The gene for chipmunk hibernation-specific protein HP-25 is expressed specifically in the liver. To understand the transcriptional regulation of HP-25 gene expression, we isolated its genomic clones, and characterized its structural organization and 5H flanking region. The gene spans approximately 7 kb and consists of three exons. The transcription start site, as determined by primer extension analysis, is located at 113 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 80 bp 5 H flanking sequence was sufficient for the liver-specific promoter activity. In a gel retardation assay using HepG2 nuclear extracts, the 5 H flanking sequence from 274 to 246 showed a shifted band. All cDNA clones isolated by a yeast one-hybrid system for a protein capable of binding to this 5H flanking sequence encoded HNF-4. HNF-4 synthesized in vitro bound to this sequence in a gel retardation assay. Furthermore, supershift assays with anti-(HNF-4) Ig confirmed that the protein in HepG2 or chipmunk liver nuclear extracts that bound to this sequence was HNF-4. When transfected into HeLa cells, HNF-4 transactivated transcription from the HP-25 gene promoter, and mutation of the HNF-4 binding site abolished transactivation by HNF-4, indicating that HNF-4 plays an important role in HP-25 gene expression.Keywords: gene organization; hibernation; promoter; regulatory element; transient transfection.Mammalian hibernation is a unique physiological adaptation that allows the sustainment of life under extremely low body temperatures. 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. Only certain small mammals, primarily in the orders Rodentia, Insectivora and Chiroptera, can undergo hibernation. During hibernation, 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]. These changes are precisely controlled and can be reverted only by internally driven mechanisms, suggesting a specific biochemical regulation [4]. There is accumulating evidence for differential gene expression during hibernation [4±9], and as most of these genes are common to all mammals, the physiological characteristics of hibernation are postulated to be controlled, in part, by the differential expression of existing genes [4]. Thus, to understand mammalian hibernation, it is of great importance to reveal how these genes are regulated during hibernation.Through comparison of plasma proteins between hibernating and nonhibernating Asian chipmunks (Tamias asiaticus), a 140-kDa protein complex was found to decrease drastically in the blood of hibernating animals [10]. This complex is composed of four proteins of 20, 25, 27, and 55 kDa . HP-20, -25, and -27 contain collagen-like Gly-X-Y repeats near the N-terminus and are highly homologous to each o...

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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 ...