Tassios, Panayotis; (1993) Control of transcription in embryonal carcinoma cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aim of this thesis was to examine the mechanisms that regulate transcription during early murine embryogenesis. F9 embryonal carcinoma (EC) cells were used as a model system, since their in vitro differentiated derivatives, the parietal endoderm-like (PE) cells, are similar, by a number of criteria, to cells of the early mouse embryo. Members of the ATF transcription factor family bind DNA through a domain rich in basic amino acid residues, adjacent to a leucine zip dimerization domain. They are required for unstimulated activity of ATF site-containing promoters but can also modulate transcription in response to diverse stimuli, such as viral /rm25-activators, intracellular cAMP, and Ca+2 ions. In this study, six ATF site-binding activities were defined in F9 EC and PE cells. They could be distinguished by their electrophoretic mobilities, their regulation during differentiation, and the effect that point mutations in or flanking the ATF site had on their ability to bind DNA. Two promoters containing ATF sites, that of the adenovirus E4 and that of the human vasoactive intestinal polypeptide (VIP) genes, bound different subsets of these activities. This correlated with these promoters' distinct transcriptional activities in vivo, since E4 was active in F9 EC cells and was further activated during differentiation, while VIP was inactive in both cell types. In addition, the regulation of transcription from the E4 promoter in vivo was reflected in a differentiation-specific change in the pattern of ATF site-binding proteins that recognized this promoter in vitro. Five human cDNAs derived from different members of the ATF family were used to measure ATF RNA levels in F9 EC and PE cells. ATF2, ATF4 and ATF6 RNA levels remained constant during F9 EC cell differentiation, whereas ATF3 RNA levels were higher in PE cells and ATF1 transcripts were undetectable in both cell types. This regulation of ATF genes contrasted with that of members of the related API transcription factor family: junD RNA levels were constant during differentiation while cjun and cfos were only expressed in PE cells. It is known that members of both the ATF and AP1 transcription factor families bind DNA as homodimers or heterodimers with members of their own or the related family. Here it was shown that heterodimerization can not only alter but also broaden DNA binding site specificity. Finally, after an ATF2-specific anti-peptide rabbit serum was raised, ATF2 protein levels were also shown to remain constant during F9 EC cell differentiation, and a similar group of nuclear polypeptides, with apparent molecular weights of 41-45 kD, was found to be present in a number of different cell types. This study has therefore shown that the regulation of ATF-dependent transcription during differentiation of EC cells can potentially occur at several levels, including ATF gene expression, as well as differential heterodimerization, binding site preference and promoter specificity of ATF proteins. The significance of these results with respect to transcriptional control during development is discussed.

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