H. Ding et al., CHARACTERIZATION OF A DOUBLE HOMEODOMAIN PROTEIN (DUX1) ENCODED BY A CDNA HOMOLOGOUS TO 3.3 KB DISPERSED REPEATED ELEMENTS, Human molecular genetics (Print), 7(11), 1998, pp. 1681-1694
Target genes for the helicase-like transcription factor (HLTF), a memb
er of the SNF/SWI family, were immunoprecipitated from HeLa chromatin
fragments with an anti-HLTF antibody. A 182 bp fragment (HEFT1) presen
ted 87% sequence identity with 3.3 kb dispersed repeats from the 4q35
D4Z4 locus linked to facioscapulohumeral muscular dystrophy (FSHD). Th
e HEFT1 loci were, however, not genetically linked to FSHD. Transfecti
on and in vitro binding studies identified within HEFT1 a promoter who
se basal activity required a GC box activated by Spl or Sp3. A 4.4 kb
homologous transcript was found mostly in human skeletal muscle and he
art. A 1.2 kb cDNA fragment was cloned that encoded a 170 amino acid p
rotein (DUX1) with two paired-type homeodomains. In vitro translated D
UX1 specifically interacted in electrophoretic mobility shift assay (E
MSA) with a P5 oligonucleotide (5'-GATCTGAGTCTAATTGAGAATTACTGTAC-3').
DUX1 co-expression activated up to 5-fold transient expression in inse
ct cells of a minimal promoter-luciferase construct fused to P5. The p
resence of 20 kDa DUX1 in vivo in rhabdomyosarcoma TE671 cell extracts
was shown by western blotting with a rabbit antiserum raised against
a DUX1 peptide, This antiserum suppressed a TE671 protein-P5 complex i
n EWSA with identical migration as the in vitro translated DUX1-P5 com
plex. Genomic PCR experiments could not identify a gene fragment linki
ng the HEFT1 and DUX1 sequences, which present one mismatch in their o
verlapping region. However, a similar gene was found in another 3.3 kb
element comprising the HEFT1 promoter and a DUX1-like open reading fr
ame. In addition, homologous gene sequences were identified in 3.3 kb
elements of the D4Z4/FSHD locus, considered until now 'junk' DNA.