The LDL1/2-HDA6 Histone Modification Complex Interacts With TOC1 and Regulates the Core Circadian Clock Components in Arabidopsis

In Arabidopsis, the circadian rhythm is associated with multiple important biological processes and maintained by multiple interconnected loops that generate robust rhythms. The circadian clock central loop is a negative feedback loop composed of the core circadian clock components. TOC1 (TIMING OF CAB EXPRESSION 1) is highly expressed in the evening and negatively regulates the expression of CCA1 (CIRCADIAN CLOCK ASSOCIATED 1)/LHY (LATE ELONGATED HYPOCOTYL). CCA1/LHY also binds to the promoter of TOC1 and represses the TOC1 expression. Our recent research revealed that the histone modification complex comprising of LYSINE-SPECIFIC DEMETHYLASE 1 (LSD1)-LIKE 1/2 (LDL1/2) and HISTONE DEACETYLASE 6 (HDA6) can be recruited by CCA1/LHY to repress TOC1 expression. In this study, we found that HDA6, LDL1, and LDL2 can interact with TOC1, and the LDL1/2-HDA6 complex is associate with TOC1 to repress the CCA1/LHY expression. Furthermore, LDL1/2-HDA6 and TOC1 co-target a subset of genes involved in the circadian rhythm. Collectively, our results indicate that the LDL1/2-HDA6 histone modification complex is important for the regulation of the core circadian clock components.


INTRODUCTION
The circadian rhythm is an endogenous oscillation widely observed in plants, animals, fungi, and cyanobacteria (Edgar et al., 2012). The plant circadian rhythm is highly associated with multiple important biological processes, and maintained by multiple interconnected loops that generate robust rhythms. The circadian clock central loop is a negative feedback loop composed of the core circadian clock components such as TOC1 (TIMING OF CAB EXPRESSION 1) and CCA1 (CIRCADIAN CLOCK ASSOCIATED 1)/LHY (LATE ELONGATED HYPOCOTYL). TOC1 is highly expressed in the evening, but low expressed at dawn (Alabadi et al., 2001). Furthermore, TOC1 was identified as a repressor of CCA1 and LHY by binding to their promoters in the evening (Gendron et al., 2012;Huang et al., 2012). In contrast, CCA1 and LHY are highly expressed in the morning, but low expressed at nightfall (Schaffer et al., 1998;Wang and Tobin, 1998;Alabadi et al., 2001). CCA1 and LHY bind to the evening element (EE) on the promoter of TOC1 to inhibit its expression (Schaffer et al., 1998;Wang and Tobin, 1998;Alabadi et al., 2001;Nagel et al., 2015). CHE (CCA1 HIKING EXPEDITION) is an evening-expressed TCP-family transcription factor, which also targets the CCA1 promoter to repress its expression. Furthermore, CCA1 and LHY were shown to repress the CHE expression by targeting the CHE promoter (Pruneda-Paz et al., 2009).
Histone modifications play important roles in the regulation of gene expression. Histone methyltransferases and demethylases determine the methylation levels, whereas histone acetylation levels are regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs or HDAs). HDACs and the H3K4 demethylase LSD1 (Lysine-Specific Demethylase 1) are the core components of the Mi2/NuRD and CoREST protein complexes in yeast and animal cells (Khochbin et al., 2001;Lee et al., 2005;Wang et al., 2009). They act co-operatively to repress gene expression in mammals (Huang et al., 2011). The interactions among the core protein components of the HDAC complexes are relatively stable and the HDAC complexes can also interact with various transcription factors under different environmental conditions (Joshi et al., 2013;Liu et al., 2014). FLD (FLOWERING LOCUS D), LDL1 (Lysine-Specific Demethylase-LIKE 1), LDL2, and LDL3 are the LSD1 homologs in Arabidopsis (Jiang et al., 2007). LDL1 and LDL2 act redundantly to regulate FLC (FLOWERING LOCUS C) by H3K4 demethylation (Jiang et al., 2007). Furthermore, Arabidopsis HISTONE DEACETYLASE 6 (HDA6) directly interacts with FLD to repress FLC, MAF4, and MAF5 by reducing H3K4 methylation (H3K4me) and H3 acetylation (H3Ac) to regulate flowering time (Yu et al., 2011). In addition, HDA6 can also interact with LDL1 and LDL2 to regulate gene expression (Hung et al., 2018).
The HDAC inhibitor TSA treated plants show delayed phases and higher amplitudes of TOC1 expression (Perales and Más, 2007). In addition, the expression of Arabidopsis CCA1, LHY, and TOC1 is specifically associated with H3Ac and H3K4me changes (Hemmes et al., 2012;Malapeira et al., 2012), indicating that the expression of the core circadian clock components is associated with H3Ac and H3K4me level changes. Our recent study indicated that CCA1 and LHY can interact with the HDAC complex containing LDL1, LDL2, and HDA6. Furthermore, the LDL1/2-HDA6 complex can be recruit by the transcription repressors CCA1 and LHY to their target genes including TOC1. Since CCA1 and LHY are low expressed at nightfall, the expression of TOC1 is increased due to the release of LDL1/2-HDA6 from the TOC1 promoter (Hung et al., 2018). In this study, we demonstrated that LDL1/2-HDA6 can also interact with TOC1 to regulate the expression of CCA1 and LHY. Furthermore, LDL1/2-HDA6 and TOC1 co-target a subset of genes involved in the circadian rhythm.
The full-length coding sequence (CDS) fragment of TOC1 was PCR-amplified and cloned into the pCR8/GW/TOPO vector (Invitrogen), and then recombined into the PK7WGF2 binary vector or 3xFLAG Gateway vector (Invitrogen 1 ). The 35S::TOC1:GFP vector was transformed into Col-0 WT or hda6/ldl1/2 by the floral dip method.

Bimolecular Fluorescence Complementation (BIFC) Assays
To generate the constructs for BiFC assays, the full-length coding sequence (CDS) fragment of TOC1 was amplified by PCR and cloned into the pCR8/GW/TOPO vector, and then recombined into the pEarleyGate201-YN (Lu et al., 2010). LDL1-YC and HDA6-YC were described in the previous studies (Yu et al., 2011;Hung et al., 2018). Constructed vectors were transformed into Arabidopsis protoplasts or tobacco (Nicotiana benthamiana) leaves for transient assays. Transformed protoplasts and tobacco leaves were then examined by confocal spectral microscope imaging system (NTU-TCS SP5, Leica 2 ).

Yeast Two-Hybrid (Y2H) Assays and Co-immunoprecipitation (Co-IP) Assays
Yeast two-hybrid assays were performed based on the instruction for the Matchmaker GAL4-based two-hybrid system 3 (Clontech). The LDL1, LDL2, and TOC1 full length cDNA fragments were sub-cloned into pGADT7 and pGBKT7 vectors. All constructs were transformed into the yeast (Saccharomyces cerevisiae) strain AH109 by the lithium acetate method, and yeast cells were grown on a minimal medium/-Leu-Trp according to the manufacturer's instructions (Clontech). Transformed colonies were grown on the medium containing X-α-gal for the α-galactosidase activity assay or minimal medium/-Leu-Trp-His (3DO) with 0.25 mM 3-amino-1,2,4-triazole (3AT).
Co-immunoprecipitation assays were performed as previously described (Yu et al., 2011). The 35S::TOC1:3xFLAG plasmid was transformed into Arabidopsis protoplasts extracted from LDL1pro::LDL1:GFP or 35Spro::GFP transgenic plants. Total proteins were than extracted from the transformed protoplasts. Anti-GFP (Santa Cruz Biotechnologies, catalog no. SC-9996; 1:3000 dilution) and anti-FLAG (SIGMA catalog no. M2; 1:3000 BiFC assays in Arabidopsis protpplasts showing interaction between LDL1/LDL2 and TOC1 in living cells. LDL1, LDL2, and TOC1 fused with the N terminus (YN) or C terminus (YC) of YFP were co-delivered into Arabidopsis protpplasts. The nucleus was indicated by mCherry carrying a nuclear localization signal. (B,C) Yeast two hybrid analysis of the interaction of LDL1/LDL2 with TOC1. LDL1-BD/LDL2-BD with CCA1-AD or LHY-AD was co-transformed into the yeast strain AH109. The transformants were plated on the SD/-Leu-Trp-His medium. (C) Quantitative α-galactosidase assays for protein-protein interaction in yeast. Bars indicate SD from three biological replicates. (D) Co-IP of the native promoter driven LDL1:GFP with TOC1 in LDL1pro::LDL1:GFP transformed Arabidopsis protoplasts. Western blot (WB) was performed with the anti-FLAG and anti-GFP antibodies.
dilution) antibodies were used as primary antibodies for Western blot. The resulting signals were detected by using a Pierce ECL Western blotting kit (Pierce 3 ).

Quantitative Real-Time PCR (qRT-PCR) Analysis
The TRIZOL reagent (Invitrogen, 15596026) was used for total RNA isolation according to the manufacturer's instructions. Total RNA treated with 2 µg of DNAse (Promega, RQ1 #M6101) were then used for cDNA synthesis (Promega, #1012891). The iQ SYBR Green Supermix solution (Bio-Rad, #170-8880) was used for real-Time quantitative PCR assays with the CFX96 real-time PCR Detection System (Bio-Rad Laboratories, Inc.). Cycling conditions were started with 95 • C/10 min, followed by 45 cycles of 95 • C/15 s, 60 • C/30 s, and then fluorescent detection, and melting curve detection (65-95 • C, incrementing 0.5 • C for 5 s, and plate reading). Each sample was normalized by calculating delta quantification cycle (Cq) to the expression of the UBQ10 (Ubiquitin10) internal control and quantified at least in triplicate. The Cq and relative expression level are calculated by the Biorad CFX Manager 3.1 based on the MIQE guidelines (Bustin et al., 2009). Supplementary  Table S1 listed the gene specific primers used for qRT-PCR. Standard deviations (SD) represent at least three technical and three biological replicates. The variance in average data is represented by standard error of the mean (SEM). The SD, SEM determination and P-value were calculated using Student's paired t-test.

Protoplast Transient Assays
The CCA1pro::LUC plasmid construct was previously described (Wang et al., 2011). For transcriptional activity assays, the 35Spro::TOC1, 35Spro::LDL1, 35Spro::HDA6, or 35Spro::GFP effector constructs were co-transformed into protoplasts with CCA1pro::LUC, and the plant samples were collected at ZT0 after 12 h. The relative activities of LUC (luciferase) reporter were standardized by activities of co-expressed Renilla LUC. Experiments were repeated at least three times for each reporter-effector combination. The dual luciferase assay reagent (Promega) was used for Firefly LUC and Renilla LUC detection.

Chromatin Immunoprecipitation (ChIP) Assays and ChIP-seq Data Analyses
Chromatin immunoprecipitation assays were accomplished as previously described (Yu et al., 2011;Hung et al., 2018). Plant seedlings were treated with 1% formaldehyde for chromatin extraction. The extracted DNA was sheared to the mean length near 500 bp by sonication, proteins, and DNA fragments were then immunoprecipitated by the H3K9K14 (Millipore, catalog no. 06-599), H3K4me3 (Milipore, catalog no. 04-745), or GFP (Abcam, catalog no. ab290) antibodies. The cross-link between DNA with immunoprecipitated proteins were reversed, and then analyzed by real-time PCR using specific primers (Supplementary Table S1). The quantification cycle(Cq) was calculated by Biorad CFX Manager 3.1 based on the MIQE guideline (Bustin et al., 2009). Percent input was calculated as 2 ∧ [Cq(IN)-Cq(IP)]X100. Each sample was quantified at least in triplicate, and normalized by calculating delta Cq to the expression of the internal control. Standard deviations (SD) represent at least three technical and three biological replicates. The variance in average data is represented by standard error of the mean (SEM). The SD, SEM determination and P-value were calculated using Student's paired t-test. ChIP-seq assays were performed based on previous research (Li et al., 2015(Li et al., , 2016Hung et al., 2018). The LDL1 ChIPseq data were deposited to NCBI-Gene Expression Omnibus (GEO) database (GSE118025) (Hung et al., 2018). The ChIP-Seq files from other research groups, GSE35952 (Huang et al., 2012) and (Kamioka et al., 2016), were downloaded from the NCBI-GEO database.

LDL1 and HDA6 Interact With TOC1 and Directly Target on CCA1 and LHY
Our recent study indicated that CCA1/LHY can interact with the LDL1/2-HDA6 complex to repress TOC1 (Hung et al., 2018). In addition, the expression of TOC1, CCA1 and LHY is also associated with H3K4me and H3 acetylation changes (Hemmes et al., 2012;Malapeira et al., 2012). We further analyzed the functional correlation between TOC1 and the LDL1/2-HDA6 complex. TOC1 directly interacted with both LDL1 and LDL2 in BiFC assays by using Arabidopsis protoplasts and Agrobacterium-infiltrated tobacco leaves. The YFP fluorescence signal was detected in nucleus of the transformed cells (Figure 1A and Supplementary Figure S1). The interaction between LDL1, LDL2, and TOC1 was further confirmed by yeast two-hybrid assays (Figures 1B,C) and Co-IP assays using Arabidopsis protoplasts ( Figure 1D and Supplementary Figure S1). Furthermore, TOC1 can also interact with HDA6 in BiFC assays (Supplementary Figures S2A,B). These results suggested that TOC1 may recruit the LDL1/2-HDA6 histone modification complex to its target genes such as CCA1 and LHY.
We further analyzed the binding of LDL1 and HDA6 to CCA1 and LHY by ChIP assays. The LDL1:GFP and HDA6:GFP transgenic plants were previously described (Yu et al., 2011;Hung et al., 2018). 14 days old plants grown under 12 h light/12 h dark condition were collected on Zeitgeber time 0 (ZT0) and ZT12. An anti-GFP antibody was used for ChIP assays, and the binding of LDL1 and HDA6 was analyzed by qPCR. We identified that both LDL1 and HDA6 can bind to the promoters of CCA1 and LHY. Furthermore, the binding of LDL1 and HDA6 to the promoters of CCA1 and LHY were significantly decreased on ZT0 compared to ZT12 (Figure 2 and Supplementary Figure S2C). The binding of LDL1 and HDA6 to the CCA1 and LHY promoters is correlated to TOC1 accumulation, since TOC1 is highly expressed at nightfall but low expressed in the morning (Alabadi et al., 2001).

TOC1 and LDL1 Co-target Genes Involved in the Circadian Rhythm
Previously, we identified the global binding sites of LDL1 by ChIP-Seq assays (Hung et al., 2018). The GO-BP (Gene Ontology_Biological Process) analysis of LDL1-targeted genes revealed that LDL1 targets on a subset of circadian rhythm genes. Furthermore, LDL1 also binds to a cluster of circadian rhythm genes regulated by CCA1 (Hung et al., 2018). In this study, we further analyzed whether the LDL1 and TOC1 also co-target genes involved in the circadian rhythm.
We compared the previously published TOC1 ChIP-Seq data (Huang et al., 2012) with the LDL1 ChIP-Seq data (Hung et al., 2018). The genome browser views by Integrative Genomics Viewer (IGV) indicated that LDL1 bound to CCA1 and LHY, and the binding peaks of LDL1 are highly correlated with the TOC1 binding regions on CCA1 and LHY promoters ( Figure 3A). Among 772 genes occupied by TOC1 (Huang et al., 2012), 195 of them are also co-occupied by LDL1 (P = 1.14e-16) ( Figure 3B). Furthermore, the genomic binding regions of TOC1 are closed to the LDL1 binding regions (Figure 3C), indicating that TOC1 and LDL1 tend to bind to the similar genome sites. GO-BP analysis also indicated that LDL1 and TOC1 cotarget on a subgroup of genes involved in circadian rhythm and response to cold ( Figure 3D). In GO-BP analysis, the ratio of the circadian genes of LDL1/TOC1 co-targeted genes is increased when compared to the LDL1-targeted genes or the TOC1targeted genes alone (Supplementary Figure S3). Interestingly, the ratio of the circadian rhythm genes is further increased in the LDL1/CCA1/TOC1 co-targeted genes (Supplementary Figure S3). Previous studies indicated that several cis-elements are enriched in the promoters of TOC1 regulated genes, including the (AG/CT) n repeat, G-box (CACGTG), Evening Element (EE)like and TCP binding site (TBS, GGCCCA) (Gendron et al., 2012;Huang et al., 2012). Similar cis-elements are also enriched in the LDL1-targeted promoter regions (Hung et al., 2018).

DISCUSSION
Arabidopsis HDA6 is a class I RPD3-like histone deacetylase associated with regulation of rRNA and transcription repression (Murfett et al., 2001;Probst et al., 2004;Earley et al., 2006;Liu et al., 2012;Yu et al., 2017). Different transcription factors can recruit HDA6 to regulate the gene expression involved in flowering, leaf development, abiotic stress response, and senescence (Wu et al., 2008;Chen et al., 2010;Yu et al., 2011;Luo et al., 2012;Liu et al., 2014). In animal and yeast cells, HDACs and LSD1 regulate gene expression cooperatively and they are both identified as the core components of Mi2/NuRD and CoREST complexes (Khochbin et al., 2001;Lee et al., 2005;Wang et al., 2009). Our recent study demonstrated that the Arabidopsis H3K4 demethylases LDL1 and LDL2 can interact with HDA6 to repress gene expression (Hung et al., 2018). The LDL1/2-HDA6 complex can also interact with CCA1/LHY and reduce H3Ac and H3K4me levels of the circadian core component TOC1 (Hung et al., 2018). Furthermore, a subset of genes involved in the circadian clock are co-targeted by LDL1 and CCA1 (Hung et al., 2018).
Arabidopsis circadian clock genes are regulated by a complicate feedback regulation network forming multiple interconnected loops. The central loop is comprised of the core clock components, such as TOC1 and CCA1/LHY (Schaffer et al., 1998;Wang and Tobin, 1998;Alabadi et al., 2001;Gendron et al., 2012;Huang et al., 2012;Nagel et al., 2015). The central loop is interlocked with the evening loop and morning loop. PRR5, PRR7, PRR9, and CCA1/LHY constitute the morning loop (Nakamichi et al., 2010;Salomé et al., 2010;Pokhilko et al., 2012), whereas PRR3, GI, ZTL (ZEITLUPE), and TOC1 comprise the evening loop (Kim et al., 2003;Mas et al., 2003b;Para et al., 2007;McClung and Gutiérrez, 2010). We found that LDL1 and CCA1 co-target to a subset of circadian genes, which are repressed by CCA1 in the morning. However, LDL1 also targets to the morning expressed circadian genes, which may not be repressed by CCA1 and LHY (Nagel et al., 2015;Kamioka et al., 2016;Hung et al., 2018). Although the binding of LDL1 on the LDL1/CCA1 co-targeted genes are reduced in the cca1/lhy mutant, their binding is not completely abolished (Hung et al., 2018). These results suggested that in addition to CCA1 and LHY, the LDL1/2-HDA6 complex may also functionally associate with other circadian clock genes. EC (Evening Complex) is FIGURE 6 | A model for LDL1/2 and HDA6 functions in the regulation of core circadian clock components. Both morning accumulated CCA1/LHY and evening accumulated TOC1 interact with the same histone modification complex containing LDL1/2 and HDA6. CCA1/LHY act as transcription repressors and recruit the histone modification complex to their target loci such as TOC1 in the morning. Meanwhile, TOC1 also recruits the histone modification complex to its targets such as CCA1 and LHY in the evening. also associated with regulation of the circadian genes, which is comprised of LUX (LUX ARRHYTHMO), ELF3 (EARLY FLOWERING3), and ELF4 (EARLY FLOWERING4) (Hazen et al., 2005;Nusinow et al., 2011). A previous study indicated that Arabidopsis HDACs are associated with PRR9 through direct interacting with TPL/TPR (TOPLESS/TOPLESS-RELATED) to regulate the expression of CCA1 (Wang et al., 2013). Further research is required to investigate the functional correlation among LDL1/2-HDA6, PRR9, and EC.
The central loop of Arabidopsis circadian clock is consisted of the core clock components including CCA1, LHY, and TOC1 (Schaffer et al., 1998;Wang and Tobin, 1998;Alabadi et al., 2001;Gendron et al., 2012;Huang et al., 2012;Nagel et al., 2015). Although CCA1 and LHY are low expressed at nightfall, they are highly induced at dawn (Schaffer et al., 1998;Wang and Tobin, 1998;Alabadi et al., 2001). Previously, we found that CCA1 interacts with LDL1 in the morning (Hung et al., 2018). The binding of LDL1 and HDA6 on promoter of TOC1 is higher in the morning but decreased in the evening (Hung et al., 2018). Furthermore, HDA6, LDL1, and LDL2 are constitutively expressed at different time periods. CCA1/LHY can therefore recruit the LDL1/2-HDA6 complex to suppress TOC1 expression at dawn (Hung et al., 2018). In this study, we found that LDL1/2 and HDA6 also interact with TOC1 to regulate the expression of CCA1 and LHY. In consistent with the fact that TOC1 is highly accumulated at nightfall (Alabadi et al., 2001), we found that the binding of LDL1 and HDA6 on the CCA1 and LHY promoters is higher in the evening but decreased in the morning. Since TOC1 is a repressor of CCA1 and LHY, the expression of CCA1 and LHY is decreased with increased TOC1 expression (Gendron et al., 2012;Huang et al., 2012). We found that histone acetylation and H3K4 methylation levels of CCA1 and LHY are decreased in TOC1-OE plants. However, the H3Ac, H3K4me and expression levels of CCA1 and LHY are significantly increased in TOC1-OE/hda6/ldl1/2 compared to the TOC1-OE plants, indicating that the LDL1/2-HDA6 complex is functionally associated with the regulation of CCA1 and LHY expression. Although the expression of TOC1 is highly increased in hda6/ldl1/2 compared to wild type, the expression of CCA1 and LHY is not decreased. It is possible that in addition to LDL1/2-HDA6, other unknown proteins may also be involved in the regulation of CCA1 and LHY expression.
Collectively, we propose a model to demonstrate how the core circadian clock components are regulated by H3K4 demethylation and histone deacetylation (Figure 6). The histone modification complex containing LDL1/2 and HDA6 can interact with both morning accumulated CCA1/LHY (Hung et al., 2018) and evening accumulated TOC1. The transcription repressors CCA1 and LHY can recruit the LDL1/2-HDA6 complex to their target loci including TOC1 in the morning (Hung et al., 2018). Furthermore, TOC1 can also recruit the histone modification complex to its targets such as CCA1 and LHY in the evening.

DATA AVAILABILITY
All datasets generated for this study are included in the manuscript and/or the Supplementary Files.

AUTHOR CONTRIBUTIONS
F-YH, KW, and YC designed the research. F-YH, F-FC, and J-HC performed the research. F-YH, CL, CC, and KW analyzed the data. F-YH and KW wrote the article.

FUNDING
This work was supported by the Ministry of Science and Technology of the Republic of China (105-2311-B-002-012-MY3 and 106-2313-B-002-003-to KW) and National Taiwan University (106R891501 and 107L893101 to KW). This work was also supported by funding from the Agriculture and Agri-Food Canada A-base and the National Science and Engineering Research Council of Canada (RGPIN/04625-2017 to YC).