Developmental activation of an episomic hsp70 gene promoter in two-cell mouse embryos by transcription factor Sp1

To investigate the control of zygotic genome expression in two-cell mouse embryos, we studied transcription factors required for transient expression of microinjected DNA constructs driven by the promoter of one of the earliest genes activated after fertilization in this system, the heat shock gene hsp70. Cis-acting elements required for hsp70 activation were first investigated by mutational analysis. Mutation of the TATA box and a proximal GC box strongly inhibited construct expression, while that of a CCAAT box had no effect. Transcription factors binding the wild-type hsp70 promoter were then titrated in vivo by coinjecting the construct with double-stranded oligodeoxyribonucleotides containing definite consensus sequences. Wild-type GC box oligonucleotides strongly inhibited construct expression, while those containing mutated GC boxes, wild-type CCAAT boxes, and heat shock elements had no effects. Finally, construct expression was challenged by coinjecting antibodies to specific transcription factors. Antibodies to factor Sp1 depressed construct expression in a dose-dependent manner, while those to Sp2, HSF1 and HSF2 were ineffective. These results pinpoint the Sp1 transcription factor as an absolute requirement for activation of the hsp70 gene promoter in two-cell mouse embryos, and make this factor a candidate for a major regulator of the onset of murine zygotic genome expression.