טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMahroum Mohammed
SubjectTranscriptional Regulation and Structure-Function Analysis
of Homothorax
DepartmentDepartment of Medicine
Supervisor Professor Adi Salzberg
Full Thesis textFull thesis text - English Version


Abstract

The homothorax (hth) gene is a pleiotropic gene which has wide effects on many aspects of fly development. One major role of Hth is the nuclear translocation of Extradenticle (Exd), a known cofactor of homeotic proteins. The hth gene is expressed throughout embryonic development in a spatially restricted pattern, which is modulated by the activity of homeotic genes. To gain a better insight into the regulation of hth expression we have isolated genomic fragments containing enhancer elements from the hth locus that drives b-galactosidase expression in chordotonal organs, peripheral glia cells, midgut, wing disc, hemocytes, salivary gland and Malpighian tubules, where the hth gene itself is expressed. Within the nucleus Hth participates in transcription regulating protein complexes with Exd, and possibly other partners, and activates the expression of downstream target genes.  It is not known whether Hth can bind DNA independently of Exd.  To address this question we generated mutant forms of Hth (deficiencies and point mutations) and tested their ability to cause phenotypes in transgenic flies. Our results suggest that in the specific contexs of distal leg, eye and antenna Hth can bind DNA and affect transcription independently of Exd. In addition Hth can affect transcription without its homeodomain by binding the DNA via a DNA binding complex. In contrast, Hth must bind DNA through its own homeodomain to affect transcription in the eye. Hth was shown to function as a transcriptional activator; however, its activation domain has not been identified. Therefore, we generated a series of deletions in Hth. The ability of these defective proteins to activate transcription of endogenous genes in vivo was tested in Xenopus laevis. Injection of RNA transcribed from native hth or XMeis3 (XMeis3 is an hth homologue from the frog Xenopus laevis) was shown to result in anterior truncations of embryos and expansion of the expression domain of krox20. We looked for a deletion construct which lost the ability to cause these phenotypes. The expression of Krox-20 was expanded in all embryos injected with deleted forms of hth RNA except for the embryos injected with hth D[HD] RNA (hth lacking the homeodomain) in which The expression was eliminated partially. This observation suggests that XMeis3, or Hth, needs its homeodomain to induce the expression of Krox-20. In addition a clear result, both in flies and in frogs, showed that the deletion of 57-residues in the C terminus did not remove a transcription activation domain, despite what had been seen in Meis 1 homolog.