractions of D4Z4 on 4qB subtelomeres do not cause facioscapulohumeral
muscular dystrophy. Am J Hum Genet 75: 1124–1130.
17. Dekker J, Rippe K, Dekker M, Kleckner N (2002) Capturing chromosome
conformation. Science 295: 1306–1311.
18. Tolhuis B, Palstra RJ, Splinter E, Grosveld F, de Laat W (2002) Looping and
interaction between hypersensitive sites in the active beta-globin locus. Mol Cell
19. Carter D, Chakalova L, Osborne CS, Dai YF, Fraser P (2002) Long-range
chromatin regulatory interactions in vivo. Nat Genet 32: 623–626.
20. van Deutekom JC, Lemmers RJ, Grewal PK, van Geel M, Romberg S, et al.
(1996) Identification of the first gene (FRG1) from the FSHD region on human
chromosome 4q35. Hum Mol Genet 5: 581–590.
21. Li K, Hodge JA, Wallace DC (1990) OXBOX, a positive transcriptional element
of the heart-skeletal muscle ADP/ATP translocator gene. J Biol Chem 265:
22. Gabriels J, Beckers MC, Ding H, De Vriese A, Plaisance S, et al. (1999)
Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD
identifies a putative gene within each 3.3 kb element. Gene 236: 25–32.
23. van Geel M, Dickson MC, Beck AF, Bolland DJ, Frants RR, et al. (2002)
Genomic analysis of human chromosome 10q and 4q telomeres suggests a
common origin. Genomics 79: 210–217.
24. Stout K, van der Maarel S, Frants RR, Padberg GW, Ropers HH, et al. (1999)
Somatic pairing between subtelomeric chromosome regions: implications for
human genetic disease? Chromosome Res 7: 323–329.
25. Kowaljow V, Marcowycz A, Ansseau E, Conde CB, Sauvage S, et al. (2007) The
DUX4 gene at the FSHD1A locus encodes a pro-apoptotic protein.
Neuromuscul Disord 17: 611–623.
26. Eivazova ER, Vassetzky YS, Aune TM (2007) Selective matrix attachment
regions in T helper cell subsets support loop conformation in the Ifng gene.
Genes Immun 8: 35–43.
27. van Overveld PG, Lemmers RJ, Sandkuijl LA, Enthoven L, Winokur ST, et al.
(2003) Hypomethylation of D4Z4 in 4q-linked and non-4q-linked facioscapu-
lohumeral muscular dystrophy. Nat Genet 35: 315–317.
28. Tawil R, Van Der Maarel SM (2006) Facioscapulohumeral muscular dystrophy.
Muscle Nerve 34: 1–15.
29. Barro M, Carnac G, Flavier S, Mercier J, Vassetzky YS, et al. (2008) Primary
myoblasts derived from the facioscapulohumeral dystrophy patients are
hypersensitive to oxidative stress and show defects upon terminal differentiation.
J Cell Mol Med in press.
30. Eivazova ER, Aune TM (2004) Dynamic alterations in the conformation of the
Ifng gene region during T helper cell differentiation. Proc Natl Acad Sci U S A
31. Keeney S, Kleckner N (1996) Communication between homologous chromo-
somes: genetic alterations at a nuclease-hypersensitive site can alter mitotic
chromatin structure at that site both in cis and in trans. Genes Cells 1: 475–489.
32. Cai S, Kohwi-Shigematsu T (1999) Intranuclear relocalization of matrix binding
sites during T cell activation detected by amplified fluorescence in situ
hybridization. Methods 19: 394–402.
Role of 4qA/B Marker in FSHD
PLoS ONE | www.plosone.org9 October 2008 | Volume 3 | Issue 10 | e3389