Characterization and functional analysis of the bidirectional promoter region of the zebrafish hsp10/60 genes

Protein folding in vivo, often requires a set of proteins called chaperones. Chaperones assist the folding of newly synthesized proteins, to obtain their active tri-dimensional structures. They may also provide adaptive response against adverse conditions such as heat shock and stress. Chaperonins 10 and 60 (Hsp10 and Hsp60) are present in the mitochondria of all cells. The Hsp10/60 machinery occurs as a complex with a 2:1 stoichiometry of Hsp10 to Hsp60. hsp10/60 genes are linked in a head-to-head manner with a short intergenic region. To investigate the bidirectionality of the hsp10/60 promoter region, we made a number of reporter constructs and tested them by microinjection into fertilized zebrafish eggs and by transfection of mammalian 293 and zebrafish ZF-4 cells. Our results showed that the promoter region is comprised of a ∼1-kb fragment that encompasses the hsp10 transcription start site, the intergenic region, and the transcription start site of hsp60. A larger fragment of about 4 kb, covering the start codon of both genes, which includes the first exon of hsp10, the first intron of hsp60, and the first two exons of hsp60, directs twice as much expression of reporter genes in the hsp60 direction as compared to the hsp10 direction. This fragment also causes strong expression in the yolk syncytial layer, particularly during the gastrula stage. Deletion of the first intron of hsp60 results in the loss of the 2:1 ratio in expression and the loss of expression in the yolk, suggesting the existence of a cis-acting element within the intronic region. This element may be responsible for maintaining protein concentrations suitable for maintaining normal stoichiometry. A 86-bp region within the first intron of hsp60 seems to include a site that is linked to the expression in the yolk. However, the region responsible for the stronger expression in the hsp60 direction could not be more precisely localized. The hsp10/60 genes show a detectable expression under normal conditions and are inducible by at least two-fold under heat shock conditions. The hsp10/60 promoter could be used as an inducible promoter in transgenic studies especially for simultaneous co-expressions.