Further studies on the DUX4, DUX4c and PITX1 genes in FSHD

Several years ago our group identified the DUX4 gene within each unit of the D4Z4
repeat array. We have demonstrated its expression at the mRNA and protein levels in
FSHD myoblasts but not in controls. We have also shown that the homologous DUX4c
gene mapped 42 kb of the D4Z4 array was expressed in control and FSHD myoblasts.
The DUX4c protein was found at higher levels in biopsies of patients with FSHD and
low D4Z4 copy number.

We performed additional studies on the DUX4 mRNA 3' ends in control and FSHD
myoblasts. The products of 3'RACE experiments were cloned and sequenced, and we
could only find DUX4 mRNAs in FSHD myoblasts. We found two introns 3' from the
DUX4 stop codon: the first one was alternatively spliced and mapped in the D4Z4 unit,
and the second one was always spliced and mapped in the pLAM region where a poly-A
addition signal was used. We could amplify by RT-PCR a 1.7-kb product covering the
full DUX4 ORF with a forward primer in the start codon region and a reverse primer 3'
of the pLAM intron in 4 FSHD myoblast lines, with increased signal upon differentiation.
Sequence analyses confirmed the DUX4 identity. These results suggested that only the
DUX4 gene from the last D4Z4 unit can express a stable mRNA, by use of pLAM
sequences.

We previously identified Pitx1 as a direct transcription target of the DUX4 protein in
vitro. The PITX1 gene (on 5q31) is specifically up-regulated in FSHD and associated to
muscle atrophy and left/right asymmetry, providing a direct link between the genetic
defect in 4q35 and the pathophysiology of the disease. In order to evaluate whether
DUX4 could also activate the endogenous Pitx1 gene we transfected C2C12 cells with
pClneo-DUX4 and performed a co-immunofluorescence staining of DUX4 and Pitx1.
Both proteins were detected in the same nuclei 24 h post-transfection, while only
background signals were observed in cells transfected with the insertless pClneo. We
similarly evaluated DUX4/PITX1 expression in FSHD primary myotubes and could
detect DUX4 positive nuclei, some of which were also stained for PITX1. By Western
blot, we detected high levels of 52-kDa DUX4 in muscle biopsies from 3 patients with
FSHD but not 2 controls. We could only detect the PITX1 protein in a severely affected
FSHD muscle biopsy.

In a functional study we transfected TE671 rhabdomyosarcoma cells with pCINeo
expression vectors for either DUX4, DUX4c, DUX1 or without insert. By
FSHD International Research Consortium Meeting. October 23, 2007, San Diego, California. ©FSH Society 2007.
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immunofluorescence we observed a strong up-regulation of PCNA (proliferating cell
nuclear antigen) 24h post-transfection in DUX4c expressing cells only. When we added a
differentiation medium (2% horse serum), DUX4c expressing cells continued to
proliferate instead of aligning to fuse into myotubes like the other transfected cells.
Together with previous data showing that DUX4c specifically induced the Myf5
transcription factor, these experiments suggest a role in the maintenance of the satellite
cell pool. We propose that as well an excess (in patients with low D4Z4 copy number) as
a reduced amount (in families where the D4Z4 deletion removes the DUX4c gene) of
DUX4c expression could affect muscle regeneration and contribute to the FSHD
pathology.