HNF-4 participates in the hibernation-associated transcriptional regulation of the chipmunk hibernation-related protein gene

Abstract

The chipmunk hibernation-related protein 25 (HP-25) is involved in the circannual control of hibernation in the brain. The liver-specific expression of the HP-25 gene is repressed in hibernating chipmunks under the control of endogenous circannual rhythms. However, the molecular mechanisms that differentially regulate the HP-25 gene during the nonhibernation and hibernation seasons are unknown. Here, we show that the hibernation-associated HP-25 expression is regulated epigenetically. Chromatin immunoprecipitation analyses revealed that significantly less hepatocyte nuclear receptor HNF-4 bound to the HP-25 gene promoter in the liver of hibernating chipmunks compared to nonhibernating chipmunks. Concurrently in the hibernating chipmunks, coactivators were dissociated from the promoter, and active transcription histone marks on the HP-25 gene promoter were lost. On the other hand, small heterodimer partner (SHP) expression was upregulated in the liver of hibernating chipmunks. Overexpressing SHP in primary hepatocytes prepared from nonhibernating chipmunks caused HNF-4 to dissociate from the HP-25 gene promoter, and reduced the HP-25 mRNA level. These results suggest that hibernation-related HP-25 expression is epigenetically regulated by the binding of HNF-4 to the HP-25 promoter, and that this binding might be modulated by SHP in hibernating chipmunks.

Figure 1. HP-25 is regulated at the transcriptional level in association with hibernation.

HP-25 primary transcript, HP-25 mRNA and albumin primary transcript were amplified by RT-PCR using total RNA extracted from the liver of nonhibernating (NHL; lanes 1–6) and hibernating chipmunks (HL; lanes 7–12). The PCR products were separated by electrophoresis on a 2% agarose gel.

Figure 2. Amount of HNF-4 bound to the HP-25 promoter increased in the nonhibernation season.

(a) Immunoblot analyses of HNF-4, USF-1, and USF-2 using nuclear extracts prepared from the liver, kidney, heart, and lung of a nonhibernating (NH) and a hibernating (H) chipmunk. (b) Immunoblot analyses of HNF-4, USF-1, USF-2, and HNF-1 were performed as in (a) using the liver of five nonhibernating (NHL; lanes 1–5) and five hibernating chipmunks (HL; lanes 6–10). (c) Binding of transcription factors to the HP-25 gene promoter was analyzed by ChIP (left panel) and ChIP-qPCR (right panel). ChIP and ChIP-qPCR were performed with chromatin from the liver, kidney, and heart of a nonhibernating chipmunk (NH) and the liver of a hibernating chipmunk (H) using the indicated antibodies, normal rabbit IgG (IgG), or no antibody (no Ab). Following DNA purification, the samples were analyzed by PCR using primer set specific for the HP-25 gene promoter region. One hundredth of four percent of the total input sample (Input) was also examined by PCR. Results are representative of more than three each of nonhibernating and hibernating chipmunks. In ChIP-qPCR, the values were normalized to the total input values, and the results are shown as the fold increase over the values for the liver of the nonhibernating chipmunk (NH). Results are means ± SEM for two independent experiments using the samples prepared from different individuals. Different letters (a–c) are significantly different at p < 0.05; one-way ANOVA with Tukey-Kramer post test. (d) ChIP (left panel) and ChIP-qPCR (middle and right panels) were performed with the indicated antibodies using chromatin from the liver of a nonhibernating chipmunk (NH) and a hibernating chipmunk (H) different from those used in (c). In ChIP-qPCR, the values were normalized as in (c). Results are means ± SEM for three independent experiments. **p < 0.01; Student’s t-test.

Figure 3. Increased active transcription histone markers on the HP-25 promoter in the liver of nonhibernating chipmunks.

(a) Comparison of the amounts of modified histones in the liver of nonhibernating and hibernating chipmunks. Immunoblotting was performed as in Fig. 2b using nuclear extracts from the liver of four nonhibernating chipmunks (NHL; lanes 1–4) and four hibernating chipmunks (HL; lanes 5–8). USF-1 was used as a loading control. (b) ChIP was performed as in Fig. 2c with chromatin from the liver of a nonhibernating chipmunk (NH) and a hibernating chipmunk (H) using the indicated antibodies and primer sets specific for the indicated gene promoter regions. (c) ChIP-qPCR was performed as in Fig. 2d with chromatin from the liver of a nonhibernating (NHL) and a hibernating chipmunk (HL) using the indicated antibodies. Results are means ± SEM for three independent experiments performed in technical triplicate. **p < 0.01. The chipmunks used in (b) and (c) are different individuals.

Figure 4. Coactivators enhance the HP-25 transcriptional activation in nonhibernating chipmunks.

(a) ChIP was performed as in Fig. 2c using the indicated antibodies. (b) ChIP-qPCR was performed as in Fig. 2d with chromatin from the liver of two nonhibernating (NHL) and two hibernating chipmunks (HL) using the indicated antibodies. Results are means ± SEM for two independent experiments performed in technical triplicate. **p < 0.01, ***p < 0.001. The chipmunks used in (a) and (b) are different individuals.

Figure 5. SHP represses HP-25 gene transcription by inhibiting HNF-4 binding to the promoter.

(a) SHP, HP-25, and albumin mRNA levels in the liver of four nonhibernating chipmunks (NHL) and four hibernating chipmunks (HL), measured by RT-qPCR. The results were normalized to the amount of mRNA of the respective genes in the NHL. The results are shown as means ± SEM. *p < 0.05. (b–g) Primary hepatocytes prepared from a nonhibernating chipmunk were transfected with a FLAG-SHP expression plasmid or an empty vector plasmid (control). In (b), whole-cell extracts from the primary hepatocytes were immunoblotted with the indicated antibodies. USF-2 was used as a loading control. In (c–e), total RNA was prepared from the primary hepatocytes (SHP or control), and the endogenous HP-25 (c), albumin (d), and HNF-4 (e) expression was assessed by RT-qPCR. The results were normalized to the amount of mRNA for each gene in control cells. Results show means ± SEM for three independent experiments. **p < 0.01. In (f and g), ChIP-qPCR was performed with chromatin from the primary hepatocytes using anti-HNF-4 (f) or anti-histone H3 (g) antibodies, and the values were normalized to the total input value. Results are shown as the fold increase over the control value. Results show means ± SEM for three independent experiments. **p < 0.01.

Figure 6. A schematic model of the hibernation-associated transcriptional regulation of the HP-25 gene based on this study.

In nonhibernating chipmunks, HNF-4 binds the HP-25 gene promoter region to activate HP-25 transcription in combination with coactivators and active histone modification marks (left side part). On the other hand, in hibernating chipmunks, HNF-4 and its coactivators dissociate from the promoter, possibly by an interaction of HNF-4 with SHP whose expression is upregulated during hibernation, and active transcription histone marks are removed, resulting in transcriptional repression of the HP-25 gene (right side part).