Elsevier

Gene

Volume 236, Issue 1, 5 August 1999, Pages 25-32
Gene

Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD identifies a putative gene within each 3.3 kb element

https://doi.org/10.1016/S0378-1119(99)00267-XGet rights and content

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the polymorphic D4Z4 locus on chromosome 4q35. In non-affected individuals, this locus comprises 10–100 tandem copies of members of the 3.3 kb dispersed repeat family. Deletions leaving 1–8 such repeats have been associated with FSHD, for which no candidate gene has been identified.

We have determined the complete nucleotide sequence of a 13.5 kb EcoRI genomic fragment comprising the only two 3.3 kb elements left in the affected D4Z4 locus of a patient with FSHD. Sequence analyses demonstrated that the two 3.3 kb repeats were identical. They contain a putative promoter that was not previously detected, with a TACAA instead of a TATAA box, and a GC box. Transient expression of a luciferase reporter gene fused to 191 bp of this promoter, demonstrated strong activity in transfected human rhabdomyosarcoma TE671 cells that was affected by mutations in the TACAA or GC box. In addition, these 3.3 kb repeats include an open reading frame (ORF) starting 149 bp downstream from the TACAA box and encoding a 391 residue protein with two homeodomains (DUX4). In-vitro transcription/translation of the ORF in a rabbit reticulocyte lysate yielded two 35S Cys/ 35S Met labeled products with apparent molecular weights of 38 and 75 kDa on SDS–PAGE, corresponding to the DUX4 monomer and dimer, respectively.

In conclusion, we propose that each of the 3.3 kb elements in the partially deleted D4Z4 locus could include a DUX4 gene encoding a double homeodomain protein.

Introduction

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy primarily characterized by the progressive weakening of muscles of the face and shoulder girdle, further extending to the proximal arms and to the legs. FSHD has a variable severity and age of onset, but by the age of 20, the penetrance of the disease is almost complete [reviewed in Padberg (1998) and Tawil et al. (1998)]. Genetic analyses have linked this disease to a polymorphic region (D4Z4) close to the telomere on the long arm of chromosome 4 (Wijmenga et al., 1992). This region consists of a variable number of tandem repeats of the 3.3 kb family, members of which are found in several locations associated with heterochromatin (on the short arm of all the acrocentric chromosomes, close to the telomeres of chromosomes 4 and 10, and in the pericentromeric region of chromosome 1; Lyle et al., 1995). In unaffected individuals, 10–100 tandem 3.3 kb repeats are found in D4Z4, whereas deletions leaving only one to eight repeats have been associated with FSHD (van Deutekom et al., 1993). A few 3.3 kb elements from control D4Z4 loci have been cloned and sequenced, showing a complex pattern with several known repeated motifs. These are LSau, a middle repetitive element associated with heterochromatin, hhspm3, a low copy number GC-rich repeat, 68 bp satellite, and a large open reading frame (ORF) encompassing two homeoboxes (Hewitt et al., 1994, Lee et al., 1995).

Two mechanisms that might explain how the deletion is linked to FSHD have been put forward. The rearrangement of the locus could disrupt or completely remove the FSHD gene, or the loss of a great number of GC-rich repeats could result in the loosening of heterochromatin causing a position effect variegation that might affect the expression of a more centromeric gene yet to be identified (Winokur et al., 1994).

We have cloned, by serendipity, a genomic fragment (HEFT1) that presents 87% identity to 3.3 kb repeats from the D4Z4 locus and contains a functional promoter. We have also cloned a homologous cDNA that hybridizes mostly on Northern blots to mRNAs from skeletal muscle and heart, and encodes a new transcription factor with two homeodomains (DUX1) (Ding et al., 1998). Recently, we isolated two new putative 3.3 kb-like genes (DUX 2 and DUX 3) with similar promoters and ORFs. None of these DUX genes is linked to FSHD since they map to the acrocentric chromosomes (Beckers et al., 1999). Alignment of the DUX genes with the known sequences of 3.3 kb repeats derived from D4Z4 loci of non-affected individuals identified in some of these a homologous promoter within the previously described ORF encompassing the homeoboxes (Hewitt et al., 1994). This promoter was followed by a translation initiation codon in the same reading frame (Ding et al., 1998). In the present study, we investigated whether the 3.3 kb repeats actually present in the D4Z4 locus after the partial deletion linked to FSHD could still include such putative DUX genes.

Section snippets

DNA analysis

The λ42 phage was described earlier (van Deutekom et al., 1993) and contains a 13.5 kb EcoRI fragment encompassing the partially deleted D4Z4 locus of a patient with FSHD. Phage DNA was purified using the QIAGEN Lambda Midi kit from overnight phage cultures that had been grown according to Sambrook et al. (1989). The EcoRI insert was subcloned into the pGEM11-Z plasmid (Promega, Leiden, The Netherlands), yielding pGEM/42. Positive clones were identified by hybridisation with probe p13E-11 (

Nucleotide sequence of the D4Z4 locus

A 13.5 kb EcoRI genomic fragment covering the partially deleted D4Z4 locus of a patient with FSHD was previously cloned in phage λ42 (van Deutekom et al., 1993). This fragment, comprising only two 3.3 kb elements, was cloned in the pGEM11-Z plasmid yielding pGEM/42 and further subcloned as EagI fragments into the pCI-neo plasmid for sequence determination. The only region that was not subcloned was a 243 bp EagI fragment of the 3.3 kb elements (starting at bp 7273 in the first element, and bp 10 570

Discussion

We have identified a putative gene comprising an ORF for a double homeodomain protein, DUX4, within each of the 3.3 kb elements that remain in the D4Z4 locus after the partial deletion associated with FSHD. These features are located within a previously identified larger ORF also encompassing the two homeoboxes but for which no promoter had been identified (Hewitt et al., 1994, Lee et al., 1995). Interestingly, the promoter that we identify in the DUX4 gene (TACAA box at bp 7385) is located

Acknowledgements

We thank Drs J.C. Kaplan and M. Jeanpierre (Groupe Hospitalier Cochin, Paris, France) for helpful discussions. This study was funded by grants from the Belgian Government (Geconcerteerde Onderzoeksacties 1996–2000), the ‘Association Française contre les Myopathies’, and ‘The Muscular Dystrophy Group’ of Great Britain. J.G. was supported by a pre-doctoral fellowship from the ‘Vlaams Instituut voor de bevordering van het wetenschappelijk-technologisch onderzoek in de industrie’ (IWT; Belgium),

References (26)

  • J.E. Hewitt et al.

    Analysis of the tandem repeat locus D4Z4 associated with facioscapulohumeral muscular dystrophy

    Hum. Mol. Genet.

    (1994)
  • J. Lee et al.

    Characterization of a tandemly repeated 3.3-kb KpnI unit in the facioscapulohumeral muscular distrophy (FSHD) gene region on chromosome 4q35

    Muscle & Nerve

    (1995)
  • R.J.L.F. Lemmers et al.

    Inter- and intrachromosomal sub-telomeric rearrangements on 4q35: implications for facioscapulohumeral muscular dystrophy (FSHD) aetiology and diagnosis

    Hum. Mol. Genet.

    (1998)
  • Cited by (281)

    View all citing articles on Scopus
    1

    Present address: Mount Sinai Hospital, Lunenfeld Institute, 600 University Avenue, Toronto, Ontario, M5G1X5, Canada.

    View full text