Fig 2: A model for LDL1/2 and HDA6 functions in the regulation of circadian clock central oscillator TOC1. The morning accumulated-CCA1/LHY interact with the histone modification complex containing LDL1/2 and HDA6. CCA1/LHY act as transcription repressors and recruit the histone modification complex to their targets loci such as TOC1. In the evening, CCA1/LHY are low expressed, and TOC1 is highly expressed because the LDL1/2-HDA6 complex is released from the TOC1 promoter.

Fig 3: LDL1-occupied sites in the genome identified by ChIP-seq analysis. (A) GO-BP annotation of LDL1 -targeted genes. Annotation terms with P-value < 0.005 were listed. (B) Overlap between CCA1 target genes (43) and LDL1 targeted genes. P = 2.45e–17 (hypergeometric distribution). (C) Distribution of distances between the total binding sites of LDL1 and CCA1. (D) (GA/TC)n, G-box, TBS and EE-like motifs were significantly enriched in the LDL1-binding sites.

Fig 4: LDL1 and LDL2 interacts with CCA1. (A) Schematic representation of deletions in LDL1 and CCA1 constructs. LDL1ΔS: deletion of 154–255aa; LDL1SW: 154–255aa; CCA1myb: 1–82aa; CCA1β: 83–608aa; CCA1βN: 83–316aa; CCA1βC: 311–608aa. SWIRM: SWIRM domain of LDL1; myb: myb-domain of CCA1. (B) BiFC assays in Arabidopsis protoplasts showing interaction of LDL1/LDL2 with CCA1 in living cells. Different regions of LDL1 and CCA1, and full-length of LDL2 were fused with the N terminus (YN) or C terminus (YC) of YFP and co-delivered into Arabidopsis protoplasts. The nucleus was indicated by mCherry carrying a nuclear localization signal. (C) Yeast two hybrid analysis of the interaction of LDL1/LDL2 with CCA1. LDL1-BD/LDL2-BD with CCA1-AD was co-transformed into the yeast strain AH109. The transformants were plated on the SD/-Leu-Trp-His medium. (D) Co-IP of LDL1:GFP with CCA1 in LDL1pro::LDL1:GFP transformed Arabidopsis. Western blot (WB) was performed with the indicated antibodies.

Fig 5: Binding of LDL1 to the CCA1/LDL1 putative co-targets. (A) Integrated Genome View of LDL1-occupied sites on the CCA1-occupied genes. LDL1 binding peaks of LDL1 and CCA1 putative co-targeted genes are shown. Pink bars indicate the CCA1-binding regions form previous published data (43). Bar summits = 50. (B, C) LDL1 binding to the promoters of CCR2, TRFL3, GI, ELF5A-1, HSFA3, SIB1, SCPL49, PRR7, PRR9 and TIC. 14 days-old seedlings grown under 12/12 light/dark were harvested on ZT0 and ZT12 (B) or ZT0 only (C). ChIP assays were performed with the anti-GFP antibody. The amount of immunoprecipitated DNA was quantified by qRT-PCR. Values represent the average immunoprecipitation efficiencies (%) against the total input DNA. Error bars correspond to standard deviations from three biological replicates. *P < 0.01, **P < 0.001 (Student's t-test).