|M.Sc Student||Savariego Dana|
|Subject||Identification of cg5804 as a Target Gene of Homothorax in|
|Department||Department of Medicine||Supervisor||Professor Adi Salzberg|
|Full Thesis text|
Homothorax (hth) encodes for a homeobox-containing transcription factor which plays multiple roles in the development of the embryo. To identify target genes of Hth a microarray experiment was conducted using two recombinant forms of Hth: vp16-hth and en-hth. Vp16-hth and en-hth encode for chimeric proteins, in which hth is fused either to a viral activation domain (Vp16) or to a repression domain (En). RNA samples prepared from embryos that ectopically expressed these chimeric proteins under the regulation of da-GAL4 were hybridized on Affymetrix fly genome chips. We expected that direct target genes of Hth should display a reduction in transcript level in RNA samples prepared from embryos that express En-Hth and an increase in transcript signal in RNA samples prepared from embryos that express Vp16-Hth as compared to wild-type embryos. The microarray results of ten genes were validated by real time PCR. Analysis of the microarray results identified 288 genes that displayed a significant fold change (≥2.5). However, only ten genes were identified that exhibited the behavior we had expected for direct target genes of Hth. Out of these ten genes the cg5804 gene carries a unique biological activity and we therefore decided to examine it as a candidate target of Hth.
I show that Hth is required to activate the expression of cg5804 in the midgut endoderm. Additionally, I provide evidence for co-localization of cg5804 and hth expression and demonstrate that the endodermal expression of Hth is required for regulating the transcription of cg5804.
Identifying the regulatory region of cg5804 will allow searching for potential binding sites of Hth in this region and test for direct binding of Hth to such sites. To this end I generated transgenic flies carrying β-gal reporter constructs fused to genomic sequences flanking the cg5804 gene. Examination of the expression pattern of these sequences identified an upstream region that responds to changes in Hth activity similarly to the endogenous cg5804 gene. Further deletion analysis of this sequence identified a 450bp fragment which contains the regulatory region of cg5804.
Hth contains an MH domain necessary for binding to Exd and a homeodomain (HD) that is required for binding to the DNA. Using truncated forms of Hth which lack either the MH or the HD domains we show that the MH domain, but not the HD, of Hth is necessary for regulating the transcription of cg5804.