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 homeo-domain. To investigate the DNA binding specificities of these two domains in SIX5, they were expressed as GST fusion proteins, both separately and together. Affinity purified recombinant proteins and cell lysates from bacteria expressing the recombinant proteins were used in gel retardation assays with double stranded oligonucleotides representing putative DNA binding sites. The putative sites included two in the promoter region of DMPK (dystrophia myotonica protein kinase ) and the previously characterised murine Six4 DNA binding site in the Na(+)/K(+) ATPase alpha 1 subunit gene ( ATP1A1 ) regulatory 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 the ARE. The recombinant protein containing both domains formed a second specific 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 which SIX5 binds, as an initial stage in the identification of genes regulated by SIX5.
Harris
S E
SE
Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Anderson College, 56 Dumbarton Road, Glasgow G11 6NU, UK.
Winchester
C L
CL
Johnson
K J
KJ
D001419:Bacteria / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D002474:Cell-Free System / Q000737:chemistry; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015894:Genome, Human; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D011993:Recombinant Fusion Proteins; C106687:SIX5 protein, human; D004247:DNA; D005982:Glutathione Transferase