FRG1P is localised in the nucleolus, Cajal bodies, and speckles.

T he highly conserved facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) was initially cloned as candidate gene of unknown function for FSHD. To explore the biological function of the FRG1 protein (FRG1P), we studied its cellular localisation in untransfected and FRG1 transfected cell lines. In interphase cells, FRG1P is localised in the dense structures of the nucleolus, in Cajal bodies, and in 60–80% of cells in nuclear speckles. A time course study revealed that FRG1P accumulates in nuclear speckles before its appearance in nucleoli, whereas the localisation in Cajal bodies remains unchanged, as does the localisation of NHPX protein. In accordance with the presence of FRG1P in these nuclear structures, transcription inhibition experiments showed an effect of RNA polymerases I and II on the localisation of FRG1P. Finally, by deletion of the predicted nuclear localisation signals of FRG1P, we demonstrated that both signals are necessary for this subnuclear localisation. FRG1P is an attractive candidate for the pathogenesis of FSHD because of its high evolutionary conservation; its transcriptional deregulation in FSHD; and its colocalisation with proteins that are defective in the neuromuscular disorders oculopharyngeal muscular dystrophy (OPMD) and spinal muscular atrophy (SMA). FRG1 (FSHD Region Gene 1, accession number L76159) was isolated as the first candidate gene for the autosomal dominant myopathy, facioscapulohumeral muscular dystrophy, in 1996. FSHD is primarily characterised by progressive weakness and atrophy of the facial, upper arm, and shoulder muscles. The FSHD locus on 4q35 contains a polymorphic repeat array consisting of 3.3 kb repeated elements (D4Z4). In control individuals, this array may vary between 11–100 units, whereas FSHD patients carry 1–10 units on one of their chromosomes 4, owing to the deletion of an integral number of D4Z4 units. It is becoming increasingly evident that FSHD is caused by a local chromatin alteration, in which contraction of the D4Z4 repeat array results in the transcriptional deregulation of genes located on 4q35 (position effect). Indeed, Gabellini et al recently claimed evidence for transcriptional upregulation of genes close to the repeat in FSHD muscle, including FRG1. In cultured cells, a transcriptional repressor complex binds to D4Z4, and partial loss of this complex leads to transcriptional upregulation of at least one 4qter gene, as observed in FSHD muscle. New expression data based on quantitative RT-PCR suggest that the expression of the 4q35 copy of FRG1 is in fact changed, although the nature of the deregulation is still controversial. FRG1P is highly conserved in vertebrates and nonvertebrates; the human protein is 97% identical to the murine protein and 46% identical to its orthologue in C elegans. Database searches have identified potential orthologues in—for example—Drosophila, tomato, Xenopus and Schizosaccharomyces pombe. As a result of multiple ectopic duplications, in humans FRG1 belongs to a multigene family, with copies on many different chromosomes. The ancestral copy is located on chromosome 4q35, and is demonstrably transcribed in all tissues tested. Additionally, this chromosome 4 copy of FRG1 is deregulated in FSHD muscle. 5 The FRG1 transcript is 1042 bp in length, and is distributed over nine exons encoding an open reading frame (ORF) of 258 amino acid residues. This ORF is coding for a 30 kDa protein, which does not show homology to known proteins. Computer algorithms predict an amino terminal nuclear localisation signal (NLS) and a carboxy terminal bipartite (BP) nuclear localisation signal in FRG1P. In fact, the amino terminal signal consists of two NLS motifs, each four residues in size. Moreover, an imperfect lipocalin motif is predicted, which is involved in transport of hydrophobic particles. Despite its impressive conservation during evolution, indicating a fundamental function, little is known about the role of FRG1P. To obtain more insight into the biological function of FRG1P and its putative involvement in FSHD pathogenesis, we used immunological and GFP based techniques to study its subcellular localisation during different cell stages in untransfected and transfected cell lines. These studies demonstrate that FRG1P colocalises with PABPN1 and SMN1, both involved in related neuromuscular disorders.