immunoprecipitation, firstly, with the a-GFP antibody to immu- noprecipitate total CCA1 protein bound to the target promoters and secondly, with the a-PSer antibody to discriminate between phosphorylated and dephosphorylated CCA1 isoforms. By virtue of doubling-up the immunoprecipitation round, we expected to get PCR amplification only if CCA1 phosphorylated isoforms were preferentially bound to the target promoters (Figure S15). Our results showed slightly above background amplification that was observed at both 22uC or 27uC and for all the promoters examined (Figure 7A and 7B). This corresponds to only a minimal fraction of the total amount of CCA1 bound to the promoters, as assayed by double round of immunoprecipitation with the anti- GFP antibody (Figure 7A and 7B). The reliability of these results is supported by the abundant CCA1 phosphorylated isoforms observed at 27uC (Figure 6D) and the efficient detection of immunoprecipitated CCA1 by the P-Ser antibody (Figure 2A and Figure 6D). In addition, the lack of amplification in the Double ChIP assays was not likely due to reduced efficiency of the technique, as Double-ChIP experiments similarly processed with a-Histone3 (a-H3) antibody followed by a-PSer immunoprecip- itation revealed clear amplified bands in all cases (Figure 7A and Figure S15). Together, the results confirmed and extend our findings suggesting that the complex regulatory interplay between CK2 and CCA1 binding activity is modulated by temperature and contributes to proper temperature compensation within the Arabidopsis circadian clock. Discussion Our results show that over-expression of CKB4 reduces the severity of CCA1-YFP-ox phenotypes while the lack of additive phenotypes in CKB4-MYC-ox/cca1-1/lhyRNAi plants indicates that CKB4 and CCA1 function in the same signalling pathway. Repression of TOC1 expression in CCA1-YFP-ox plants was alleviated by over-expression of CKB4, which assigns an important role for CKB4 modulating one of the main feedback Clock Temperature Compensation in Arabidopsis loops described in Arabidopsis [58]. Pharmacological treatment with specific CK2 inhibitors or the use of plants expressing a CK2 inducible dominant mutant reflected the involvement of CK2 activity in this regulation, rather than a holoenzyme-independent function of the CK2 regulatory subunit [64] . In human cells, treatment with DRB or DMAT also results in long-period phenotypes [19-21]. Similarly, decreased CK2 activity causes long-period behavioral rhythms in Drosophila [22,23]. These results highlight a remarkable conservation of CK2 circadian function among very different organisms including plants, insects and mammals. Based on studies with other circadian systems, it was plausible to assume that phosphorylation of CCA1 by CK2 might mediate changes in CCA1 accumulation or subcellular localization [12]. However, our results assigned a role for CK2 in the control of CCA1 binding activity rather than protein accumulation or localization. Our studies also revealed that CCA1 regulates the expression of morning- and evening-expressed genes most likely by direct binding to their promoters. Therefore, by antagonizing this binding, CK2 can precisely modulate CCA1 regulation of circadian gene expression. The binding of CCA1 to the LIGHT- HARVESTING CHLOROPHYLL A/B1*3 (LHCB1*3) promoter was shown to be increased by CK2 [25,26]. However, the authors also reported that in CKB3-ox plants, the induction of the LHCB1*1 gene was reduced compared to WT (Sugano et al., 1999). In our studies, we observed that CK2 antagonizes CCA1 binding to the promoters of the oscillator genes. This function consistently fits with the CKB and CCA1 inverse correlation between phenotypes and expression. Evidence that CK2 phosphorylation decreases protein binding to target promoters was also provided for the bZIP transcription factor HY5 [65]. Our binding analyses were consistent with the Double-ChIP results showing that CCA1 dephosphorylated isoforms were preferentially bound to the promoters of CCA1 target genes. An altered ratio of phosphor- ylated/dephosphorylated isoforms by increased or decreased CK2 activity is fully consistent with the circadian phenotypes observed Figure 7. Analysis of CCA1 isoforms bound to the promoters of the oscillator genes. (A) Double ChIP assays of CCA1 isoform binding to the promoters of TOC1, LUX, PRR7 and PRR9 in CCA1-YFP-ox/CKB4-MYC-ox plants entrained under LD cycles at 22uC and transferred to LL conditions at 22uC or 27uC. Samples were taken after 35 h under LL. The first immunoprecipitation was performed with a-GFP antibody followed by a second round with a-PSer or with a-GFP. The combinations of a-H3/a-H3 antibodies or a-H3/a-Pser antibodies were used as controls. The experiments were performed at least twice with similar results to those shown here. (B) Q-PCR analysis of CCA1 isoform binding by double ChIP assays. Data are shown as means 6 SD relative to the maximum value of two independent experiments (*** p-value,0.001). doi:10.1371/journal.pgen.1001201.g007 PLoS Genetics | www.plosgenetics.org 9 November 2010 | Volume 6 | Issue 11 | e1001201