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. 2004 Jul;24(14):6278-87.
doi: 10.1128/MCB.24.14.6278-6287.2004.

Circadian and light-induced transcription of clock gene Per1 depends on histone acetylation and deacetylation

Yoshihisa Naruse  1 , Kentaro Oh-hashi, Norio Iijima, Midori Naruse, Hideyo Yoshioka, Masaki Tanaka
Affiliations

Circadian and light-induced transcription of clock gene Per1 depends on histone acetylation and deacetylation

Yoshihisa Naruse et al. Mol Cell Biol. 2004 Jul.

Abstract

Circadian clock genes are regulated through a transcriptional-translational feedback loop. Alterations of the chromatin structure by histone acetyltransferases and histone deacetylases (HDACs) are commonly implicated in the regulation of gene transcription. However, little is known about the transcriptional regulation of mammalian clock genes by chromatin modification. Here, we show that the state of acetylated histones fluctuated in parallel with the rhythm of mouse Per1 (mPer1) or mPer2 expression in fibroblast cells and liver. Mouse CRY1 (mCRY1) repressed transcription with HDACs and mSin3B, which was relieved by the HDAC inhibitor trichostatin A (TSA). In turn, TSA induced endogenous mPer1 expression as well as the acetylation of histones H3 and H4, which interacted with the mPer1 promoter region in fibroblast cells. Moreover, a light pulse stimulated rapid histone acetylation associated with the promoters of mPer1 or mPer2 in the suprachiasmatic nucleus (SCN) and the binding of phospho-CREB in the CRE of mPer1. We also showed that TSA administration into the lateral ventricle induced mPer1 and mPer2 expression in the SCN. Taken together, these data indicate that the rhythmic transcription and light induction of clock genes are regulated by histone acetylation and deacetylation.

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Figures

FIG. 1.
FIG. 1.
Rhythmic histone acetylation of mPer1 and mPer2 in fibroblast cells and the liver. (A) The structure of the mPer1 gene is illustrated, along with the region for PCR amplification in the ChIP assay. Soluble chromatin preparations from NIH 3T3 cells treated with 50% horse serum were immunoprecipitated with anti-acetylated histone H3 or H4 antibodies (α-AcH3 and α-AcH4, respectively) and analyzed by semiquantitative PCR. Aliquots of chromatin obtained before immunoprecipitation were also analyzed (input). Representative PCR results are shown in the lower three panels. The data in the upper panel were quantified with NIH Image and plotted as the change in relative acetylation and relative mRNA level. Data shown were confirmed in two independent experiments. (B) Cross-linked chromatin from liver was immunoprecipitated with anti-acetylated histone H3 or H4 antibodies and then analyzed by semiquantitative PCR with primers in the mPer1 promoter. (C) The same ChIP assay as in panel B was performed with primers in the mPer2 promoter. The expression of mPer1, mPer2, mCry1, and the control G3PDH gene is also shown in panels A and B.
FIG. 2.
FIG. 2.
mCRY1 represses basal transcription and the HDAC inhibitor TSA relieves this repression. (A) Schematic diagrams of the effector and reporter constructs used in the transient transfection assays. (B) NIH 3T3 cells were transfected with reporter constructs pGL-S10PR5GB (200 ng) and pRL-TK (50 ng) as internal controls as well as increasing amounts of plasmids expressing G4DBD-fused mCRY1 (G4mCRY1) (2.5, 25, and 100 ng), G4DBD-fused mPER1 (G4mPER1) (2.5, 25, and 100 ng), fused NRSF (G4NRSF) (100 ng) as a positive control, or G4DBD alone (100 ng) as a negative control. (C) NIH 3T3 cells were transfected with the same reporter as in panel A and equal amounts of G4DBD fused to each protein. After 24 h, the cells were treated with TSA (100 ng/ml) for an additional 24 h. (D) An experiment similar to that in panel C with a reporter containing the simian virus 40 promoter instead. The luciferase activities of all experiments are expressed as the mean ± standard error of the mean of at least three independent experiments performed in duplicate.
FIG. 3.
FIG. 3.
mCRY1 interacts with mSin3B, HDAC1, and HDAC2 in vitro and in vivo. (A) In vitro-translated 35S-labeled BMAL1, full-length mSin3B, and various deletion mutants of mSin3B were incubated with an immobilized fusion protein containing GST and mCRY1 or NRSF (amino acids 1 to 153) (N). Input protein (I) (20% of total), protein bound with GSTmCRY1 (C), protein bound by GSTNRSF-N (N), and protein bound with GST alone (G) were analyzed by SDS-PAGE. BMAL1 and GST-NRSF (N) were positive controls. A schematic representation of the mSin3B interaction domain with mCRY1 is also shown. (B) In vitro-translated 35S-labeled mHDAC1, mHDAC2, and mHDAC3 were incubated with an immobilized fusion protein containing GST and mCRY1. The input (I) shown is 20% of the amount used in each incubation. (C) COS7 cells were transfected with expression plasmids encoding Flag-tagged mCry1 (Flag-mCry1) and mSin3B, and cell extracts were immunoprecipitated (IP) with anti-Flag M2 antibodies, followed by immunoblotting analyses with antibodies against mSin3B, HDAC1, HDAC2, and Flag. Input (5% cell extracts) was applied to ascertain the positions of the blotted proteins. (D) mCRY1 is bound to the mPer1 E-box after serum shock in NIH 3T3 cells. After serum shock, NIH 3T3 cells were cross-linked and immunoprecipitated with antibody against mCRY1. Precipitated DNA was then amplified with primers flanking the second mPer1 E-box with a semiquantitative PCR method. Input represents signals derived with input chromatin as template. Immune complexes were Western blotted and probed for mCRY1. (E) mCRY1 association with mSin3B in serum-induced NIH 3T3 cells. Cross-linked extracts were immunoprecipitated with an antibody against mCRY1 or with immunoglobulin G at 20 and 28 h after serum shock in NIH 3T3 cells. The immune complexes were Western blotted and probed for mCRY1 and mSin3B. The input shown is 5% of the total extracts.
FIG. 4.
FIG. 4.
TSA induces endogenous mPer1 gene expression and acetylation of histones at the mPer1 promoter. (A) NIH 3T3 cells were incubated with TSA for the duration indicated, and expression levels of various mRNAs were determined by semiquantitative RT-PCR (26, 28). Five clock genes, mPer1, mPer2, mPer3, mCry1, and mCry2, as well as a constitutively expressed control gene, G3PDH, are shown. (B) The data in panel A were quantified with NIH Image and are shown graphically. (C) Soluble chromatin preparations from NIH 3T3 cells treated with TSA for 1 h were immunoprecipitated with anti-acetylated histone H3 or H4 antibodies (α-AcH3 and α-AcH4, respectively). ChIP samples were analyzed by semiquantitative PCR with primers from the promoter (transcription start site region) of the mPer1 gene. (D) Semiquantitative PCRs were carried out three times per experiment shown in panel C and are plotted as the average change in acetylation (mean ± standard error of the mean). (E) TSA can shift the phase of the rhythmic expression of mPer1 and mCry1 in fibroblast cells. NIH 3T3 cells at 4 h after serum shock were incubated with TSA or vehicle (control) for 1 h, and then the expression levels of mPer1 and mCry1 mRNA were determined by RT-PCR. The lower graph shows the data in the upper panel after quantification with NIH Image.
FIG. 5.
FIG. 5.
Light induction of histone H3 and H4 acetylation in the mPer1 and mPer2 promoters and binding of CREB and pCREB to the CRE site on the mPer1 locus in the SCN. Mice were exposed to a 600-lux light pulse for 30 min at CT16. Six brains were collected at each time point, 0, 5, 10, 30, 60, 90, and 180 min after the light pulse, and processed for the ChIP assay. Cross-linked chromatin from SCNs was immunoprecipitated with anti-acetylated histone H3 or H4, anti-CREB, and anti-pCREB antibodies and then analyzed by semiquantitative PCR with primers for the mPer1 (A), mPer2 (B), or mPer1 CRE site (C) promoters. Aliquots of chromatin obtained before immunoprecipitation were also analyzed (input).
FIG. 6.
FIG. 6.
mPer1 and mPer2 induction by TSA in the SCN. TSA or vehicle was administered at CT16. By in situ hybridization with digoxigenin-labeled probes, mPer1 and mPer2 mRNAs were demonstrated in the whole SCN 1 h after TSA treatment. OC, optic chiasm; V, third ventricle. Bars, 100 μm.
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