The tree squirrel HP-25 gene is a pseudogene

The gene for the hibernation-specific protein HP-25 is expressed in the liv er in hibernating species of the squirrel family (chipmunk and ground squir rel), but not in a nonhibernating species (tree squirrel). To investigate w hy the HP-25 gene is not expressed in the tree squirrel, we isolated the tr ee squirrel HP-25 gene and compared its gene structure and promoter activit y with that of the chipmunk. The tree squirrel HP-25 gene is composed of th ree exons, and the gene structures are conserved between the tree squirrel and chipmunk. However, the tree squirrel HP-25 gene has an insertional muta tion of 13 nucleotides in exon 2 that disrupts the ORE In the chipmunk HP-2 5 gene, the 80-bp 5' flanking sequence is sufficient for the liver-specific promoter activity, and HNF-4, which binds to the sequence from nucleotides -67 to -51, is involved in its transcriptional regulation. In contrast, th e corresponding tree squirrel 5' flanking sequence had almost no promoter a ctivity in HepG2 cells, and HNF-4 did not bind to the corresponding region of the tree squirrel HP-25 gene. Furthermore, a tree squirrel-type G to A m utation at -57 in the chipmunk HP-25 gene promoter context abolished its bi nding to and transactivation by HNF-4. Thus, the point mutation in the HNF- 4-binding site is likely to be involved in the lack of HP-25 gene expressio n in the tree squirrel.