Facioscapulohumeral dystrophy is considered an epigenetic disorder [24
]. Abnormalities in the expression of candidate genes such as ANT1
, and in the transcription of the D4Z4 repeat, have been reported in FSHD patients [24
], but the chromatin features of the FSHD locus have not been studied in detail.
We found that the upregulation of FRG1
in FSHD patients is a gain of function mechanism that could explain the autosomal dominant inheritance of the disease, and that it is revealed only when myogenic differentiation triggers the remodeling of the locus. FRG1P is a nuclear protein that is thought to be involved in RNA processing [12
]. Slight differences in the cell levels of regulatory proteins such as FRG1P may affect a number of factors and have multiple effects on cell physiology. For instance, the alternative splicing of muscle-specific genes is abnormally regulated in mice overexpressing FRG1
and showing an FSHD phenotype [21
], and so inappropriate regulation of FRG1
during the early phases of muscle differentiation may have serious effects on the formation of muscle fiber. We suggest that FRG1
misregulation in a specific window of muscle differentiation may contribute to FSHD, although it cannot be considered the only molecular defect causing the FSHD phenotype: for example, the transcription of DUX4 recently observed in FSHD myoblasts [14
] may contribute to the manifestation of FSHD.
In order to investigate the molecular basis of the FRG1 transcriptional alteration, we made a detailed analysis of the chromatin structure of two DNA regions residing in the FSHD locus in a human model of myogenic differentiation: the candidate gene FRG1 and the D4Z4 array, to which the genetic mutation underlying the disease has been mapped. These two DNA regions were studied at different levels of the epigenome, from DNA methylation and histone code modifications to higher order structures. In this regard, it is important to point out the intrinsic limitation of molecularly analyzing repetitive DNA elements, and so we used the complementary approaches of ChIP and 3D-FISH analysis to gain insights into the chromatin structure of D4Z4.
The analyses showed that the FSHD locus undergoes chromatin remodeling during myogenic differentiation. In normal myoblasts, the FRG1 gene is repressed and its promoter physically interacts with the D4Z4 array; upon differentiation, the Polycomb complex dissociates from the FRG1 promoter and the FRG1 gene is expressed. Like the FRG1 promoter, D4Z4 chromatin also shows the presence of the Polycomb complex and H3K27me3 in myoblasts, and their loss in myotubes; moreover, D4Z4 and the FRG1 promoter physically interact in myoblasts, whereas this chromatin loop is relaxed upon myogenic differentiation.
These data support the hypothesis that the 4q D4Z4 array may have a regulatory effect on FRG1
expression, which we suggest is due to their physical association in the nucleus. It has recently been demonstrated that Polycomb occupancy can repress transcription by maintaining a series of long-range chromatin interactions that are lost when mammalian cells differentiate [39
], and so it would be interesting to investigate directly the involvement of the Polycomb repressor complex as a mediator of the FRG1
-D4Z4 chromatin loop in myoblasts.
Chromatin characterization of FRG1 and the D4Z4 array in FSHD myoblasts revealed a reduction in H3K27me3 on the contracted D4Z4 allele, and a kinetic analysis of Polycomb dissociation during differentiation that was very similar to that observed in the control cells. The reduction in H3K27me3 may be due to the decrease in the number of D4Z4 units or to hypomethylation of the residual repeats. Furthermore, the early expression of FRG1 in differentiating FSHD myoblasts may indicate that muscle cells, like their non-muscle counterparts, require the recruitment of additional factors in order to activate FRG1 expression.
In our cell system, the regulation of FRG1 expression therefore seems to be preferentially conditioned by the chromatin structure of the region (that is to say, the strength of the loop between the FRG1 promoter and the D4Z4 array related to its chromatin structure). We found a slight reduction in the frequency of loop formation between the D4Z4 array and the FRG1 promoter in FSHD myoblasts in comparison with control cells. D4Z4 contraction in FSHD cells may qualitatively alter the repressive effect of this chromatin loop affecting the correct timing of FRG1 expression. It is possible that relaxed looping in the presence of protein factors that may induce further changes in chromatin conformation and/or more efficient transcription allows the expression of the FRG1 gene. Nonetheless, the observed reduction in the frequency of loop formation between the D4Z4 array and the FRG1 promoter in FSHD myoblasts is too small to certainly infer its involvement in the misregulation of FRG1 gene expression, and thus further experiments are required to link macrosatellite contraction and gene expression.
Pirozhokova et al
] published a 3C analysis of the FSHD locus and described the formation of loops between DUX4c and the FRG1
promoter. We detected the same loop in myoblasts, although the frequency of the interaction was one order of magnitude lower than that of the loop between FRG1
and D4Z4 sequences. The same authors also found a second loop between a telomeric element downstream of the D4Z4 array (the 4qA/B marker) and the FRG1
promoter only in FSHD myoblasts, and suggested that this element may enhance the transcription of the gene [40
]. As we did not detect FRG1
up regulation in FSHD myoblasts, we suggest that the interaction with the 4qA/B marker have the proposed effect of transcription enhancement on FRG1
expression only when myogenic differentiation is triggered. Our data, together with data of Petrov et al
. and Pirozhokova et al
. indicate that the tridimensional structure of the FSHD region is functional for the expression of the FRG
1 gene, and probably more than one sequence elements (for example, D4Z4, DUX4c, MAR region) could contribute to the fine regulation of gene expression [40
Finally, our model may also explain the manifestation of FSHD in the absence of D4Z4 contraction, as in the case of phenotypic FSHD in which a wild-type 4q D4Z4 array is strongly hypomethylated [9
]. In this case, D4Z4 hypomethylation may impair the Polycomb recruitment that leads to a reduction in H3K27 trimethylation, the same molecular defect that we observed in contracted 4q alleles.