HNF‐4 plays a pivotal role in the liver‐specific transcription of thechipmunk HP‐25 gene

Abstract

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...

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

“…Mouse USF-1, USF-2a, USF-2b, and chipmunk HNF-4 were synthesized from pcDNA3/FLAG-mUSF-1, pcDNA3/FLAGmUSF-2a, pcDNA3/FLAG-mUSF-2b (Fujii et al, 2006), and pcDNA3/HNF-4 (Kojima et al, 2000), respectively, using an in vitro transcription/translation system (Promega). The following double-stranded oligonucleotides were used as probes: CM25G-173/-144, containing the chipmunk HP-25 gene sequence from − 173 to − 144 (5′-GGGGACAGTGACCACGTAAGGGGCA-GGGTT-3′); CM25G-159/-129, containing the chipmunk HP-25 gene sequence from − 159 to − 129 (5′-GTAAGGGGCAGG-GTTCATGTGTTAGGAGA-3′); and a mutant oligonucleotide, CM25G-159/-129TSmut (5′-GTAAGGGGCAGGGTTCAgcT-GTTAGGAGA-3′, in which lower-case letters indicate base substitutions); CM25G-163/-129, containing the chipmunk HP-25 gene sequence from − 163 to − 129 (5′-ACCACGTAAGGG-GCAGGGTTCATGTGTTAGGAGA-3′); and CM25G-77/-43, containing the chipmunk HP-25 gene sequence from −77 to − 43 (5′-GGAACTTTCCTCTGATTAAAGTCCAACCAG-GACA-3′).…”

“…The construction of pCM25G-80/luc and pCM25G-260/luc was described previously (Kojima et al, 2000). Three mutant constructs, pCM25G-260(USFmut-1)/luc, pCM25G-260(USFmut-2)/luc, and pCM25G-260(USFmut-3)/luc, carrying the tree squirrel-type base substitutions in the first, second, or both potential USF-binding sites (CAC → TGT from −161 to −159 and TG → GC from −141 to −140), respectively, were constructed by PCR from pCM25G-260/luc.…”

“…In previous studies, we showed that there are two positively acting transcriptional regulatory regions in the 5′-flanking region of the HP-25 gene, one that extends from − 260 to − 80 and another from − 80 to − 59, and we found that hepatocyte nuclear factor-4 (HNF-4) binds to the proximal regulatory region and plays a pivotal role in the liver-specific transcription of the HP-25 gene (Kojima et al, 2000). In this study, we found two potential upstream stimulatory factor (USF)-binding sites in the distal regulatory region, and investigated the role of USF in HP-25 gene promoter activity.…”

Analysis of gene structures and promoter activities of the chipmunk α1-antitrypsin-like genes

“…Nuclear extracts were prepared from HepG2 or COS-7 cells as described (Kojima et al, 2000). The oligo probes ML-83/-54, MM-79/-54, and MS-77/-53 contained the sequence from nucleotides À 83 to À 54 of the CM55-ML gene, from nucleotides À 79 to À 54 of the CM55-MM gene, and from nucleotides À 77 to À 53 of the CM55-MS gene, respectively.…”

“…For competition assays, a 25-fold molar excess of ML-83/-54, MM-79/-54, MS-77/-53, ML-83/-54mut, MM-79/-54mut, or MS-77/-53mut was used as a competitor, and for super-shift experiments, a rabbit polyclonal HNF-1 antibody (Santa Cruz Biotechnology) was included in the binding reactions. The samples were subjected to electrophoresis through a nondenaturing 5% polyacrylamide gel, and autoradiographed as described (Kojima et al, 2000).…”