Stage-Specific Regulation of Murine Hsp68 Gene Promoter in Preimplantation Mouse Embryos

Abstract

In early mouse embryos, the major inducible heat shock gene, hsp68, is spontaneously and transiently activated at the two-cell stage and becomes heat-inducible around blastocyst stage. We have probed mouse embryo's ability to activate the promoter of this gene during preimplantation development by expression analysis of DNA constructs containing a reporter lacZ gene driven by hsp68 (hsp70A1) 5'-regulatory sequences of various length: (i) a full-length promoter (construct phsplacZ); (ii) a heat shock element (HSE)-deleted promoter (p delta 1hsplacZ); and (iii) a minimal, proximal promoter (p delta 2hsplac Z). When analyzed in transfected L-cells, phsplacZ was heat-inducible, while neither p delta 1hsplacZ nor p delta 2hsplacZ was. Developmental activity of the full-length construct was first analyzed after genome integration in transgenic embryos and found to follow endogenous hsp68 expression in terms of spontaneous activation at the 2-cell stage, down-regulation at the 4-cell stage, and acquisition of heat inducibility at the 16/32-cell stage. In transient expression experiments, injected phsplacZ, p delta 1hsplacZ, and p delta 2hsplacZ were expressed at similar levels by 2-cell embryos, independently of construct topology and injection stage. At the 4-cell stage, however, phsplacZ and p delta 1hsplacZ were expressed at similar levels, while p delta 2hsplacZ was inactive. Only phsplacZ became heat-inducible in late morulas. We conclude that in early mouse embryos, developmental activity of episomic hsp68 promoter depends on proximal sequences at the 2-cell stage and on putative enhancer sequences at the 4-cell stage, while HSEs appear dispensable during early cleavage.

TCL1 participates in early embryonic development and is overexpressed in human seminomas

“…Normal ZGA in these embryos was evidenced by the expression of phspLacZ, a DNA construct directed by the hsp70.1 gene promoter (18), which is spontaneously activated during ZGA at the early 2-cell stage in the mouse (19,20) (Fig. 2D).…”

Evidence for the Involvement of Mouse Heat Shock Factor 1 in the Atypical Expression of the HSP70.1 Heat Shock Gene during Mouse Zygotic Genome Activation

“…Since the initial two-dimensional gel electrophoresis analyses from Bensaude et al (3), numerous studies using different approaches have focused on the latter question (4,10,17,25,29,33,34,48). Recent studies (4,5,8,46) have revealed spontaneous transient expression of HSP70 lacZ or Luc hybrid genes at the two-cell stage, demonstrating that the sequences driving the spontaneous expression of HSP70 at the two-cell stage are localized within the HSP70 regulatory region included in these constructs. Furthermore, using this approach, Bevilacqua et al (5) have suggested that HSE which are bound by HSF are not involved in this spontaneous expression, since they obtained the same results with a mutant form in which the two proximal HSE sequences of the HSP70A1 promoter were deleted.…”

Oogenetic and zygotic gene expression directing early bovine embryogenesis: A review

“…In embryos, enhancers are believed to relieve transcriptional repression which may result from particular components of chromatin structure. Reporter gene studies in both the mouse and the rabbit suggest the requirement for an enhancer begins at the 2-cell stage and continues with cleavage, although in the rabbit this requirement becomes even more restrictive for only particular enhancer sequences at the 8-to 16-cell stage (Wiekowski et al, 1991;Christians et al, 1994;Bevilacqua et al, 1995;Henery et al, 1995). Experiments to elucidate the mechanism underlying zygotic genome activation in bovine embryos have yet to be performed.…”