Epigenetic suppression of mouse Per2 expression in the suprachiasmatic nucleus by the inhalational anesthetic, sevoflurane.
BACKGROUND
We previously reported that sevoflurane anesthesia reversibly suppresses the expression of the clock gene, Period2 (Per2), in the mouse suprachiasmatic nucleus (SCN). However, the molecular mechanisms underlying this suppression remain unclear. In this study, we examined the possibility that sevoflurane suppresses Per2 expression via epigenetic modification of the Per2 promoter.


METHODS
Mice were anesthetized with a gas mixture of 2.5% sevoflurane/40% oxygen at a 6 L/min flow for 1 or 4 h. After termination, brains were removed and samples of SCN tissue were derived from frozen brain sections. Chromatin immunoprecipitation (ChIP) assays using anti-acetylated-histone antibodies were performed to investigate the effects of sevoflurane on histone acetylation of the Per2 promoter. Interaction between the E'-box (a cis-element in the Per2 promoter) and CLOCK (the Clock gene product) was also assessed by a ChIP assay using an anti-CLOCK antibody. The SCN concentration of nicotinamide adenine dinucleotide (NAD(+)), a CLOCK regulator, was assessed by liquid chromatography-mass spectrometry.


RESULTS
Acetylation of histone H4 in the proximal region of the Per2 promoter was significantly reduced by sevoflurane. This change in the epigenetic profile of the Per2 gene was observed prior to suppression of Per2 expression. Simultaneously, a reduction in the CLOCK-E'-box interaction in the Per2 promoter was observed. Sevoflurane treatment did not affect the concentration of NAD(+) in the SCN.


CONCLUSIONS
Independent of NAD(+) concentration in the SCN, sevoflurane decreases CLOCK binding to the Per2 promoter E'-box motif, reducing histone acetylation and leading to suppression of Per2 expression.
Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan ; Department of Anesthesiology and Pain Medicine, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
Keisuke
K
Mori
Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
Norio
N
Iijima
Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
Shimpei
S
Higo
Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
Satoko
S
Aikawa
Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan ; Department of Anesthesiology and Pain Medicine, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
Izumi
I
Matsuo
Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
Ken
K
Takumi
Department of Anesthesiology and Pain Medicine, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
Atsuhiro
A
Sakamoto
Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
Hitoshi
H
Ozawa
D056930:ARNTL Transcription Factors / Q000378:metabolism; D000107:Acetylation / Q000187:drug effects; D018685:Anesthetics, Inhalation / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001921:Brain / Q000378:metabolism; D056926:CLOCK Proteins / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D044127:Epigenesis, Genetic / Q000187:drug effects*; D015870:Gene Expression / Q000187:drug effects; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics; D006657:Histones / Q000378:metabolism; D008297:Male; D008738:Methyl Ethers / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009243:NAD / Q000378:metabolism; D056950:Period Circadian Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077149:Sevoflurane; D013493:Suprachiasmatic Nucleus / Q000378:metabolism*; D013997:Time Factors
D056930:ARNTL Transcription Factors; D018685:Anesthetics, Inhalation; C538961:Bmal1 protein, mouse; D018841:HSP90 Heat-Shock Proteins; D006657:Histones; D008738:Methyl Ethers; C513312:Per2 protein, mouse; D056950:Period Circadian Proteins; D009243:NAD; D000077149:Sevoflurane; D056926:CLOCK Proteins