The tree squirrel HP‐25 gene is a pseudogene

Abstract

The gene for the hibernation-specific protein HP-25 is expressed in the liver in hibernating species of the squirrel family (chipmunk and ground squirrel), but not in a nonhibernating species (tree squirrel). To investigate why the HP-25 gene is not expressed in the tree squirrel, we isolated the tree squirrel HP-25 gene and compared its gene structure and promoter activity with that of the chipmunk. The tree squirrel HP-25 gene is composed of three exons, and the gene structures are conserved between the tree squirrel and chipmunk. However, the tree squirrel HP-25 gene has an insertional mutation of 13 nucleotides in exon 2 that disrupts the ORF. In the chipmunk HP-25 gene, the 80-bp 5 0 flanking sequence is sufficient for the liver-specific promoter activity, and HNF-4, which binds to the sequence from nucleotides 267 to 251, is involved in its transcriptional regulation. In contrast, the corresponding tree squirrel 5 0 flanking sequence had almost no promoter activity 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 mutation at 257 in the chipmunk HP-25 gene promoter context abolished its binding 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 expression in the tree squirrel.Keywords: gene organization; hibernation; promoter; regulatory element; transient transfection.For most mammals that maintain a high steady body temperature throughout their adult lives, lowering the body temperature brings about dysfunction of physiological systems, resulting in death. However, certain small mammals can undergo hibernation, during which the body temperature drops to below 10 or even 5 8C [1]. The heart and breathing rates also fall [2], and the metabolic rate is reduced to only a few percent of the euthermic level [3]. Thus, mammalian hibernation is considered to be a unique physiological adaptation that allows life to be sustained under extremely low body temperatures.In the squirrel family, some species, such as the chipmunk (Tamias asiaticus ) and 13-lined ground squirrel (Citellus tridecemlineatus ), hibernate; other species, such as the tree squirrel (Callosciurus caniceps ), do not. The chipmunk hibernation-specific proteins have been identified as components of a 140-kDa protein complex in the blood that decreases drastically in hibernating chipmunks [4]. The level of this 140-kDa complex varies during the hibernation process: it starts to decrease before the onset of hibernation, stays at the reduced level during hibernation, and starts to increase just before the cessation of hibernation [4]. This complex contains four proteins, HP-20, HP-25, HP-27, and HP-55. The first three are homologous proteins (with < 45% amino-acid sequence identity with each other) with a collagen-like domain near the N-terminus [4,5]. In the chipmunk and 13-lined ground squirrel, the HP-20, HP-25, and HP-27 genes are expressed specifically in the liver and are ...

Mammalian Hibernation: Cellular and Molecular Responses to Depressed Metabolism and Low Temperature

“…Recent work on the plasma proteins HP-25 (150,151), HP-20 (195), and HP-27 (196) has focused on the liver-specific regulation of their expression at the transcriptional level.…”

Adaptations to environmental extremes by multicellular organisms

“…Recent work on the plasma proteins HP-25 (Kojima et al 2000(Kojima et al , 2001, HP-20 (Ono et al 2001) and HP-27 (Ono et al 2003) has focused on the liver-specific regulation of their expression at the transcriptional level. At the protein level, myoglobin appears to increase in skeletal muscle as animals prepare for and undergo hibernation.…”

USF is involved in the transcriptional regulation of the chipmunk HP-25 gene

“…Since the tree squirrel HP-25 gene has two base substitutions in the corresponding sequence (CAgcTG, in which the base substitutions are shown by lower-case letters) (see Fig. 1B) (Kojima et al, 2001), we examined whether these base substitutions would affect the binding of USF. The chipmunk sequence with the tree squirreltype base substitutions (CM25G-159/-129TSmut) did not form a complex with USF (Fig.…”