Xin-Jian He's laboratory reveals dual recognition of H3K4me3 and DNA by a plant-specific subunit of ISWI chromatin-remodeling complex regulating the flowering time

On April 30, 2020, Xin-Jian He’s laboratory published a research article entitled "Dual recognition of H3K4me3 and DNA by the ISWI component ARID5 regulates the floral transition in Arabidopsis" online in Plant Cell. This study identified a plant-specific subunit of the ISWI chromatin-remodeling complex and revealed that the subunit recognizes both the histone H3K4me3 modification and DNA and thereby regulates the floral transition in Arabidopsis.

ATP-dependent chromatin-remodeling proteins regulate the packaging, unwrapping, and mobilization of nucleosomes and thereby affect DNA replication, DNA repair, and transcription. Imitation Switch (ISWI) is one of four families of ATP-dependent ISWI proteins typically associate with 1 to 3 accessory subunits and form multiple ISWI complexes in eukaryotes. Accessory subunits of the ISWI complexes either regulate nucleosome mobilization or facilitate the association of the ISWI complexes with specific chromatin sites. InArabidopsis thaliana, there are two closely related ISWI chromatin-remodeling proteins, CHR11 and CHR17 (CHR11/17). Loss-of-function of CHR11/17 results in pleiotropic developmental defects and early flowering. To date, only DDT-domain proteins RTL1 and RTL2 (RTL1/2) have been reported to be accessory subunits of ISWI complexes in Arabidopsis. It remains to be determined whether the Arabidopsis ISWI complexes contain any other accessory subunits and how the accessory subunits function in the complexes.

Xin-Jian He's laboratory identify components of the ISWI complexes inArabidopsis thalianaand found that the Arabidopsis ISWI chromatin-remodeling complexes includes three types, which are named CRA (CHR11 / 17-RLT1 / 2/2 ARID5), CDM (CHR11 / 17-DDP1 / 2 / 3-MSI3) and CDD (CHR11 / 17) -DDR1 / 3/4/5 / DDW1) (Figure 1A, 1B). The CRA-type ISWI complex contains a plant-specific subunit named ARID5. As shown by previous studies, thechr11/17andrlt1/2mutant plants showed small stature, reduced fertility, and early flowering. The phenotype ofrlt1 / 2mutant is weaker than that ofchr11/17mutant. The study showed that the developmental and flowering-time phenotype of thearid5mutant is similar to therlt1/2mutant, suggesting that ARID5 is functionally related to the other subunits of the CRA-type ISWI complex. RNA-seq analysis indicated that ARID5 and other subunits of the CRA-type ISWI complex co-regulate the expression levels of multiple critical flowering-time genes, supporting the notion that ARID5 functions as a subunit of the ISWI complex to regulate flowering time by co-regulating the expression of the flowering-time genes.

Figure 1. Identification of the Arabidopsis ISWI complexes. (A) The interaction network of three subtypes of ISWI complexes detected by IP-MS. (B) The interaction network of ISWI complex subunits detected by Y2H.

ARID5 contains a putative ARID domain and its adjacent PHD domain. We found that the ARID domain of ARID5 can bind to double-stranded DNA containing AT; the PHD domain specifically recognizes the histone modification H3K4me3 but does not recognize unmodified histones or other modified histones ( Figure 2A-2E). By collaborating with Dr. Jiamu Du’s laboratory from Southern University of Science and Technology, the study obtained a ternary complex formed by ARID5, DNA, and H3K4me3 and revealed its high-resolution crystal structure. By structural analysis, the study showed that the trimethyllysine of H3K4me3 is accommodated by an aromatic cage formed by PHD finger residues Trp688 and Trp697. In addition, both the PHD and ARID domains can bind to the amino acid residues around H3K4 through hydrogen-bonding interactions, thereby enhancing the binding of ARID5 to the H3K4me3 histone peptide (Figure 3A-3E). The ARID domain of ARID5 mainly binds to the phosphate group on the DNA backbone. In addition, the ARID residue T648 specifically recognizes the central AT dinucleotide pairs to achieve the sequence preference.

Figure 2. ARID5 binds to DNA and H3K4me3. (A, B) The binding of ARID5 to AT-containing DNA as determined by EMSA. (C) Analysis of the binding of ARID5 to H3K4me3 by pull down assays. (D) The binding of the ARID-PHD cassette to DNA as tested by ITC. (E) The binding of the ARID-PHD cassette to H3K4me3 as tested by ITC.

Figure 3. Structure of the ARID5 ARID-PHD cassette in complex with an H3K4me3 peptide and DNA. (A,B) Overall structure of the ARID5 ARID-PHD cassette in complex with an H3K4me3 peptide and an AT-containing DNA duplex. (C) The interactions between the ARID and PHD domains with the interacting residues highlighted in stick. The hydrogen bonds are shown in dashed silver lines. (D,E) The specific recognition of H3A1, H3R2 (D), H3K4me3, H3Q5, and H3T6 (E) by AIRD5. The interacting residues are highlighted in stick. The hydrogen bonding interactions are highlighted by dashed silver lines.

ChIP-seq indicated that the wild-type ARID5 are enriched in the gene body proximal to the transcription start site; the ARID5 pattern on the genic region was similar to the H3K4me3 pattern (Figure 4A), supporting the notion that H3K4me3 is necessary for the association of ARID5 with chromatin. ChIP-qPCR showed that the association of ARID5-∆PHD and ARID5-ARID-M with ARID5 target loci was markedly decreased, indicating that both PHD and ARID domains are required for the association of ARID5 with chromatinin vivo. By complementation testing, the study demonstrated that both the ARID and PHD domains of ARID5 are necessary for the regulation of development and floral transition (Figure 4B), suggesting that the binding of ARID5 to chromatin through the ARID and PHD domain are involved in the regulation of development and flowering time. ChIP-PCR indicated that the ISWI catalytic subunit CHR11 can associate with the ARID5-binding loci and the binding is significantly reduced by thearid5mutation. In summary, this study identified a plant-specific ISWI subunit and demonstrated that dual recognition of H3K4me3 and DNA by the subunit regulates the development and floral transition.

Figure 4. ARID5 binds to H3K4me3-enriched genic regions and regulates the floral transition. (A) The distribution pattern of ARID5 and H3K4me3 signals on ARID5-enriched genes. (B) Morphological phenotypes of WT,arid5, and transgenic plants expressing ARID5-WT, ARID5-ΔPHD and ARID5-ARID-M in thearid5background.

Dr. Lian-Mei Tan from Xin-Jian He’s laboratory and Dr. Rui Liu from Jiamu Du’s laboratory in Southern University of Science and Technology are co-first authors of this study. Dr. Xin-Jian He from National Institute of Biological sciences, Beijing and Dr. Du Jiamu from Southern University of Science and Technology are co-corresponding authors of this study. The study was supported by the Chinese Ministry of Science and Technology, the National Natural Science Foundation of China, the Beijing Municipal Government, and the Shenzhen Municipal Government.