c-ABL gene expression and spermatogenesis : investigations into the possible role of octamer transcription factors

The work described in this thesis aims at the elucidation of mechanisms that govern cellular differentiation events in male germ cell development (spermatogenesis), especially during the post-meiotic phase (spermiogenesis). and in embryonal carcinoma cells. Chapter II describes the molecular characterization of a variant c-abl mANA (TSabl) that is specifically expressed at high levels during spermiogenesis and was suggested to play an important role in this proces. The TSabl mANA is transcribed from the proximal of the two c-abl promoters and is alternatively processed resulting in a removal of most of the 3'UTA, without an effect on the coding capicity of the mANAs. The high levels of this shortened mANA in post-meiotic male germ cells could be due to two not mutually exclusive mechanisms, i.e. a higher mANA stability or/and continued transcription of the gene during the later phases of spermiogenesis. Chapter Ill describes experiments that tried to address the question whether the TSabl mANA has a longer half life as a consequence of the removal of most of the 3'UTA. In chapter IV a preliminary analysis of the c-abl promoter is presented, using DNAsel footprinting and gel retardation assays. The results of these experiments hinted at the possibility that there exists a testis specific octamer binding factor that could be involved in the haploid specific regulation of gene expression. This stimulated us to undertake the experiments described in chapter V that aimed at the cloning of testis specific cDNAs encoding octamer binding factors. We show that the POU domain gene Oct2 is highly expressed in spermatogenic cells, generating two transcripts through a mechanism of alternative processing and/or promoter usage. This chapter closes with a discussion of testis specific gene expression. The temporally regulated expression of a family of octamer binding factors during 'neuronal 'differentiation of P19 EC cells is described in chapter VI. One factor, Oct6, is expressed in a bi-phasic pattern, suggesting that it might play a role at different stages of development. This factor is further characterized by cloning of the cognate eDNA and was found to be the mouse homologue of the rat Tst-1 POU gene [29]. This gene is highly expressed in rat testis but not in mouse testis (this thesis). Chapter VII describes the functional mapping of the protein domains involved in transcriptional activation and DNA binding. In chapter VIII the genomic organization of the Oct6 gene is described. Furthermore, we present an initial characterization of the Oct6 promoter, to begin to address the important question of how this transcriptional regulator is regulated itself. In the last chapter some aspects of the Oct6 gene are discussed in relation to its possible function in differentiation, drawing on examples from other members of the POU domain gene family