Rhythmic oscillation of histone acetylation and methylation at the Arabidopsis central clock loci

Abstract

Circadian clock genes are regulated by a transcriptional-translational feedback loop. In Arabidopsis, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) transcripts are highly expressed in the morning. Translated LHY and CCA1 proteins repress the expression of TIMING OF CAB EXPRESSION 1 (TOC1), which peaks in the evening. TOC1 protein induces expression of LHY and CCA1, forming a negative feedback loop which is believed to constitute the oscillatory mechanism of the clock. The rhythmic oscillation of mouse clock genes mPERIOD 1 (mPER1) and mPER2 has been correlated with regular alteration of chromatin structure through histone acetylation/deacetylation. However, little is known about the relationship between the transcriptional activity of Arabidopsis clock genes and their chromatin status. Here, we report that histone H3 acetylation (H3Ac) and H3 lysine 4 tri-methylation (H3K4me3) levels at LHY, CCA1, and TOC1 are positively correlated with the rhythmic transcript levels of these genes, whereas H3K36me2 level shows a negative correlation. Thus, our study suggests rhythmic transcription of Arabidopsis clock genes might be regulated by rhythmic histone modification, and it provides a platform for future identification of clock-controlling histone modifiers.

Fig. 1

Expression of LHY, CCA1, and TOC1 transcripts in wild-type (WT) Arabidopsis grown in 12L12D. WT seedlings were grown under 12L12D conditions for 7 days and harvested every 4 h for 1 day. Expression was analyzed by RT-PCR. UBQ10 was included as an expression control.

Fig. 2

Histone modifications within LHY, CCA1, and TOC1 chromatin in 12L12D. (A) ChIP assay of LHY chromatin with H3Ac-, H4K4me3-, H4Ac-, and H3K36me2-specific antibodies. WT seedlings were grown in 12L12D for 7 days and harvested at ZT0 and ZT12 (A-C). ZT means time (h) after lights on. “Input” indicates chromatin before immunoprecipitation. “Mock” refers to control samples lacking antibody. ACTIN 1 was used as a control chromatin. The gray boxes in the front indicate exonic 5′ untranslated regions (UTRs), and the rear gray boxes represent 3′ UTRs (AC). Labeled lines indicate regions amplified in ChIP-PCRs using specific primers (see “Materials and Methods”; A-C). (B) ChIP assay of CCA1 chromatin. (C) ChIP assay of TOC1 chromatin.

Fig. 3

Expression of LHY, CCA1, and TOC1 transcripts in WT seedlings grown in LL. (A) RT-PCR analysis of LHY, CCA1, and TOC1 expression in LL. WT seedlings were grown under 12L12D conditions for 7 days and then released to constant light at Time 0. Plants were harvested every 4 h for 2 days in LL and used for RNA extraction and RT-PCR. UBQ10 was included as an expression control. (B) Quantification of LHY, CCA1, and TOC1 expression in (A). Each transcript level was normalized by the corresponding transcript level of UBQ10 before plotting.

Fig. 4

Oscillation of H3Ac within LHY, CCA1, and TOC1 chromatin in LL. (A) ChIP-PCR analysis of H3Ac within LHY, CCA1, and TOC1 chromatin. WT seedlings were entrained under 12L12D conditions for 7 days and subsequenttly released to LL conditions. Plants were harvested every 4 h for 2 days in LL and used for ChIP using H3Ac-specific antibody. ACTIN 1 was used as a control chromatin. Regions tested and primers used for PCR are those shown in Fig. 2. (B) Quantification of the H3Ac levels in (A). Each H3Ac level was normalized by the level of corresponding input before plotting.

Fig. 5

Oscillation of H3K4me3 within LHY, CCA1, and TOC1 chromatin in LL. (A) ChIP-PCR analysis of H3K4me3 levels within LHY, CCA1, and TOC1 chromatin. (B) Quantification of the H3K4me3 levels in (A). Experimental conditions were as described in Fig. 4, with H3K4me3-specific antibody in ChIP.

Fig. 6

Oscillation of H3K36me2 within LHY, CCA1, and TOC1 chromatin in LL. (A) ChIP-PCR analysis of H3K36me2 levels within LHY, CCA1, and TOC1 chromatin. (B) Quantification of the H3K36me2 levels in (A). Experimental conditions were as described in Fig. 4, with H3K36me2-specific antibody in ChIP.