Functional analysis of the homeodomain protein SIX5

SIX5 (previously known as myotonic dystrophy associated homeodomain protein -DMAHP) is a member of the SIX [sine oculis homeobox (Drosophila) homologue ] gene family which encodes proteins containing a SIX domain adjacent to a homeodomain. To investigate the DNA binding specificities of these two doma ins in SIX5, they were expressed as GST fusion proteins, both separately an d together. Affinity purified recombinant proteins and cell lysates from ba cteria expressing the recombinant proteins were used in gel retardation ass ays with double stranded oligonucleotides representing putative DNA binding sites. The putative sites included two in the promoter region of DMPK (dys trophia myotonica protein kinase) and the previously characterised murine S ix4 DNA binding site in the Na+/K+ ATPase alpha 1 subunit gene (ATP1A1) reg ulatory element (ARE), None of the recombinant proteins showed any affinity for the two putative sites in DMPK, However, the two recombinant proteins containing the homeodomain both formed at least one specific complex with t he ARE. The recombinant protein containing both domains formed a second spe cific complex with the ARE, assumed to be a dimer complex. Finally, a whole genome PCR-based screen was used to identify genomic DNA sequences to whic h SIX5 binds, as an initial stage in the identification of genes regulated by SIX5.