Regulation of promoter occupancy during activation of cryptobiotic embryos from the crustacean Artemia franciscana.

Artemia franciscana embryos can suspend their development and metabolism at the gastrula stage to enter a state of cryptobiosis, forming cysts. Embryonic development and metabolism can be resumed under favorable environmental conditions to give rise to free-swimming larvae or nauplii. The mechanisms that mediate these processes are not completely known. Here, we report our studies of the mechanisms that regulate transcriptional activation upon exiting cryptobiosis. Regulatory regions of several A. franciscana gene promoters were identified. Functional analyses in mammalian cells allowed the identification of transcriptional activator regions in the Actin302 promoter and in promoter 2 of the sarco/endoplasmic reticulum Ca(2+)-ATPase-encoding gene. These regions were shown to specifically bind protein factors from nuclear extracts of A. franciscana nauplii by means of electrophoretic mobility shift assays. Several protein-binding regions were also detected by DNase I protection analysis in the promoters of the genes encoding the alpha1 subunit of Na(+)/K(+)-ATPase, actin 302 and sarco/endoplasmic reticulum Ca(2+)-ATPase. Specific DNA-binding proteins in nauplius nuclear extracts were detected for all the promoter regions analyzed. These proteins were either not present in cyst nuclear extracts or were present in much smaller concentrations. Three of the five regions analyzed also bound proteins present in cyst nuclear extracts. These data indicate that transcriptional activation upon exiting cryptobiosis in A. franciscana involves the expression/activation of DNA-binding transcription factors that are not present in cyst nuclei

Transactivation domains are not functionally conserved between vertebrate and invertebrate serum response factors.

The transcription factor serum response factor (SRF) regulates expression of growth factor-dependent genes and muscle-specific genes in vertebrates. Homologous factors regulate differentiation of some ectodermic tissues in invertebrates. To explore the molecular basis of these different physiological functions, the functionality of human, Drosophila melanogaster and Artemia franciscana SRFs in mammalian cells has been compared in this article. D. melanogaster and, to a lesser extend, A. franciscana SRF co-expression represses the activity of strong SRF-dependent promoters, such as those of the mouse c-fos and A. franciscana actin 403 genes. Domain-exchange experiments showed that these results can be explained by the absence of a transactivation domain, functional in mammalian cells, in D. melanogaster and A. franciscana SRFs. Both invertebrate SRFs can dimerize with endogenous mouse SRF through the conserved DNA-binding and dimerization domain. Co-expression of human and A. franciscana SRFs activate expression of weaker SRF-dependent promoters, such as those of the human cardiac alpha-actin gene or an A. franciscana actin 403 promoter where the SRF-binding site has been mutated. Mapping of A. franciscana SRF domains involved in transcriptional activation has shown that the conserved DNA-binding and dimerization domain is neccessary, but not sufficient, for promoter activation in mammalian cells.