DUX4c Is Up-Regulated in FSHD. It Induces the MYF5
Protein and Human Myoblast Proliferation
Euge ´nie Ansseau1., Dalila Laoudj-Chenivesse2., Aline Marcowycz1., Alexandra Tassin1", Ce ´line
Vanderplanck1", Se ´bastien Sauvage1, Marietta Barro2, Isabelle Mahieu1, Axelle Leroy1, India Leclercq1,
Ve ´ronique Mainfroid3, Denise Figlewicz4, Vincent Mouly5, Gillian Butler-Browne5, Alexandra Belayew1,
Fre ´de ´rique Coppe ´e1*
1Laboratory of Molecular Biology, University of Mons-Hainaut, 6, Mons, Belgium, 2INSERM ERI 25 Muscle et Pathologies, CHU A. de Villeneuve, Montpellier, France,
3Eppendorf Array Technologies, Namur, Belgium, 4Department of Neurology, University of Michigan, Ann Arbor, Michigan, United States of America, 5Institute of
Myology, Platform for human cell culture, Paris, France
Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contractions of the D4Z4 repeat array in
4q35. We have previously identified a double homeobox gene (DUX4) within each D4Z4 unit that encodes a transcription
factor expressed in FSHD but not control myoblasts. DUX4 and its target genes contribute to the global dysregulation of
gene expression observed in FSHD. We have now characterized the homologous DUX4c gene mapped 42 kb centromeric of
the D4Z4 repeat array. It encodes a 47-kDa protein with a double homeodomain identical to DUX4 but divergent in the
carboxyl-terminal region. DUX4c was detected in primary myoblast extracts by Western blot with a specific antiserum, and
was induced upon differentiation. The protein was increased about 2-fold in FSHD versus control myotubes but reached 2-
10-fold induction in FSHD muscle biopsies. We have shown by Western blot and by a DNA-binding assay that DUX4c over-
expression induced the MYF5 myogenic regulator and its DNA-binding activity. DUX4c might stabilize the MYF5 protein as
we detected their interaction by co-immunoprecipitation. In keeping with the known role of Myf5 in myoblast accumulation
during mouse muscle regeneration DUX4c over-expression activated proliferation of human primary myoblasts and
inhibited their differentiation. Altogether, these results suggested that DUX4c could be involved in muscle regeneration and
that changes in its expression could contribute to the FSHD pathology.
Citation: Ansseau E, Laoudj-Chenivesse D, Marcowycz A, Tassin A, Vanderplanck C, et al. (2009) DUX4c Is Up-Regulated in FSHD. It Induces the MYF5 Protein and
Human Myoblast Proliferation. PLoS ONE 4(10): e7482. doi:10.1371/journal.pone.0007482
Editor: Patrick Callaerts, Katholieke Universiteit Leuven, Belgium
Received June 2, 2009; Accepted September 17, 2009; Published October 15, 2009
Copyright: ? 2009 Ansseau 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 study was funded by the ABMM (Belgium; A.B.), AFM (France; A.B. #8598 and D. L-C. AFM-CNRS), F.N.R.S. (Belgium; A.B. #3.4623.01), FSH Society
(A.B. #DR-01) and MDA (A.B. # 3209 and F.C. # 3453). Additional support of the Fischer-Shaw Family (USA; A.B.) and Mr. D. Frenzel (Germany; A.B.). E.A., A.M. and
A.T. held pre-doctoral fellowships of the FRIA (Belgium), S.S. of the AFM, and M.B. of the MRT (France). D. L-C. and I.L. were AFM post-doctoral fellows. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
" These authors also contributed equally to this work.
OMIM #158900) is the third most frequent hereditary disease
of muscle, affecting one individual in 20,000 and is associated with
contractions of a repeat array in the subtelomeric 4q35 region
[1–3]. In non-affected individuals the array comprises 11–100
tandem copies of a 3.3-kilobase (kb) element named D4Z4. In
patients, only 1–10 D4Z4 copies are left, and the disease is usually
more severe with shorter repeat arrays [4–7]. It is chromosome-4
specific since contractions of the homologous repeat array in 10q26
do not cause FSHD . Additional features are also needed on
chromosome 4 besides the array contraction since neither the 4qB
allele  nor some 4qA polymorphic alleles arelinked to FSHD .
Several genes identified in the region proximal to D4Z4 might
contribute to the full FSHD phenotype (reviewed in ). Various
mechanisms were proposed to explain how the deletion might
activate their expression in FSHD muscles (reviewed in ; ).
A FSHD-related nuclear matrix attachment site (FR-MAR) was
recently mapped in the locus  that establishes a first chromatin
loop containing the D4Z4 array and a second 150-kb loop
containing FRG1 (FSHD region gene 1) [14,15], TUBB4Q (a
tubulin pseudogene)  and FRG2 (FSHD region gene 2) . In
addition a very potent transcriptional enhancer was found in the
59-part of the D4Z4 unit (). The FR-MAR has an enhancer
blocking activity that is efficient in human control myoblasts and
non-muscle cells. However in FSHD the FR-MAR is weakened so
that the repeat array and its neighbouring genes are brought into a
single chromatin loop where the D4Z4 enhancer might up-
regulate transcription of any gene ([13,17]) including the DUX4
(double homeobox) gene that our group mapped inside each D4Z4
element . Among those genes, FRG1 was recently shown to be
involved in muscle development in Xenopus , and transgenic
mice that strongly over-express FRG1 in their skeletal muscles
present a muscular dystrophy, suggesting an involvement in the
PLoS ONE | www.plosone.org1October 2009 | Volume 4 | Issue 10 | e7482
Our group has shown that DUX4 although initially considered
as a pseudogene, was expressed in FSHD but not control primary
myoblasts . DUX4 over-expression alters emerin distribution,
and induces caspases 3/7 activation leading to death of established
cell lines . The DUX4 protein is a transcription factor that can
activate or repress numerous genes. Its expression in C2C12 cells
recapitulates key features of the FSHD molecular phenotype,
including repression of MyoD and its target genes, diminished
myogenic differentiation, repression of glutathione redox pathway
components, and sensitivity to oxidative stress . One
additional DUX4 target is the paired-like homeodomain tran-
scription factor 1 gene (Pitx1) on chromosome 5, that is specifically
up-regulated in muscles of patients with FSHD as compared to 11
other neuromuscular disorders . The PITX1 protein in turn
activates genes involved in skeletal muscle atrophy that is a
hallmark of FSHD . Together these studies provided a link
between the genetic defect at D4Z4 (activating DUX4 gene
expression) and the pathophysiology of FSHD muscles and thus
demonstrated a major role for DUX4 and PITX1 in the disease.
The evolutionary conservation of the DUX4 ORF since
placental mammals and the presence of several functional
paralogues in rodents lend support to a defined function for
double homeodomain proteins . We have identified a human
DUX4 homologue that we named DUX4c (centromeric) mapping
42 kb proximal of the D4Z4 array, next to the FRG2 gene (Fig. 1A).
DUX4c is inside a truncated and inverted solitary D4Z4 unit 
(locus D4S2463) mapped in 4q35 at the proximal limit of
homology with 10q26 (Fig. 1A) .
A previous functional study indicated that the DUX4c protein
could interfere with differentiation of mouse C2C12 cells . We
have now characterized the endogenous DUX4c gene located near
the FSHD locus and shown it was functional. We could detect its
expression in human muscle cells (biopsies and primary myoblasts)
at RNA and protein levels, and found it was up-regulated in
FSHD. DUX4c over-expression could activate human myoblast
proliferation and inhibit their differentiation in vitro. This process is
most probably caused through induction of the MYF5 myogenic
regulator that is up-regulated by DUX4c.
Characterization of the DUX4c gene
We have identified the DUX4c gene by analysis of a published
genomic sequence (GenBank accession no. AF146191) containing
FRG1, TUBB4Q and FRG2 (Fig. 1A). Due to the high GC content,
we have confirmed its sequence on two different genomic fragments
(GenBank accession no. AY500824). DUX4c is identical on a large
part of its coding and proximal sequences (including the two
homeoboxes) to its DUX4 homologue in D4Z4. The DUX4c ORF is
located in a single exon and extends over 1,125 bp as compared to
1,275 for DUX4. Both genes are identical in a 1,137 bp fragment
starting 111 bp 59 from their common start codon, except for three
mismatches outside the double homeoboxes.
The DUX4c gene contains a functional promoter
A modified TATAA box (TACAA), and a GC box binding Sp1
mediate basal activity of the DUX4 promoter . DUX4c presents
similar elements at slightly shifted positions, and an additional GC
box (Supplemental Fig. S1). In order to evaluate the DUX4c
promoter activity, we fused a 477-bp PstI/EagI upstream fragment
to the luciferase reporter gene in pGL3 and generated pDUX4c-
LUC (Fig. 1B). The luciferase activity was assayed 24 h after
transfection of C2C12 (mouse myoblast), TE671 (human rhabdo-
myosarcoma) or HeLa cells. As compared to the promoter-less
vector (Luc), the luciferase activity was increased 6- and 11-fold in
C2C12 and TE671 cells, respectively, but only 1.8-fold in HeLa
cells (p,0.01) (Fig. 2).
The DUX4c protein is up-regulated in FSHD primary
myoblasts and biopsies
Conceptual translation of the DUX4c gene yields a 374-residue
protein identical to DUX4 (424 residues) in the double homeo-
and most of the carboxyl-terminal domains (positions 1–342). The
last 32 residues in DUX4c only present 40% identity with DUX4
(Supplemental Fig. S2). The DUX4 and DUX4c proteins
presented apparent molecular weights (MW) of 52 and 47 kDa,
Figure 1. Localization of the DUX4 and DUX4c genes. (A) Schematic representation of the 4q35 subtelomeric region with the D4Z4 repeat array
and the SLC25A4 (previously known as ANT1) , ALP , FRG1 , TUBB4Q  and FRG2  genes. DUX4 maps within each D4Z4 element 
and DUX4c within an isolated inverted D4Z4 unit at the D4S2463 locus. S/MAR and FR-MAR: nuclear scaffold/matrix attachment regions, . Upper
line: 4q35/10q26 limit of homology . (B) Enlargement (inverted orientation) of the 7.5-kb fragment that contains DUX4c with part of the FRG2
gene. The DUX4c ORF is boxed, with the homeoboxes in black. The promoter GC boxes, the putative variant TATA box (CATAA) and polyadenylation
signal are indicated. Numbering from the EcoRI site (GenBank acc. no. AY500824).
DUX4c in FSHD
PLoS ONE | www.plosone.org2October 2009 | Volume 4 | Issue 10 | e7482
respectively, in PAGE-SDS upon expression of their ORF by
transcription-translation in vitro (Fig 3A). In order to investigate
DUX4c expression in human muscle cells we raised a rabbit
antiserum against a carboxyl-terminal peptide present in DUX4c
but not in DUX4 (underlined in Supplemental Fig. S2).
This antiserum revealed a 47-kDa protein in Western blot of
both control and FSHD primary myoblasts, but at a higher
intensity in the later (Fig. 3B, left panel, lanes F1, F2). This band
was specific since it disappeared upon competition with the
DUX4c immunogenic peptide (Fig. 3B, right panel) or when the
cells were transfected with a siRNA targeting the DUX4c 39UTR
(Supplemental Fig S3). During differentiation (2 and 6 days after
confluence), the DUX4c protein progressively accumulated in
both control and FSHD myoblasts (Fig. 3C).
This antiserum also detected DUX4c by immunofluorescence
in the nuclei of primary myotubes, as expected for a homeodo-
main protein (Fig. 3D; a, enlarged in a’; 2-day differentiation). The
punctuate labelling increased during differentiation (b, enlarged in
b’; 6-day differentiation) and was lost upon competition with the
DUX4c peptide (c). A myoblast subpopulation not included in
myotubes also presented labelled nuclei (b, white circle).
We then compared DUX4c expression in control, FSHD or
Duchenne muscular dystrophy (DMD). Since DUX4c was induced
upon differentiation we performed Western blots on extracts of
homogenous myotube populations (6-day differentiation) to
quantify the protein (Fig 3E) relative to the a-tubulin loading
control: a 1.5- to 2-fold increase was seen in FSHD as compared to
control or DMD cells (Fig. 3F).
We similarly analysed DUX4c expression in proteins extracted
from human muscle biopsies (Fig 4A.) The biopsies were taken in
FSHD unaffected quadriceps (Q) or deltoid (D) except for one
FSHD affected trapezius (T). Control biopsies were taken in the
same muscles of non-affected individuals. The DUX4c signal was
quantified relative to the cytochrome C loading control by
densitometric analysis of this Western blot and of another one
not shown (Fig. 4B). An increased DUX4c level was shown in all
FSHD samples with the highest ones detected in the samples
derived from patients with low D4Z4 copy numbers (5 or 6;
Fig. 4B), except for the F7 sample corresponding to the affected
trapezius muscle that presented important necrosis and fat
accumulation (data not shown). We observed a progressive
DUX4c increase associated to decreasing D4Z4 copy numbers.
One of the samples was derived from a patient homozygous for the
4q35 deletion (5 and 7 D4Z4 units) and presented a similar
DUX4c level as those found in the biopsies derived from patients
with 5 but not 7 D4Z4 units. The nature of the 4q alleles (A or B)
in this patient is unknown but only one allele might be pathogenic
as reported for two other homozygous patients with FSHD .
DUX4c levels were also higher in DMD compared to control
biopsies, in contrast to the levels observed in myoblast cultures.
DUX4c over-expression induces MYF5 and cell
We investigated whether DUX4c might affect myogenic factor
activities with ELISA-based assays that detect trans-factors upon
binding to their immobilized DNA target (TransAm, Active
Motif). We prepared nuclear extracts of human TE671 cells
transfected with pCIneo vectors expressing DUX4c, DUX4 or the
shorter DUX1 protein (a non-4q35 homologue limited to the
homeodomains ) or with the insert-less vector. Both DUX4c
and DUX4 decreased MYOD1 at 24 h (not shown) and 48 h as
well as the MEF2 family members evaluated at 48 h (Fig. 5A–B).
DUX4c uniquely induced MYF5 binding activity at 24 and 48 h
(Fig. 5C, p,0.001). A Western blot demonstrated that MYF5 was
induced at the protein level and reached 5- to 6-fold 48 h after
transfection (Fig. 5D). This induction was dose-dependent as
shown in cells transfected with a doxycyclin-inducible DUX4c
expression vector (Fig 5E).
Intriguingly in another study, the Myf5 mRNA was down-
regulated in mouse C2C12 cells expressing an inducible DUX4c
cassette . To evaluate whether this discrepancy was caused by
species differences we transfected C2C12 cells with pCINeo-DUX4c.
Again the Myf5 protein was induced by DUX4c as compared to
cells transfected with the insert-less pCINeo vector. Moreover, we
could confirm the dose-dependence between DUX4c and Myf5
levels as observed in human muscle cells (Fig 5F).
We then evaluated whether the MYF5 protein might interact
with DUX4c. TE671 cells were transfected with pCINeo-DUX4c
and protein extracts prepared 48 h later. The immuno-precipitate
obtained with an anti-MYF5 serum was loaded on a PAGE-SDS
gel and a Western blot was performed with the anti DUX4c
serum, showing the expected 47-kDa protein. The protein
interaction was confirmed when the immuno-precipitaion was
performed with the anti-DUX4c serum and the Western blot with
the anti-MYF5 serum (Fig 5G).
As MYF5 expression and MYOD1 down regulation are related
to the maintenance of the muscle satellite cell pool (reviewed by
[30,31]), we investigated whether DUX4c protein expression
affected cell proliferation in human TE671 cells or immortalized
primary myoblasts . DUX4c induced a 2–3 fold higher
proliferation rate than two controls (DUX1 or the insert-less
pCINeo vector, p,0.005) as determined with a colorimetric MTT
assay 24 and 48 h post transfection. The cells expressing DUX4
had a decreased proliferation rate as expected from its toxicicity
. These data were confirmed by total protein quantification
and detection of two proliferation markers i.e. PCNA and cyclin
A, in the DUX4c expressing cells (Fig. 6A,C).
We switched the different transfected TE671 cells to a low
serum medium to induce differentiation. Four days later, all the
cells showed cytoplasmic extensions and high immunofluorescence
staining for desmin, an early myogenic differentiation marker,
except for the DUX4c expressing cells. These were numerous and
smaller and only had a weak labelling for desmin (Fig. 6B).
Similarly, at a later time (8-day differentiation) troponin T,
another marker of myoblast differentiation, was undetected in
Figure 2. Transcriptional activity of the DUX4c gene. Transcrip-
tional activity of the DUX4c promoter. HeLa, C2C12and TE671 cells were
transfected with pGL3 vectors containing the luciferase reporter gene
either promoterless (black bars) or fused to the DUX4c (white bars) or
DUX4 (striped bars) promoter. Luciferase activity was measured 24 h
post-transfection and expressed relative to the activity of the
promoterless vector set to 1. The means and standard errors are
DUX4c in FSHD
PLoS ONE | www.plosone.org3 October 2009 | Volume 4 | Issue 10 | e7482
human immortalized myoblasts transfected with pCI-neoDUX4c
while it was clearly induced in the other transfected cells (Fig 6D).
In conclusion, forced DUX4c expression induced MYF5 and
cell proliferation suggesting a role in myoblast proliferation during
Characterization of the endogenous DUX4c mRNAs
In the last part of this study, we wanted to characterize the
DUX4c mRNAs expressed in human myoblasts. Optimal 59 and
39 RACE conditions were established on mouse C2C12 cells
transfected with the DUX4c genomic clones to avoid the
background generated by hundreds of homologous human DUX
genes [2,29,33–35] (see Supporting Information S1 and Supple-
mental Fig. S1). A 59RACE performed on total RNAs of human
primary myoblasts detected 2 and 1 initiation sites in control and
FSHD cells, respectively, that might result from the use of either a
GC or the variant TATAA box (Fig. 7A). A single DUX4c mRNA
end (identical to the most frequent end observed in transfected
mouse cells; position 2629) was detected by 39RACE on total RNA
of FSHD myoblasts (Fig 7B). The oligo-dT adapter used for the
RT step of the 39RACE suggested that the mRNAs were
polyadenylated although no poly-A addition signal could be
identified on the gene.
Since the DUX4c mRNA ends we mapped were flanking the
complete ORF we selected primers to detect the full size mRNA
by reverse transcription (RT) with a DUX4c-specific primer
followed by PCR (Fig 7C, Supplemental Table S1). The RT-
PCR was first performed on total RNAs of C2C12 cells transfected
Figure 3. DUX4c protein expression in myoblasts. (A) Transcription/translation in vitro in a rabbit reticulocyte lysate in the presence T7 RNA
polymerase and [35S]-cysteine with genomic fragments encoding DUX4 (lane 1) or DUX4c (lane 2) cloned in pCIneo. 52 kDa-DUX4 (white arrow) and
47 kDa-DUX4c (black arrow) are detected by autoradiography after 10% PAGE-SDS. (B–C) 30 mg total proteins extracted from primary myoblast were
analysed by 4–12% PAGE-SDS and Western blot with the indicated primary antibodies, appropriate secondary antibodies coupled to HRP and the ECL
kit. a-Tubulin was the loading control. (B) Competition: a 5-fold excess of DUX4c antigenic peptide was pre-incubated (+) or not (2) with the serum
raised against DUX4c or cadherin as indicated. (C) Extracts were prepared either from proliferating myoblasts or 2 (d2) or 6 (d6) days after induction of
differentiation. (D) DUX4c (red) was detected by immunofluorescence in nuclei of myoblasts and myotubes 2 (d2) and 6 (d6) days after inducing
differentiation. a’ and b’ correspond respectively to two enlarged nuclei from d2 and d6 (white boxes in a and b). The labeling is weakened after
competition with the immunogenic peptide (c). Troponin T (green) is a myotube differentiation marker. Myoblasts not fused to myotubes express
DUX4c (red nuclei) but not troponin T. Bar corresponds to 20 mm. (E) 30 mg protein extracted from primary myotubes were analyzed by Western blot
as in (C). (F) Densitometric scanning of the film shown in (E): DUX4c expression levels were normalized to a-Tubulin (relative absorbance units). C:
control, F: FSHD, D: DMD.
DUX4c in FSHD
PLoS ONE | www.plosone.org4 October 2009 | Volume 4 | Issue 10 | e7482
with the DUX4c genomic fragment and yielded the expected 1265-
bp fragment (Fig. 7D, lane 3) that was missing upon RT omission
or in cells transfected with the insertless vectors (data not shown).
A sample not treated with DNase I provided a PCR positive
control (lane 1). The 1265-bp RT-PCR product was detected at a
very low intensity in a control and an FSHD myoblast line both in
proliferation (lanes 4 and 7), and after differentiation to myotubes
(lanes 6 and 9). It was absent upon RT omission (lanes 5 and 8).
The RT-PCR products were cloned in pCR4 and the E. coli
colonies were screened by hybridization with a DUX4c specific
probe (see Material and Methods), yielding 30–50% positives.
Several of these clones were analyzed and their full cDNA
sequences were found identical to DUX4c in either control or
In aggregate, these data demonstrated that the DUX4c gene
could be transcribed from its natural promoter into RNAs
covering its entire ORF, and that such mRNAs were expressed
in FSHD and control myoblasts.
In the present study, we have characterized the DUX4c gene
mapped near the FSHD locus and have demonstrated it expressed
a protein in human myoblasts and muscle biopsies.
Our demonstration of DUX4c mRNA expression confirmed
that this 4q35 gene was in a chromatin structure compatible with
transcription as suggested by previous studies: its promoter was
found associated with acetylated histone H4  and it interacted
with RNA polymerase II at slightly higher level than non-
transcribed sequences . Moreover, DUX4c is brought in the
same chromatin loop as the enhancer-containing D4Z4 repeats in
FSHD myoblasts [13,17]; Fig. 1A) and this enhancer directly
interacts with the DUX4c promoter . However, these
publications had failed to detect DUX4c mRNAs by RT-PCR.
Our present study is the first report detecting the endogenous
DUX4c mRNA and protein of human muscle cells. Similarly to the
homologous DUX4 mRNA, the DUX4c sequence is extremely GC-
rich and difficult to retro-transcribe and amplify. We have recently
listed the critical technical points to amplify these low-abundance
and GC rich mRNAs, and highlighted the need for a higher
temperature and a gene-specific primer during the RT step 
(see Materials and Methods). An additional problem in the study
by Alexiadis et al  is that the forward primer used to
specifically amplify DUX4c hybridized at position 777–800, i.e.
upstream of some of the 59 ends found in the present study.
Indeed, our 59RACE data suggested that transcription could be
driven as well by the variant TATAA box as by several GC boxes
in the DUX4c promoter.
In the DUX4c gene, a single exon comprised the full ORF, and
we found a spliced out intron in the 39 untranslated region (UTR)
of some mRNAs. No polyadenylation signal could be found on the
gene and the DUX4c mRNAs presented heterogeneous 39 ends.
Such a structure was reported for histone genes that use the U7
snRNA to process the mRNA ends (37). However, the 39RACE
was done with an oligo-dT primer suggesting a polyadenylation of
the DUX4c mRNAs.
The studies of gene transcription in 4q35 are complicated by the
fact these genes belong to families with functional or pseudogene
homologues in multiple locations of the human genome. No
transcript could be detected for TUBB4Q , some studies found
increased FRG1 and FRG2 mRNAs in FSHD samples [16,39],
others did not [36,40]. The only protein expression data were
reported for DUX4 present within each D4Z4 repeated element
 and for ANT1 (also called SLC25A4) located 4.9 Mb from the
locus. The ANT1 protein was strongly upregulated in contrast to
its mRNA, suggesting a posttranscriptional regulation [36,41].
Y. Vassetsky’s group showed that an enhancer located in D4Z4
interacts directly with the DUX4c promoter as a result of changes
in chromatin looping caused by the D4Z4 array contraction .
In agreement with this observation, we detected that the DUX4c
protein was increased in extracts of FSHD muscles. Moreover, a
progressive DUX4c accumulation was associated to decreasing
D4Z4 copy numbers, with DUX4c expression reaching 10-fold the
control value when the pathogenic allele only presented 5 D4Z4
units (F8–F10 Fig. 4B). A similar activation pattern was previously
shown for the flanking FRG2 gene that belongs to the same
chromatin loop as DUX4c . The DUX4c increase was
observed in non-affected muscles of patients suggesting it was an
early event in the disease progression. DUX4c expression could
therefore be considered as a sensor of chromatin structure in
FSHD. It would be very interesting to evaluate DUX4c expression
in biopsies of the few reported patients with a D4Z4 deletion
removing DUX4c (see below) or of asymptomatic individuals with
D4Z4 deletion to confirm this hypothesis.
No link between DUX4c expression and D4Z4 copy number
could be observed in myoblasts. However the only primary
myoblast line with 5 units we used derived from the F7 biopsy
taken in an affected trapezius, not in a non-affected quadriceps like the
other samples. The relationship between DUX4c expression and
D4Z4 copy number in muscle biopsies is in agreement with a
transcriptional inhibitory role of the D4Z4 element  and should
be further evaluated in additional samples from different muscles
and in patients with lower D4Z4 copy numbers. We could not
correlate DUX4c protein expression and residual D4Z4 copy
Figure 4. DUX4c protein expression in muscle biopsies. (A)
30 mg protein extracted of muscle biopsies were analyzed by Western
blot as in Fig. 3, except that cytochrome C was the internal loading
control. (B) Densitometric scanning of the Western blot shown in (A)
and of additional samples (not shown): DUX4c expression levels were
normalized to cytochrome C (relative absorbance units). Samples are
indicated C1 to C4 for controls, F1 to F10 for FSHD, and D1 to D4 for
DMD as well as the D4Z4 copy numbers of the FSHD patients. The
biopsied muscle is indicaded (D, Q: non-affected deltoid or quadriceps;
T*: affected trapezius). F10 also has a D4Z4 array contraction on the
second 4q35 allele (+7).
DUX4c in FSHD
PLoS ONE | www.plosone.org5 October 2009 | Volume 4 | Issue 10 | e7482
number (5 to 8) to clinical disease severity in the present study. This
is in agreement with previous data for which such a correlation
could not be established for D4Z4 arrays larger than 3 units [7,42].
The DUX4c protein has not been observed by other groups in
proteome studies of FSHD muscles [41,43] most probably because
its very high pI (11.1) was not reached during the isoelectrofocalisa-
Forced DUX4c expression in human muscle cells induced the
MYF5 protein and its DNA-binding activity. This transcription
factor is known to inhibit myoblast differentiation [44,45].
Furthermore, DUX4c expression inhibited MYOD1 DNA-
binding activity and prevented cell differentiation following serum
withdrawal, as reported for Myf5+/MyoD2myoblasts  and in
iC2C12-DUX4c cells . The later study reported a down-
regulation of the Myf5 mRNA following DUX4c induction. In
contrast, in the present report, we observed a dose-dependent
induction of the Myf5 protein as well in human TE671 as in
mouse C2C12 cells expressing DUX4c. In addition, we found an
interaction between DUX4c and Myf5 that might lead to a
stabilisation of the later protein. Indeed Myf5 degradation is
known to be controlled by specific posttranslational modifications
 and it is therefore possible that deregulation of the mRNA
does not lead to change in the protein levels.
Besides an interference with myoblast differentiation that was
also described by Bosnakovski et al (2008), we found that DUX4c
over-expression in human cells led to an increased proliferation
rate by MTT assay, PCNA and cyclin A labelling. This
phenomenon could be due to MYF5 protein accumulation since
its absence was shown to reduce the proliferation rate of satellite
cell derived myoblasts [47,48]. The DUX4c increase in DMD
Figure 5. DUX4c over-expression induces MYF5. (A–C) TE671 cells were transfected with the indicated pCIneo vectors. Nuclear extracts were
deposited in triplicate on a plate where the MYOD1, MEF2 or MYF5 specific DNA target was immobilized. The DNA-bound protein was detected by
ELISA (TransAm assay). Relative absorbances are given relative to the insertless pCIneo sample arbitrarily set to 1. Three independent experiments (1
to 3) made in triplicate are presented. (D) TE671 nuclear extracts were prepared 48 h after transfection as above and 30 or 15 (*) mg were analyzed by
10% PAGE-SDS and Western blotting with a serum raised against MYF5 or actin (internal control). NT: non transfected cells. (E) same as in D but
transfection was with pAC1M2-DUX4c and DUX4c expression induced by doxycycline (o to1000 ng). (F) Mouse C2C12 cells were transfected with the
indicated pCIneo vectors. Total protein extracts were prepared 24 or 48 h later and 40 mg were analysed by Western blot with a serum raised against
MYF5 or DUX4c as in D. (G) 40 mg nuclear extracts of TE cells transfected with the indicated vectors were subjected to immunoprecipitation with the
anti-DUX4c or the anti-MYF5 serum. The immunoprecipitate was analysed by Western blot with the anti-DUX4c or anti-MYF5 serum as in D.
DUX4c in FSHD
PLoS ONE | www.plosone.org6 October 2009 | Volume 4 | Issue 10 | e7482
muscle biopsies (Fig. 4A–B) could be related to the higher
regeneration rate reported for this disease . The DMD
biopsies used in the present study indeed contained newly formed
fibres still presenting centrally located nuclei (data not shown). The
DUX4c up-regulation in the DMD biopsies (Fig. 4B) could be
related to the increased satellite cell proliferation in comparison to
control muscles where satellite cells were quiescent. In keeping
with this idea, no difference in DUX4c expression was found
between DMD and control myoblasts that were both derived from
activated satellite cells (Fig 3E). In contrast to FSHD, DMD
muscles do not present D4Z4 contraction (see Supplemental Table
S2) therefore DUX4c up-regulation could only be related to
increased muscle regeneration.
The up-regulation of DUX4c expression upon myoblast
differentiation and its inhibitory effect on this very process appear
contradictory. However one could hypothesize that DUX4c has
different function in myoblasts or in myotubes according to its
interaction with different protein partners. Indeed its MYF5
partner which is involved in proliferation is only expressed in
myoblasts but not in myotubes . Moreover the observed
change of DUX4c nuclear localization during differentiation is in
favour of a functional switch.
Functional studies performed in parallel on the homologous
DUX4c and DUX4 proteins have shown several differences
despite their high similarity. Both proteins share an identical
double homeodomain and specifically bind the Pitx1 promoter but
DUX4 activates its transcription at a stronger level . DUX4
does indeed present a carboxyl terminal region partly missing in
DUX4c that mediates strong transcriptional activation [51,52].
Forced DUX4c expression in TE671 cells did not induce caspases
3/7 activity nor cell death as shown for DUX4  (A.
Marcowycz, unpublished data). Moreover, we found that the
MYF5 induction was unique to DUX4c expression.
The DUX4c gene is only present on chromosome 4 where it
defines the proximal limit of homology with chromosome 10, and
our results suggest it plays a role in FSHD that is uniquely
associated with array contractions in 4q35 . However, a
deletion extending from the D4Z4 repeat array to include FRG2
and DUX4c was reported in some families with FSHD 
suggesting that neither gene could cause the disease. A transvec-
tion effect resulting from a misbalance of chromatin and
transcription factors at 4q35 and unrelated loci following the
deletion  was proposed to explain that the FRG2 mRNA
expressed in FSHD myoblasts originated mostly from the
homologous FRG2 gene on chromosome 10, not 4 . A similar
mechanism in trans might also occur between the two chromosome
4 alleles in FSHD cells, activating DUX4c on the non-affected one.
This could be tested in muscle biopsies of patients with an
extended 4q35 deletion removing DUX4c on one allele. Never-
theless, in most affected families, both DUX4c and FRG2 are
present and could contribute to the penetrance and severity of the
disease. Alternatively, since we found DUX4c expression in
control myoblasts and muscle biopsies, it is possible that a single
allele deletion could also have pathological consequences.
We have demonstrated functionality of the DUX4c gene in spite
of a variant TATAA box, an intron-less ORF and the lack of a
poly-A addition signal. If the other 3.3-kb repeated elements
scattered on other chromosomes could similarly be expressed, the
human genome might have to be expanded with hundreds of
additional DUX genes. We have previously characterized other
actively transcribed DUX genes on the acrocentric chromosomes
. One DUX gene with introns was proposed to have generated
Figure 6. DUX4c over-expression induces cell proliferation. (A) PCNA was detected by immunofluorescence (red) 24 h post transfection with
the pCIneo vectors indicated. The picture of DUX4c expressing cells was exposed 1.4 sec versus 2.7 sec for the other panels to visualize the cells. (B)
The cells were switched 24 h post transfection to a differentiation medium, and observed 4 days later by phase contrast microscopy (left panels).
Early differentiation was evaluated by desmin detection (green, right panels). (C) Cyclin A (green) and DUX4/4c (red) were detected by
immunofluorescence 24 h post transfection of human immortalized myoblasts with the indicated pCIneo-vectors. (D) Human immortalized myoblasts
were transfected with the indicated pCIneo-vectors and switched to differentiation medium 48 h later. Troponin T was detected by
immunofluorescence 8 days later. Bars correspond to 20 mm.
DUX4c in FSHD
PLoS ONE | www.plosone.org7 October 2009 | Volume 4 | Issue 10 | e7482
multiple retrotransposed pseudogenes with reported EST on
autosomal chromosomes . Four putative DUX genes were
reported in the pericentromeric region of the Y chromosome
[34,54]. Together with our protein expression data on DUX4 and
DUX1 [21,29] this result bears on the general questions of how to
define a gene versus a pseudogene, and of what can be considered
as junk or ‘‘func’’ (functional) DNA [55,56].
FSHD is a complex disease associated with a chromatin change
affecting the expression of several genes. However to date only two
proteins (i.e. ANT1 and DUX4) were shown to be up-regulated
from FSHD candidate genes. Although DUX4 activation strikingly
recapitulates key features of the FSHD molecular phenotype
[21,23], other 4q35 genes could also contribute to the heteroge-
neity of the FSHD phenotype . This could be the case for
FRG1 that is implicated in muscle development and angiogenesis
[19,58] but at the present time no data on FRG1 protein
expression in FSHD muscle is available. The present study
demonstrated that the DUX4c protein is over-expressed in FSHD
muscle and could therefore contribute to the development of the
disease. Moreover, we have uncovered a putative role for DUX4c
in muscle regeneration that should be further investigated in
injured or atrophic muscles of healthy individuals, and in muscles
of patients with different neuro-muscular pathologies.
Materials and Methods
Muscle biopsies (see Supplemental Table S2) were performed
according to a procedure approved either by the University of
Rochester Research Subjects Review Board (reference number
RSRB#8567) or current ethical and legislative rules of France as
described  (ref number 050503). Written informed consent was
obtained from all subjects, as directed by the ethical committee of
either institution. In addition, the uses of this material have been
approved by the ethics committee of the University of Mons-
Hainaut (ref number A901).
Mammalian Cell Cultures
C2C12 and TE671 cells were grown in DMEM, 1%
gium) and 10% fetal calf serum (PAA Laboratories) at 37uC
under 5% CO2. HeLa cells were grown in DMEM-F12
(Cambrex) supplemented as above. The primary myoblast
cultures were established as described  and grown in DMEM
with 10% FCS and 1% Ultroser G (BioSepra, Cergy-Pontoise,
France). For differentiation, the cells were either grown to
confluence or the medium was replaced by DMEM supplement-
ed by 2% horse serum (PAA laboratories, Pasching, Austria) as
indicated. Immortalized myoblasts were grown and differentiat-
ed as reported (Zhu et al) except that 1% Ultroser G was used
instead of HGF during proliferation.
A 477-bp PstI/EagI fragment corresponding to the DUX4c
promoter was fused to the luciferase reporter gene in pGL3
(Promega, Leiden, The Netherlands) yielding pGL3-DUX4c. The
pGL3control has the SV40 promoter/enhancer (Promega). A 3-kb
EcoRI fragment containing the DUX4c natural gene was cloned in
pENTR1A (Invitrogen, Carlsbad, CA), yielding p3 kb-DUX4c. The
1.2-kb DUX4c ORF was cloned into pCIneo (Promega) or pAC1M2
(for doxycyclin induction  yielding pCIneo-DUX4c or pAC1M2-
DUX4c. All the constructs were confirmed by sequence determi-
nation and are detailed in Supporting Information S2.
Figure 7. Characterization of the DUX4c mRNA. (A) Schematic
representation of the DUX4c promoter with the transcription start sites
(arrows and positions) identified by 59 RACE (primer indicated) on RNA
extracted from control and FSHD myoblasts. (B) Top: Schematic
representation of the p7.5 kb-DUX4c insert (see Supporting Information
S2) close to its 39 cloning site, showing the stop codon, the putative
poly-A addition signal, two purine-rich (86 and 83%) regions (black
boxes) and the primers used in 39RACE (arrows, #350 and 351). Bottom:
Mapping of the multiple 39 ends and alternative splicing detected in the
39RACE products. These were derived from RNAs of either C2C12 cells
transfected with p7.5 kb-DUX4c or FSHD primary myoblasts (*). (C)
Schematic representation of the DUX4c ORF with the homeoboxes
(black boxes) and the primers used for RT-PCR. (D) Amplification of the
DUX4c mRNA was performed on total RNA extracted from FSHD (F24) or
control primary myoblasts (C29) either in proliferation (lanes 4 and 7) or
differentiated to myotubes (diff.). RNA samples were incubated (+) or
not (2) with DNase I, and reverse transcriptase (RT) as indicated. The
PCR products were analysed by electrophoresis on a 1%-agarose gel
and stained with ethidium bromide. As a positive control (lane 3), RT-
PCR was performed on RNA of C2C12 cells transfected with p3 kb-
DUX4c in FSHD
PLoS ONE | www.plosone.org8 October 2009 | Volume 4 | Issue 10 | e7482
Transient luciferase expression
Either 105C2C12, 26105TE671 or 46105HeLa cells were
seeded in each well of 6-well plates and grown overnight.
Transfections were performed with 1.6 mg reporter plasmid and
16 ng phRL-SV40 (internal control) per well with either FuGENE6
(Roche Diagnostics, Mannheim, Germany) for TE671 cells or
Lipofectamin2000 (Invitrogen) for C2C12 and HeLa cells. Cells
were lysed 24 h later with the dual luciferase assay system
(Promega) and activity measured on a Packard LumiCount
(PerkinElmer). The firefly luciferase reporter plasmids were
derived from pGL3 (Promega) and contained either no insert
(pGL3-Basic), the DUX4c or DUX4 promoter . Experiments
were done 3 times in triplicate with 2 different preparations for
each plasmid (n=18 for each point). The DUX4c promoter was
about 40- and 350-fold less active in muscle and HeLa cells,
respectively, than the SV40 promoter/enhancer (pGL3-Control, not
RT-PCR, 59 and 39 RACE
Total RNA was extracted and DNase-treated as described
previously . RT was done on 2.5 mg freshly prepared RNA
with primer # 167 (all the primers sequences are given in
Supplemental Table S1) and 200 U of SuperScript III with a
procedure for high secondary structure . 8 ml cDNA were used
for PCR with primers # 49 and # 167 and the conditions were
3 min at 94uC, followed by 1 min at 94uC, 1 min at 68uC with a
1uC decrease at each cycle, and 2 min at 68uC for 4 cycles,
followed by 31 cycles of 1 min at 94uC, 1 min at 64uC, 2 min at
68uC with 5 sec/cycle increment during elongation. The RT step
of the 59 and 39 RACE was performed with 10 or 2 mg of total
DNase-treated RNA, respectively, with the RLM-RACE kit
(Ambion, Austin, TX). DUX4c primers #68 and #73 (59RACE)
or #350 and #351 (39RACE) were used for the nested PCR. The
products were cloned in pCR4 (TOPO TA kit, Invitrogen),
amplified in E. coli and sequenced with the CEQ 2000 (Beckman
Antibodies against DUX4c
A rabbit antiserum was raised against a 16-residue peptide
(underlined in Supplemental Fig. S2) specific of the DUX4c
carboxyl-terminal domain. This peptide was chosen by accessibil-
ity prediction programs, synthesized, coupled to KLH and injected
into rabbits. The resulting antisera were purified by affinity
chromatography on the immobilized peptide (Eurogentec, Sera-
Transcription/translation in vitro
Aliquots of rabbit reticulocyte lysate (TNT kit, Promega) were
incubated with pCIneo vectors in the presence of 30 mCi [35S]-
cysteine (Amersham Biosciences, Roosendaal, The Netherlands)
and T7 RNA polymerase. 10 ml of the products were denatured in
XT sample buffer with reducing agent (Bio-Rad, Hercules, CA)
and analysed by PAGE-SDS. The gel was incubated 30 min in
Amplify (Amersham Biosciences), air dried and submitted to
Whole cell extracts of myoblast primary cultures were obtained
by lysis in 50 mM Tris pH 7.0, 50 mM NaCl, 0.1% Nonidet P40,
1 mM DTT and protease inhibitors, were separated by PAGE-
SDS and electrotransferred onto a PVDF or nitrocellulose
membrane according to the manufacturer (Amersham Bioscienc-
es). The Western blot was incubated with the rabbit anti-DUX4c
(1:1000) or anti-Myf5 (1:500, C-20, Santa Cruz Biotechnology,
Santa Cruz, CA) sera followed by secondary antibodies coupled to
HRP and the ECL kit (Amersham Biosciences).
For standardization, the membranes were stripped and
immunostaining was performed with primary antibodies raised
against either a-tubulin (mAb, Sigma-Aldrich, Saint Louis, MO),
pan-cadherins (rabbit serum, Sigma-Aldrich), cytochrome C
(rabbit serum, Santa Cruz Biotechnology) or actin (rabbit serum,
Sigma-Aldrich) as indicated.
1.56106TE671 cells were seeded in a 75-cm2flask, grown and
transfected with pCIneo plasmids. Whole cell extracts were prepared
24 h later using sonication in 500 ml lysis buffer follow by
centrifugation 5 min at 16.000 g to discard cell membranes.
Immunoprecipitation was performed on 800 mg total extract with
the mouse monoclonal 9A12 antibody directed against DUX4 and
cross-reacting with DUX4c (1:5, Dixit 2007) or rabbit anti-MYF5
(1:100, Santa Cruz Biotechnologies) serum in 1 ml IP buffer in the
presence of protein G-agarose (Fermentas) or protein A-Sepharose
(Amersham Biosciences) respectively as described by the manufac-
turers. The final pellet was heated 5 min at 95uC in 30 ml loading
buffer with reducing agent (Fermentas), and centrifuged 5 min at
16,000 g. The supernatant was analysed by 12% PAGE-SDS
followed by electrotransfer to a PVDF membrane as above, and
Western blot was performed with the anti-MYF5 serum (1:500) or
the 9A12 antibody (1:1000) followed by secondary antibodies
coupled to HRP (Amersham Biosciences) and revealed as above.
1.56105TE671 cells were seeded on coverslips in 6-well plates
and transfected 24 h later with 1 mg plasmid DNA as indicated.
After 24 h, the cells were fixed in 4% paraformaldehyde or
Carnoy (desmin detection). Immunostaining was performed by
standard procedures as detailed in Supporting Information S2
with anti-DUX4c (1:50) or anti-PCNA (PC10, 1:40, Dako,
Glostrup, Denmark) serum or anti-desmin (DE-R-11, 1:50, Dako)
or anti-cyclin A (1:50, BD Transduction Laboratories, Erembo-
degem, Belgium) antibodies. As a control, the anti-DUX4c serum
was preincubated 2 h with a 5-fold molar excess of the DUX4c
immunogenic peptide. The anti-IgG secondary antibodies were
either coupled to FITC or biotinylated (Dako, Amersham
Biosciences) and incubated with streptavidin-Texas-Red (Vector
Laboratories, Burlingame, CA). The primary myoblasts (on
collagen-coated dishes) were incubated with anti-DUX4c and
anti-troponin T (1:100, JLT-12, Sigma) followed by Alexa
secondary antibodies (goat anti-mouse 488 and anti-rabbit 555,
1.56105TE671 cells were seeded in 6-well plates, grown
overnight and transfected with 1 mg plasmid DNA. The CellTiter
96 non-radioactive cell proliferation assay (Promega) was used 24
or 48 hours after transfection as described by the manufacturer.
Experiments were done in triplicate.
Myogenic factor activities
1.26106TE671 cells were seeded in a 75-cm2flask, grown
overnight, transfected with 10 mg of either pCIneo-DUX plasmid and
collected 48 h later. The cell lysates were prepared as described in
Supporting Information S2 and were deposited on an ELISA plate
where a specific DNA target was immobilized (TransAm kit,
ActiveMotif, Carlsbad, CA). A specific rabbit antiserum was added,
DUX4c in FSHD
PLoS ONE | www.plosone.org9October 2009 | Volume 4 | Issue 10 | e7482
followed by a secondary antibody coupled to HRP, and a substrate
yielding a product with absorbance at 450 nm.
Statistical significance was evaluated by the Student t test.
Supporting Information S1
Found at: doi:10.1371/journal.pone.0007482.s001 (0.05 MB
Supporting Information S2
Found at: doi:10.1371/journal.pone.0007482.s002 (0.04 MB
Supporting Materials and Methods
Found at: doi:10.1371/journal.pone.0007482.s003 (0.06 MB
Found at: doi:10.1371/journal.pone.0007482.s004 (0.05 MB
Biopsies and myoblast lines.
cells. Alignment of the DUX4c and DUX4 promoter sequences
(GenBank accession nos AY500824 and AF117653). The
numberings start at the 59EcoRI sites. The variant TATAA
boxes are underlined with brackets, the putative E boxes double
underlined, the GC boxes are boxed, and the translation
initiation codons circled. The broken arrows indicate the
transcription start sites experimentally determined for DUX4
(CoppA˜?e et al, 2004) and DUX4c. The later ones were
identified by 59RACE on RNA extracted from C2C12 cells
transfected with p3 kb-DUX4c (a) and p7.5 kb-DUX4c (b). At
each start site, the consensus initiator sequences is shown in low
cases (c/t c a n t/a c/t c/t). The primers (dotted line) used in a
chromatin immunoprecipitation study of acetylated histone H4 in
4q35 (Jiang et al, 2003) map in DUX4c.
Found at: doi:10.1371/journal.pone.0007482.s005 (1.38 MB TIF)
Characterization of DUX4c mRNA in transfected
proteins. The DUX4c protein sequence was derived from pSK-
DUX4c (integrating variations mentioned in GenBank accession
no. AY500824)and theDUX4
#AF117653. The identical double homeodomains are boxed.
The arrows indicate polymorphic residues: either valine or
isoleucine at position 229 in both DUX4c and DUX4; either
alanine or proline at position 272 in DUX4c, but only proline in
DUX4. The peptide used to generate a specific rabbit antiserum
against DUX4c is underlined.
Found at: doi:10.1371/journal.pone.0007482.s006 (2.56 MB TIF)
Sequence alignment of the DUX4c and DUX4
silencing. Human muscle TE671 cells were transfected (siPORT
NeoFX, Ambion) or not (-) with 20 nmol of siRNA either targeting
the DUX4c 39UTR, an unrelated genomic sequence (unrl), or a
sequence not found in the human genome (negative control, n.c.)
(Ambion). They were either transfected 5 h later (Fugene 6) with
the pCIneo-DUX4c expression vector (DUX4c) or not (NT).
Protein extracts were prepared 72 h later and analysed by Western
blot with the rabbit anti-DUX4c antiserum as in Fig 3. Actin
(antibody from Sigma) was used as a loading control.
Found at: doi:10.1371/journal.pone.0007482.s007 (2.71 MB TIF)
Downregulation of DUX4c expression by a RNA
We thank J.E. Hewitt (University of Nottingham, United Kingdom), and
S.M. Van der Maarel (University of Leiden, The Netherlands) for genomic
clones, L. Tenenbaum and A. Chtarto (University of Brussels, ULB,
Belgium) for the pAC1M2 vector, the platform for human cell culture from
the Institute of Myology (Paris, France) for the immortalized myoblasts. We
thank the patients for muscle biopsies, and the ‘‘Amis FSH Europe’’ for
their constant support.
Conceived and designed the experiments: EA DLC AM AT CV SS MB IL
VM AB FC. Performed the experiments: EA DLC AM AT CV SS MB IM
AL IL FC. Analyzed the data: EA DLC AM AT CV SS MB IM AL IL
VM FC. Contributed reagents/materials/analysis tools: DLC MB DF VM
GBB. Wrote the paper: EA AB FC.
1. Hewitt JE, Lyle R, Clark LN, Valleley EM, Wright TJ, et al. (1994) Analysis of
the tandem repeat locus D4Z4 associated with facioscapulohumeral muscular
dystrophy. Hum Mol Genet 3: 1287–1295.
2. Lyle R, Wright TJ, Clark LN, Hewitt JE (1995) The FSHD-associated repeat,
D4Z4, is a member of a dispersed family of homeobox-containing repeats,
subsets of which are clustered on the short arms of the acrocentric chromosomes.
Genomics 28: 389–397.
3. Wijmenga C, Hewitt JE, Sandkuijl LA, Clark LN, Wright TJ, et al. (1992)
Chromosome 4q DNA rearrangements associated with facioscapulohumeral
muscular dystrophy. Nat Genet 2: 26–30.
4. Lunt PW, Jardine PE, Koch MC, Maynard J, Osborn M, et al. (1995) Correlation
between fragment size at D4F104S1 and age at onset or at wheelchair use, with a
possible generational effect, accounts for much phenotypic variation in 4q35-
facioscapulohumeral muscular dystrophy (FSHD) [published erratum appears in
Hum Mol Genet 1995 Jul;4(7):1243-4]. Hum Mol Genet 4: 951–958.
5. Ricci E, Galluzzi G, Deidda G, Cacurri S, Colantoni L, et al. (1999) Progress in
the molecular diagnosis of facioscapulohumeral muscular dystrophy and
correlation between the number of KpnI repeats at the 4q35 locus and clinical
phenotype. Ann Neurol 45: 751–757.
6. Tawil R, Forrester J, Griggs RC, Mendell J, Kissel J, et al. (1996) Evidence for
anticipation and association of deletion size with severity in facioscapulohumeral
muscular dystrophy. The FSH-DY Group. Ann Neurol 39: 744–748.
7. van Overveld PG, Enthoven L, Ricci E, Rossi M, Felicetti L, et al. (2005)
Variable hypomethylation of D4Z4 in facioscapulohumeral muscular dystrophy.
Ann Neurol 58: 569–576.
8. Lemmers RJ, van der Maarel SM, van Deutekom JC, van der Wielen MJ,
Deidda G, et al. (1998) Inter- and intrachromosomal sub-telomeric rearrange-
ments on 4q35: implications for facioscapulohumeral muscular dystrophy
(FSHD) aetiology and diagnosis. Hum Mol Genet 7: 1207–1214.
9. 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.
10. Lemmers RJ, Wohlgemuth M, van der Gaag KJ, van der Vliet PJ, van
Teijlingen CM, et al. (2007) Specific Sequence Variations within the 4q35
Region Are Associated with Facioscapulohumeral Muscular Dystrophy.
Am J Hum Genet 81: 884–894.
11. van der Maarel SM, Frants RR, Padberg GW (2007) Facioscapulohumeral
muscular dystrophy. Biochim Biophys Acta 1772: 186–194.
12. van der Maarel SM, Frants RR (2005) The D4Z4 repeat-mediated pathogenesis
of facioscapulohumeral muscular dystrophy. Am J Hum Genet 76: 375–386.
13. Petrov A, Pirozhkova I, Carnac G, Laoudj D, 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 U S A 103: 6982–6987.
14. 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.
15. van Geel M, van Deutekom JCT, van Staalduinen A, Lemmers RJLF,
Dickson MC, et al. (2000) Identification of a novel beta-tubulin subfamily with
one member (TUBB4Q) located near the telomere of chromosome region 4q35.
Cytogenetics and Cell Genetics 88: 316–321.
16. Rijkers T, Deidda G, van KS, van GM, 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: 826–836.
17. Petrov A, Allinne J, Pirozhkova I, 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 Research
DUX4c in FSHD
PLoS ONE | www.plosone.org10 October 2009 | Volume 4 | Issue 10 | e7482
18. Gabriels J, Beckers MC, Ding H, De Vriese A, Plaisance S, et al. (1999) Download full-text
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.
19. Hanel ML, Wuebbles RD, Jones PL (2008) Muscular dystrophy candidate gene
FRG1 is critical for muscle development. Dev Dyn 238: 1502–1512.
20. Gabellini D, D’Antona G, Moggio M, Prelle A, Zecca C, et al. (2006)
Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1. Nature
21. 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:
22. 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.
23. Bosnakovski D, Xu Z, Gang EJ, Galindo CL, Liu M, et al. (2008) An isogenetic
myoblast expression screen identifies DUX4-mediated FSHD-associated molec-
ular pathologies. EMBO J 27: 2766–2779.
24. Dixit M, Shi R, Sutherland M, Munger S, Chen YW (2007) Characterization of
a tet-repressible muscle-specific Pitx1 transgenic mouse model as an animal
model of FSHD. Faseb J 21: 870.8.
25. Clapp J, Mitchell LM, Bolland DJ, Fantes J, Corcoran AE, et al. (2007)
Evolutionary conservation of a coding function for D4Z4, the tandem DNA
repeat mutated in facioscapulohumeral muscular dystrophy. Am J Hum Genet
26. Wright TJ, Wijmenga C, Clark LN, Frants RR, Williamson R, et al. (1993) Fine
mapping of the FSHD gene region orientates the rearranged fragment detected
by the probe p13E-11. Hum Mol Genet 2: 1673–1678.
27. 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.
28. Bosnakovski D, Lamb S, Simsek T, Xu Z, Belayew A, et al. (2008) DUX4c, an
FSHD candidate gene, interferes with myogenic regulators and abolishes
myoblast differentiation. Exp Neurol.
29. Ding H, Beckers MC, Plaisance S, Marynen P, Collen D, et al. (1998)
Characterization of a double homeodomain protein (DUX1) encoded by a
cDNA homologous to 3.3 kb dispersed repeated elements. Hum Mol Genet 7:
30. Buckingham M (2007) Skeletal muscle progenitor cells and the role of Pax genes.
C R Biol 330: 530–533.
31. Holterman CE, Rudnicki MA (2005) Molecular regulation of satellite cell
function. Semin Cell Dev Biol 16: 575–584.
32. Zhu CH, Mouly V, Cooper RN, Mamchaoui K, Bigot A, et al. (2007) Cellular
senescence in human myoblasts is overcome by human telomerase reverse
transcriptase and cyclin-dependent kinase 4: consequences in aging muscle and
therapeutic strategies for muscular dystrophies. Aging Cell 6: 515–523.
33. Beckers MC, Gabriels J, van der Maarel SM, De Vriese A, Frants RR, et al.
(2001) Active genes in junk DNA? Characterization of DUX genes embedded
within 3.3 kb repeated elements. Gene 264: 51–57.
34. Kirsch S, Weiss B, Miner TL, Waterston RH, Clark RA, et al. (2005)
Interchromosomal segmental duplications of the pericentromeric region on the
human Y chromosome. Genome Res 15: 195–204.
35. Booth HA, Holland PW (2007) Annotation, nomenclature and evolution of four
novel homeobox genes expressed in the human germ line. Gene 387: 7–14.
36. Jiang G, Yang F, van Overveld PG, Vedanarayanan V, van der Maarel SM,
et al. (2003) Testing the position effect variegation hypothesis for facioscapu-
lohumeral dystrophy by analysis of histone modification and gene expression in
subtelomeric 4q. Hum Mol Genet 12: 2909–2921.
37. Alexiadis V, Ballestas ME, Sanchez C, Winokur S, Vedanarayanan V, et al.
(2007) RNAPol-ChIP analysis of transcription from FSHD-linked tandem
repeats and satellite DNA. Biochimica et Biophysica Acta-Gene Structure and
Expression 1769: 29–40.
38. 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: e3389.
39. 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.
40. 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.
41. 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.
42. Butz M, Koch MC, Muller-Felber W, Lemmers RJ, van der Maarel SM, et al.
(2003) Facioscapulohumeral muscular dystrophy. Phenotype-genotype correla-
tion in patients with borderline D4Z4 repeat numbers. J Neurol 250: 932–937.
43. Celegato B, Capitanio D, Pescatori M, Romualdi C, Pacchioni B, et al. (2006)
Parallel protein and transcript profiles of FSHD patient muscles correlate to the
D4Z4 arrangement and reveal a common impairment of slow to fast fibre
differentiation and a general deregulation of MyoD-dependent genes. Proteo-
mics 6: 5303–5321.
44. Kitzmann M, Carnac G, Vandromme M, Primig M, Lamb NJ, et al. (1998) The
muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific
expression in muscle cells. J Cell Biol 142: 1447–1459.
45. Yamane A, Amano O, Urushiyama T, Nagata J, Akutsu S, et al. (2004)
Exogenous hepatocyte growth factor inhibits myoblast differentiation by
inducing myf5 expression and suppressing myoD expression in an organ culture
system of embryonic mouse tongue. Eur J Oral Sci 112: 177–181.
46. Doucet C, Gutierrez GJ, Lindon C, Lorca T, Lledo G, et al. (2005) Multiple
phosphorylation events control mitotic degradation of the muscle transcription
factor Myf5. BMC Biochem 6: 27.
47. Gayraud-Morel B, Chretien F, Flamant P, Gomes D, Zammit PS, et al. (2007) A
role for the myogenic determination gene Myf5 in adult regenerative
myogenesis. Dev Biol 312: 13–28.
48. Ustanina S, Carvajal J, Rigby P, Braun T (2007) The myogenic factor Myf5
supports efficient skeletal muscle regeneration by enabling transient myoblast
amplification. Stem Cells 25: 2006–2016.
49. Haslett JN, Sanoudou D, Kho AT, Bennett RR, Greenberg SA, et al. (2002)
Gene expression comparison of biopsies from Duchenne muscular dystrophy
(DMD) and normal skeletal muscle. Proc Natl Acad Sci U S A 99: 15000–15005.
50. Nicolas N, Mira JC, Gallien CL, Chanoine C (1998) Localization of Myf-5,
MRF4 and alpha cardiac actin mRNAs in regenerating Xenopus skeletal
muscle. C R Acad Sci III 321: 355–364.
51. Coppe ´e F, Matte ´otti C, Ansseau E, Sauvage S, Leclercq I, et al. (2004) The
DUX gene family and FSHD. In: Cooper D, Upadhyaya M, eds (2004)
Facioscapulohumeral Muscular Dystrophy (FSHD): Clinical Medicine and
Molecular Cell Biology. Oxford: Bios Scientific Publisher Ltd. pp 117–134.
52. Kawamura-Saito M, Yamazaki Y, Kaneko K, Kawaguchi N, Kanda H, et al.
(2006) Fusion between CIC and DUX4 up-regulates PEA3 family genes in
Ewing-like sarcomas with t(4;19)(q35;q13) translocation. Hum Mol Genet.
53. Lemmers RJ, Osborn M, Haaf T, Rogers M, Frants RR, et al. (2003) D4F104S1
deletion in facioscapulohumeral muscular dystrophy: phenotype, size, and
detection. Neurology 61: 178–183.
54. Schmidt J, Kirsch S, Rappold GA, Schempp W (2009) Complex evolution of a
Y-chromosomal double homeobox 4 (DUX4)-related gene family in hominoids.
PLoS ONE 4: e5288.
55. Castillo-Davis CI (2005) The evolution of noncoding DNA: how much junk,
how much func? Trends Genet 21: 533–536.
56. Dmitriev P, Lipinski M, Vassetzky YS (2009) Pearls in the junk: dissecting the
molecular pathogenesis of facioscapulohumeral muscular dystrophy. Neuro-
muscul Disord 19: 17–20.
57. Pandya S, King WM, Tawil R (2008) Facioscapulohumeral dystrophy. Phys
Ther 88: 105–113.
58. Wuebbles RD, Hanel ML, Jones PL (2009) FSHD region gene 1 (FRG1) is
crucial for angiogenesis linking FRG1 to facioscapulohumeral muscular
dystrophy-associated vasculopathy. Dis Model Mech 2: 267–274.
59. Chtarto A, Yang X, Bockstael O, Melas C, Blum D, et al. (2007) Controlled
delivery of glial cell line-derived neurotrophic factor by a single tetracycline-
inducible AAV vector. Experimental Neurology 204: 387–399.
60. Bouju S, Pietu G, Le CM, Cros N, Malzac P, et al. (1999) Exclusion of muscle
specific actinin-associated LIM protein (ALP) gene from 4q35 facioscapulohu-
meral muscular dystrophy (FSHD) candidate genes. Neuromuscul Disord 9:
DUX4c in FSHD
PLoS ONE | www.plosone.org11October 2009 | Volume 4 | Issue 10 | e7482