Early Transcriptional Activation of the Hsp70.1 Gene: INTRODUCTION
Fertilization in mammals activates a developmental clock that leads the newly formed embryo to progressively shift from the maternal to the zygotic genome expression (zygotic genome activation, ZGA). In the mouse, this process is initiated very soon after fertilization and de novo synthesis of RNA is consistently observed for the first time during the S phase of the one-cell stage. Mouse zygotes, in fact, are transcriptionally competent and their transcriptional machinery, including RNA polymerases I, II, and III, is functional. Transcription at this stage, however, has several peculiar features that make it very different from that which will occur after the first embryo cleavage. In fact, it takes place while the zygotic genome has not yet been assembled into chromatin, as originally indicated by the finding that plasmids injected in the male pronucleus of one-cell embryos are transcribed irrespective of the presence of an enhancer, unless the DNA is assembled into chromatin by injection of purified H1 histones. In addition, transcription is more prominent in the male pronucleus than in the female one, in agreement with the higher levels of hyperacetylated H4 histones and DNA demethylase activity present in this pronucleus. It is also temporally and causally linked to DNA replication, appearing at the onset of the first embryonic S phase and being strongly decreased by inhibition of DNA synthesis with aphidicolin. An intriguing feature of onecell embryo transcription is that it is possibly uncoupled from translation until the first embryonic G2 phase, while it does not appear that nascent RNA chains are rapidly degraded and/or display splicing deficiencies. Even though such transcription-translation uncoupling does not have a known molecular basis, it raises the question of the functional significance of the one-cell embryo’s transcriptional activity. In this regard, it has been proposed that the expression of one-cell embryo genes is opportunistic and promiscuous and, thus, that it is merely dependent on unregulated transcription factor accessibility to open male gene promoters during protamine-histone exchange.
During the G2 phase of the one-cell stage and/or the transition to the two-cell stage, the male and female genomes equally undergo a process of generalized assembly into chromatin, concomitant with the de novo synthesis of histones H2A, H2B, and H1 and extensive histone H4 deacetylation, which eventually results in the appearance of novel locally acting mechanism(s) of chromatin remodeling at the early two-cell stage. At this stage, in fact, expression of plasmids driven by a weak promoter requires the presence of an enhancer, unless the embryo chromatin is opened by histone deacetylase inhibitors. The process of zygotic genome packaging into chromatin then continues through the two-cell stage and is accompanied by the progressive appearance of more and more stringent transcriptional regulation mechanism(s), that is virtually completed by the four-cell stage. The early two-cell stage, however, marks the onset of diploid embryo genome activation with the transcription of a limited set of zygotic genes. Some of these genes, including the transcription-requiring complex, the heat shock genes hsp70.1 and hsp25, the translation initiation factor elF-2A, and a few others, are expressed transiently, whereas other genes, including the RING finger protein genes Rnf33 and Rnf35 and the Mhc gene, continue to be expressed during subsequent early embryo development.
Among the early and transiently activated genes, previous work performed in our laboratory was focused on the transcriptional regulation of hsp70.1 in the early two-cell embryos. We demonstrated that the spontaneous expression of this gene at this developmental stage is driven by transcription factor Sp1 and a murine GAGA box-binding factor (mGAF), whereas heat shock factor 1 (HSF1) is likely to be responsible for a stress-elicited hyperactivation of the endogenous hsp70.1 genes in the in vitro-cultured embryos. Heat shock-dependent activation of hsp70 was also reported in two-cell bovine embryos.
In contrast with the information presently available on embryo transcriptional regulation taking place after the zygotic genome has been assembled into chromatin, molecular mechanisms that regulate the transient transcriptional wave occurring in the one-cell embryos are still unknown. In this article, we have addressed this issue by taking advantage of the unexpected finding that, in addition to the spontaneous expression at the early two-cell stage, the hsp70.1 gene is also transiently transcribed in the in vitro-cultured, but not the in vivo-maintained, one-cell mouse embryo. We have compared the molecular regulation of hsp70.1 in the one-cell embryos with that occurring in heat-shocked dictyate oocytes derived from preantral ovarian follicles and report here that the hsp70.1 activation in one-cell embryos is mediated by HSF1 and is triggered by the osmotic stress to which embryos are subject in consequence of isolation from the tubal fluid environment. As far as we know, hsp70.1 thus represents the earliest endogenous gene physiologically expressed after fertilization that has been identified in mammals. The tight regulation of hsp70.1 at the one-cell stage indicates that stringent transcriptional regulation mechanisms are already present very soon after fertilization.