The chromatin remodeling factor OsINO80 promotes H3K27me3 and H3K9me2 deposition and maintains TE silencing in rice

Du, Kangxi; Wu, Jiabing; Wang, Jiachen; Xie, Wenhao; Yin, Liufan; Li, Xiang; Li, Chao; Dong, Aiwu

doi:10.1038/s41467-024-55387-4

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Published: 30 December 2024

The chromatin remodeling factor OsINO80 promotes H3K27me3 and H3K9me2 deposition and maintains TE silencing in rice

Nature Communications volume 15, Article number: 10919 (2024) Cite this article

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Abstract

The INO80 chromatin remodeling complex plays a critical role in shaping the dynamic chromatin environment. The diverse functions of the evolutionarily conserved INO80 complex have been widely reported. However, the role of INO80 in modulating the histone variant H2A.Z is controversial. Moreover, whether INO80 helps regulate heterochromatin remains unknown. Here, we characterize the regulatory effects of OsINO80 on protein-coding genes and transposable elements (TEs) in rice. Upon OsINO80 overexpression in rice, we found three types of OsINO80-occupied regions with different chromatin signatures: type I (enriched with H2A.Z), type II (enriched with H3K9me2), and type III (deficient in H2A.Z/H3K9me2). Loss of OsINO80 results in a decrease in H3K27me3, but not H2A.Z, at type I regions as well as a decrease in H3K9me2 at type II regions, which correlates with TE activation and transposition. Our findings reveal that OsINO80 facilitates H3K27me3 establishment, promotes H3K9me2 deposition, and maintains TE silencing.

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Introduction

The chromatin structure is dynamically regulated by many epigenetic factors in response to a variety of cellular and environmental signals. Among the epigenetic regulators, chromatin remodeling complexes use the energy generated by ATP hydrolysis to alter the contacts between histones and DNA and modify nucleosome positions and compositions, and regulate genome architecture and gene transcription^1,2,3,4. Of the four types of chromatin remodeling complexes, the INO80 subfamily members (SWR1 and INO80), are the most evolutionarily conserved in eukaryotes⁵. INO80, the catalytic subunit of the INO80 complex, plays important roles in influencing transcriptional regulation as well as DNA replication and repair^6,7,8. In budding yeast, INO80 mediates the exchange of H2A.Z-H2B for H2A-H2B and promotes the eviction of H2A.Z from chromatin⁹, but some studies in yeast showed that the deletion of INO80 does not affect the distribution of H2A.Z in the genome^10,11,12. Human INO80 reportedly removes H2A.Z from chromatin during homologous recombination^13,14. In contrast, mouse INO80 promotes H2A.Z occupancy in the bivalent promoter regions of primed pluripotent stem cells¹⁵. In murine spermatocytes, INO80 facilitates the incorporation of H2A.Z at poised promoters¹⁶. Similarly controversial findings have also been reported for plants. Arabidopsis INO80 (AtINO80) is responsible for maintaining genomic stability¹⁷. Recent studies revealed that AtINO80 has plant-specific functions and is critical for linking endogenous and environmental stimuli with H2A.Z incorporation or eviction, especially in response to light and temperature changes^{18,19,20,21,22}. However, whether INO80 contributes to H2A.Z eviction or incorporation and whether changes to H2A.Z are directly or indirectly related to the loss of INO80 remain to be clarified.

The relationship between the INO80 chromatin remodeling complex and histone lysine methylation was recently reported. The methylation of distinct histone lysine residues is catalyzed by a group of SET ___domain-containing histone lysine methyltransferases (HKMTases), and the methylation of H3K4/H3K36 and H3K9/H3K27 is generally associated with transcriptional activation and repression, respectively^23,24. In Arabidopsis, the H3K4 methyltransferases ARABIDOPSIS TRITHORAX 4/5 (ATX4/5) are co-purified with the N-terminal ___domain of AtINO80 and function in concert with other accessory subunits to facilitate H3K4 trimethylation (H3K4me3) and transcriptional activation²⁵. Similarly, WD40-REPEAT 5a (WDR5a), a component of the COMPASS-like complex in Arabidopsis, directly interacts with AtINO80 complex and functions with transcription elongation factors to promote H3K4me3 deposition and RNA polymerase II-mediated transcript elongation²¹. Yeast INO80 prevents the invasion of euchromatin into heterochromatin by blocking H3K79 methylation²⁶. Mouse INO80 facilitates the deposition of H3K27me3 at the promoters with bivalent histone modifications by directly interacting with Suppressor of Zeste 12 (SUZ12), a subunit of the PRC2 complex^15,16. Unfortunately, the links between INO80 and H3K9 methylation (heterochromatin mark) remain unknown.

Histone H3K9 methylation and DNA methylation play key roles in transposable element (TE) silencing in higher eukaryotes^27,28. As key contributors to the generation of genomic novelty and variability, TEs are ubiquitous in eukaryotes^29,30. Strictly controlling TE activation is essential for maintaining genome stability³¹. Accordingly, despite the high proportion of TEs within eukaryotic genomes, organisms have evolved silencing mechanisms that repress TE activation. Methylated DNA and H3K9me2 are crucial for TE silencing in plants^32,33,34. In Arabidopsis, the histone methyltransferases KRYPTONITE (KYP)/SUVH4, SUVH5, and SUVH6 bind to methylated DNA at CHG (H denotes A, T, or C) and CHH sites to establish H3K9me2. Moreover, the DNA methyltransferases CHROMOMETHYLASE3 (CMT3) and CMT2 recognize H3K9me2 to establish CHG and CHH methylations, respectively, thereby forming a self-reinforcing loop between non-CG DNA methylation and H3K9 methylation^35,36. In addition, DECREASE IN DNA METHYLATION 1 (DDM1), a chromatin remodeling protein in Arabidopsis, provides METHYLTRANSFERASE 1 (MET1), CMT2, and CMT3 access to heterochromatin, leading to DNA methylation and H3K9me2 and the stable silencing of TEs^37,38. Thus, multiple epigenetic mechanisms cooperatively maintain TE silencing in plants.

In this study, we identified the previously uncharacterized features of INO80 affecting the regulation of H2A.Z occupancy, deposition of H3K9me2, and regulation of TEs in rice. Our RNA sequencing (RNA-seq) analysis indicated that knocking down OsINO80 results in the aberrant transcription of a group of protein coding genes as well as TEs. Additionally, the genome-wide mapping of OsINO80-enriched sites upon OsINO80 overexpression in rice by chromatin immunoprecipitation and sequencing (ChIP-seq) revealed that OsINO80-occupied regions include both euchromatic and heterochromatic regions. Interestingly, the loss of OsINO80 directly led to a decrease in H3K27me3 deposition, but not H2A.Z deposition, in coding genes and a decrease in H3K9me2 deposition in TE regions, correlating with TE activation and transposition.

Results

Characterization of OsINO80 occupancy in the rice genome

The controversy regarding the effect of INO80 on H2A.Z regulation in eukaryotes inspired us to investigate the presence of INO80 throughout the genome of plants. A previous study confirmed that OsINO80 (LOC_Os03g22900) is the sole homolog of INO80 in rice²². The introduction of OsINO80-YFP can rescue the lethal phenotype and partially rescue the growth defects of homozygous osino80-/- rice plants at the T1 generation²², and restore the transgenic plants to the wild-type phenotypes in the subsequent generations, indicating that OsINO80-YFP functions as the endogenous OsINO80 protein in planta. We thus performed a ChIP-seq analysis by using pUBI::OsINO80-YFP transgenic rice plants in the wild-type (WT) Nipponbare background with a nearly five-fold overexpression of OsINO80 (Supplementary Fig. 1), with the wild-type and pUBI::YFP transgenic plants serving as controls, to identify the chromatin regions occupied by OsINO80 (Supplementary Fig. 2 and Supplementary Table 1). An analysis of the ChIP-seq data revealed 43,677 reliable OsINO80-occupied peaks, which corresponded to 34,086 annotated genes, reflecting the distribution of OsINO80 across gene body regions (Supplementary Fig. 2a, b). More specifically, OsINO80 was enriched at the coding region, especially the first exon of its binding genes (Supplementary Fig. 2c).

Considering OsINO80 interacts directly with the histone variant H2A.Z²² and is associated with histone methylations²⁵, to characterize the OsINO80-occupied regions (putative OsINO80 targets), we screened for the presence of H2A.Z and different histone modifications (Supplementary Table 1), including marks related to the activation and repression of transcription. The putative OsINO80 targets were classified into the following three types: type 1 (T1) regions were enriched with euchromatin marks, including H2A.Z, H3K4me2, H3K4me3, H3K27me3, H3K36me3, and H2A monoubiquitination (H2Aub) modifications; type 2 (T2) regions were enriched with the heterochromatin mark H3K9me2; and type 3 (T3) regions were deficient in H2A.Z and H3K9me2 (Fig. 1a and Supplementary Fig. 3). The analysis of DNA bases at the putative OsINO80 targets revealed a similar C/G base frequency (Fig. 1b). The CG and CHG methylation levels were relatively low at the T1 regions, which is in line with the anti-correlation between H2A.Z and DNA methylation^39,40, and high CG and CHG methylation levels were detected at the T2 regions (Fig. 1c). An intermediate DNA methylation level was observed for the T3 regions (Fig. 1c). Considered together, our results suggest that OsINO80 binds directly to both euchromatic and heterochromatic regions in rice.

**Fig. 1: Three types of OsINO80-occupied regions upon *OsINO80* overexpression.**

Loss of OsINO80 does not affect the global distribution of H2A.Z

Because OsINO80 occupancy is correlated with H2A.Z enrichment at T1 putative OsINO80 targets (Fig. 1a), we examined whether OsINO80 participates in the incorporation or eviction of H2A.Z in rice. Thus, we performed an H2A.Z ChIP-seq analysis of the WT control and the OsINO80 knockdown mutant p35S::OsINO80-RNAi-2 (osino80-2)²², with H3 ChIP-seq as an internal control (Supplementary Table 1). The detected H2A.Z peaks (FDR ≤ 0.05) overlapped the peaks identified in a previous study⁴¹ (Supplementary Fig. 4a). The ChIP-seq analysis showed that there were no obvious differences in the overall distribution of H2A.Z along the genome between the osino80-2 mutant and the WT control (Fig. 2a and Supplementary Fig. 5a, b). However, the knockdown of OsINO80 resulted in 1349 peaks with increased and 1417 peaks with decreased H2A.Z deposition (generated by DiffBind (v3.6.5)⁴², p ≤ 0.05) (Supplementary Fig. 5c), indicating that the loss of OsINO80 affected H2A.Z deposition at a number of loci (less than 10% of H2A.Z occupied peaks, Supplementary Fig. 4a). The RNA-seq analysis (Supplementary Table 2 and Supplementary Fig. 6a) detected 1435 and 974 genes that were respectively expressed at higher and lower levels (|log₂(fold-change)| ≥ log₂(1.5) and p ≤ 0.05) in the osino80-2 mutant than in the WT control (Supplementary Fig. 6b). Additionally, the OsINO80 transcript level decreased significantly in the osino80-2 mutant, whereas the transcription of LOC_Os03g22890 (i.e., a gene adjacent to OsINO80) was unaffected (Supplementary Fig. 6c). Furthermore, there were no obviously overall changes in the H2A.Z occupancy of the upregulated or downregulated genes in the osino80-2 mutant (Supplementary Fig. 7a). We further analyzed the transcript levels of genes with significantly altered H2A.Z (|log₂(fold-change)| ≥ log₂(1.5) and p ≤ 0.05) in the osino80-2 mutant compared to WT (Supplementary Fig. 7b, c), and conducted enrichment analysis⁴³ to investigate the relationship between altered H2A.Z levels and transcriptional changes in the osino80-2 mutant compared to WT (Supplementary Fig. 7d, e). Both increased and decreased H2A.Z was significantly correlated with upregulated genes, while increased H2A.Z was only significantly associated with downregulated genes in the osino80-2 mutant (Supplementary Fig. 7d, e).

**Fig. 2: Loss of *OsINO80* does not cause global H2A.Z changes.**

To further clarify the effect of OsINO80 on H2A.Z occupancy, we compared the enrichment of H2A.Z between T1 and random regions enriched with H2A.Z, but not OsINO80. The basal H2A.Z levels were significantly higher for the T1 regions than for the random H2A.Z-enriched regions in both the WT and osino80-2 mutant plants (Fig. 2b). However, the gene-by-gene heatmaps showed that the H2A.Z occupancy at the T1 regions, as well as the random H2A.Z-enriched regions, did not obviously change in the osino80-2 mutant compared with the WT control (Fig. 2c, d). To provide more comprehensive information of OsINO80 function related to H2A.Z, we further performed an H2A ChIP-seq analysis in the WT and osino80-2 mutant plants, but no significant change of H2A distribution was detected in the osino80-2 mutant compared with the WT control (Supplementary Fig. 8). Meanwhile, we categorized putative OsINO80 targets into nine groups based on H2A.Z enrichment at the TSS and gene body regions, respectively. Each group of putative OsINO80 targets displayed no significant change of H2A.Z enrichment in the osino80-2 mutant compared to the WT plants (Supplementary Fig. 9). Accordingly, peaks with increased or decreased H2A.Z occupancy were detected in the osino80-2 mutant (Supplementary Fig. 5c), but OsINO80 did not contribute to these H2A.Z changes, supporting the notion that OsINO80 may not be required for the incorporation or removal of H2A.Z in rice.

Loss of OsINO80 causes decrease in H3K27me3 within T1 regions

The link between INO80 and histone modifications in plants^21,25 compelled us to characterize OsINO80 in terms of its effect on gene transcription. More specifically, we explored the changes in various histone modifications, including H3K4me2, H3K4me3, H3K27me3, H3K36me3, and H2Aub. The ChIP-seq analysis of the osino80-2 mutant (Supplementary Table 1) showed that the loss of OsINO80 resulted in a significant decrease in the H3K27me3 and H2Aub level, but no obvious changes to the H3, H3K4me2, H3K4me3, and H3K36me3 levels in the genome (Supplementary Fig. 10). The decrease in H3K27me3 level in the osino80-2 mutant was not likely due to the transcriptional changes of the genes related to H3K27me3 establishment (Supplementary Fig. 11a).

Moreover, the basal levels of H3K4me2, H3K4me3, H3K27me3, and H2Aub were significantly higher at the T1 regions than at the random H2A.Z-enriched regions (without OsINO80 enrichment) in both the WT and osino80-2 mutant plants (Fig. 3a). However, compared with the random control genes, the T1 regions had significantly decreased H3K27me3 levels, weakly decreased H2Aub levels, and slightly increased H3K4me2 and H3K4me3 levels in the osino80-2 mutant (relative to the corresponding WT levels) (Fig. 3a). Consistent with these findings, the gene-by-gene heatmaps showed that the H3K27me3 occupancy within the T1 regions was clearly lower in the osino80-2 mutant than in the WT control (Fig. 3b); a similar difference was not detected in the random H2A.Z-enriched regions (Fig. 3c). The distribution patterns of OsINO80 and H3K27me3 decrease (in the osino80-2 mutant compared with the WT control) were highly consistent, and the H3K27me3 decrease was more pronounced within putative OsINO80 target genes compared to those without OsINO80 enrichment (Fig. 3 and Supplementary Figs. 9 and 12), supporting that OsINO80 promotes the establishment of H3K27me3 in the rice genome. Consistent with that H3K27me3 is associated with gene repression in eukaryotes, a significant correlation was found between the decrease in H3K27me3 within T1 regions and the upregulated genes in the osino80-2 mutant (relative to the expression level in the WT control) (Supplementary Fig. 13).

**Fig. 3: Loss of *OsINO80* leads to a decrease in the H3K27me3 level.**

Loss of OsINO80 causes decrease in H3K9me2 within T2 regions

Unlike the T1 regions, the T2 OsINO80-occupied regions were enriched with H3K9me2, a constitutive heterochromatin mark in plants⁴⁴, suggesting that the INO80 remodeling complex may modify heterochromatin. To elucidate the effect of OsINO80 on heterochromatin, we performed a ChIP-seq analysis of H3K9me2 in the osino80-2 mutant, which revealed a global decrease in H3K9me2 distribution in the genome of the osino80-2 mutant (compared with the WT control) (Fig. 4a). The RNA-seq analysis indicated that the possible histone H3K9 methyltransferase-encoding genes were similarly transcribed in the osino80-2 mutant and the WT control (Supplementary Fig. 11b). We identified 22,425 peaks for H3K9me2 enrichment (FDR ≤ 0.05) in the WT rice plants, with a substantial overlap with the published H3K9me2 ChIP-seq data (Supplementary Fig. 4b)⁴⁵.

**Fig. 4: Loss of *OsINO80* results in decreased H3K9me2 levels and the activation of TEs.**

The ChIP-seq analysis also showed that the H3K9me2 levels were significantly higher within the T2 regions than in the random H3K9me2-enriched regions (without OsINO80 enrichment) in the WT and osino80-2 mutant plants (Fig. 4b). Importantly, the H3K9me2 peaks with OsINO80 enrichment (T2 regions) but not those without OsINO80 enrichment (random control H3K9me2 peaks) showed significant decrease in H3K9me2 in the osino80-2 mutant compare with the WT control (Fig. 4c–e), supporting that OsINO80 functions in promoting H3K9me2 deposition in rice. Considering the direct association between H3K9me2 and non-CG DNA methylation in plants³⁴, we performed a bisulfite sequencing (BS-seq) analysis and constructed single-nucleotide resolution maps of cytosine methylation in the osino80-2 mutant and WT control (Supplementary Table 3). The genome-wide DNA methylation levels in the CG, CHG, and CHH contexts were almost unchanged in the osino80-2 mutant (compared with the WT control) (Supplementary Fig. 14). Our results support that the loss of OsINO80 leads to a significant decrease in H3K9me2 within OsINO80-occupied regions, but no obvious change in DNA methylation in rice.

OsINO80 represses TE activation and transposition

Because H3K9me2 at CHG and CHH sites maintains TE silencing in plants³³ and OsINO80 promotes H3K9me2 deposition, we speculated that OsINO80 and TE repression may be linked. To determine whether OsINO80 participates in TE regulation, we used RepeatMasker to compare homologous repeats in the rice genome and comprehensively identify TEs⁴⁶. On the basis of their transposition mechanisms, TEs may be divided into the following three classes: Class I elements (retrotransposons), including long terminal repeat (LTR) retrotransposons and non-LTR retrotransposons, are transposed in a “copy-and-paste” manner; Class II elements (DNA transposons) are transposed in a “cut-and-paste” manner; and Class III elements (Helitron transposons) are transposed via a rolling-circle transposition process³¹. We found that Gypsy, Copia, MULE-MuDR, CMC-EnSpm, and Helitron are prevalent in rice TE families (Supplementary Fig. 15), which is consistent with previously reported findings⁴⁷, reflecting the integrity of our identified TEs. Thus, we compared the osino80-2 mutant with the WT control to screen for differentially expressed TEs (|log₂(fold-change)| ≥ log₂(1.5) and adjusted p ≤ 0.05). The comparison detected 579 and 240 TEs that were upregulated and downregulated in the osino80-2 mutant, respectively (Supplementary Fig. 16). Only the upregulated TEs in the osino80-2 mutant were significantly overrepresented among the TEs with decreased H3K9me2 levels (Supplementary Fig. 17), indicating that OsINO80 is associated with the repression of TE activation, consistent with its role in promoting H3K9me2 deposition.

To examine whether the activation of TEs in the osino80-2 mutant results in TE transposition, we performed a DNA sequencing analysis (Supplementary Table 4). To avoid the possibility of tissue culture-induced TE transpositions^48,49,50, a previously reported transgenic rice line (COM1)⁵¹ was used as a control. The transposition events of 579 upregulated TEs in the osino80-2 mutant were examined to determine whether transposon transcriptional activation leads to transposition. A total of 12 putative transposition events involving TEs from four families (MULE-MuDR, Gypsy, Helitron and TcMar-Stowaway) were identified in the osin80-2 mutant (Fig. 4f and Supplementary Table 5), reflecting the contribution of OsINO80 to the activation of TE transpositions. One out of twelve putative TE transposition events were confirmed by PCR (Fig. 4g). Together, these results indicate that OsINO80 contributes to maintaining TE silencing, possibly by promoting H3K9me2 deposition in rice.

Discussion

Chromatin remodeling complexes are conserved in various species (e.g., from yeast to mammals). Their multiple functions related to the modification of chromatin structures benefit from the functional diversity of different complexes and multiple components of each complex⁵². Although many of the components and broad functions of chromatin remodeling complexes have recently been characterized, the distributions of these complexes throughout the genome remain relatively unknown. In the current study, we focused on the catalytic subunit of the INO80 complex in rice and examined the distribution of OsINO80 in the genome. We also determined that OsINO80 promotes the establishment of H3K27me3, but not H2A.Z, within euchromatin. More importantly, we revealed that OsINO80 promotes H3K9me2 deposition within heterochromatic loci.

Many studies have confirmed the association between INO80 and H2A.Z, but in contrast to the conserved function of the SWR1 complex, which incorporates H2A.Z in yeast, animal, and plant genomes, there is controversy regarding the potential effects of INO80 on H2A.Z deposition/eviction. In this study, we showed that H2A.Z is more enriched in T1 OsINO80-occupied regions than in random control genes. Additionally, the loss of OsINO80 does not lead to obvious H2A.Z changes within the T1 regions, indicating that OsINO80 does not directly regulate the deposition or eviction of H2A.Z in rice. Unlike our findings in rice, the deletion of mouse INO80 in the primed pluripotent stem cells and spermatocytes results in a clear decrease in H2A.Z levels at INO80-binding sites^15,16, and the H2A.Z levels in the random H2A.Z-enriched regions lacking INO80 also decreased following the deletion of INO80 in mouse (Supplementary Fig. 18). Whether the differences in these observations are due to the diversity in the functions of INO80 or H2A.Z in plants and animals, together with the effect of OsINO80 on H2A.Z eviction/deposition under environmental stresses, need to be determined in future investigations.

Our ChIP-seq analysis of the osino80-2 mutant and the WT control indicated that the loss of OsINO80 leads to a significant decrease in H3K27me3 levels, but a slight increase in H3K4me3 levels, at the OsINO80-occupied regions within euchromatin (T1 regions) (Fig. 3a), which is inconsistent with the findings of earlier studies that showed AtINO80 promotes H3K4me3 establishment^21,25 and inhibits H3K27me3 deposition¹⁸ in Arabidopsis, but is consistent with the results of other studies that indicated INO80 promotes H3K27me3 establishment by recruiting the PRC2 complex in mouse^15,16. Earlier research in animals demonstrated that INO80 can interact with SUZ12, but not with the catalytic subunit of Enhancer of Zeste Homolog 2 (EZH2) in the PRC2 complex^16,53. Therefore, the mechanism underlying the positive effects of INO80 on H3K27me3 establishment in plants remains to be investigated.

Notably, we observed that OsINO80 facilitates the deposition of H3K27me3 in euchromatin and the deposition of H3K9me2 in heterochromatic loci. The T2 OsINO80-occupied regions were enriched with H3K9me2 and methylated DNA (Fig. 1). There are very few reports describing the relationship between INO80 and heterochromatin. For example, in yeast, SILENT INFORMATION REGULATOR3 (SIR3) binds to heterochromatin regions and helps to recruit ACTIN-RELATED PROTEIN 5 (ARP5), which is a subunit of the INO80 complex, to prevent DISRUPTOR OF TELOMERIC SILENCING 1 (Dot1) from methylating H3K79²⁶. Our reanalysis of the ChIP-seq data for INO80 in the naïve and primed mouse pluripotent stem cells^15,16 suggested that a class of mouse INO80-binding sites is enriched with the heterochromatin marker H3K9me3 (Supplementary Fig. 18a). Hence, INO80 may also bind to heterochromatic loci in animals. In addition, Arabidopsis proteins that may interact with INO80 were recently identified^21,25. The possible interacting proteins detected via affinity purification and a mass spectrometry-based analysis²⁵ included the histone methyltransferase SU(VAR)2 (SUVR2). The Medicago truncatula homolog of this protein (MtSUVR2) reportedly functions as a methyltransferase that converts H3K9me1 to H3K9me2/3 in vitro⁵⁴. Therefore, the regulation of H3K9me2 distribution by OsINO80 within heterochromatic regions may be mediated by H3K9 histone methyltransferases. In addition, high density of nucleosomes promotes the deposition of H3K27me3 and H3K9me3^55,56. Therefore, OsINO80 could possibly affect the establishment of H3K27me3 and H3K9me2 by regulating the nucleosome density in rice.

The knockdown of OsINO80 resulted in decreased H3K9me2 levels in heterochromatic loci and the activation of a set of TEs, which is in accordance with the decreased fertility of the osino80-2 mutant. The substantial activation of TEs may help to explain the embryonic lethality of the OsINO80-knockout mutant²². Chromatin remodeling complexes regulate TEs in both animal and plant species^37,57. A recent study on Arabidopsis showed that DDM1 mediates TE silencing by depositing H2A.W in heterochromatin⁵⁸. We observed that the specific DDM1 residues involved in the binding to H2A.W are conserved in OsINO80 (Supplementary Fig. 19), suggesting that OsINO80 and DDM1 may similarly regulate TE silencing. Transposable elements play key roles in genome function and evolution. Moreover, TE silencing is largely maintained by epigenetic mechanisms. Thus, the novel finding that OsINO80 maintains TE silencing and promotes H3K9me2 establishment is important for future investigations conducted to elucidate the epigenetic mechanisms underlying the coordinated effects of chromatin remodeling, histone methylation, and DNA methylation that maintain genome integrity.

Methods

Plant materials and growth conditions

The OsINO80 knockdown mutant and transgenic plants overexpressing OsINO80-YFP in the Oryza sativa. L. ssp. japonica cv. Nipponbare background used in this study have been described²². Rice plants were grown in paddies under natural conditions at the following two locations with different latitudes: Shanghai under long-day conditions and Sanya under short-day conditions. In addition, 14-day-old rice seedlings grown on agar-solidified Murashige & Skoog (MS) medium (M0222; Duchefa, Haarlem, the Netherlands) containing 3% w/v sucrose in artificial growth chambers under short-day conditions (10-h light at 30 °C/14-h dark at 28 °C) were used for the RNA-seq, ChIP-seq, BS-seq, and DNA-seq analyses as well as for DNA extractions.

PCR assay

To validate new transposon insertion sites, genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen). The PCR amplifications were performed using a transposon-specific primer and a primer flanking the new insertion site (Supplementary Table 6). All PCR amplifications were completed using the Ex-Taq enzyme (TaKaRa, Japan).

Real-time quantitative PCR

Total RNA was extracted from 100 mg of 14-day-old whole seedlings tissue with an RNAprep pure Plant Kit (Tiangen Biotech, Beijing, China). 1 ug RNA was used for the synthesis of cDNA with PrimeScript^TM RT reagent Kit (TaKaRa, Japan). qRT-PCR reactions were carried out on CFX_Connect^TM (BIO-RAD) with TB Green^TM Premix Ex Taq^TM II (TaKaRa, Japan). Primers are listed in Supplementary Table 6. Each group data comprised three biological replicates and Ubiquitin (LOC_Os01g22490) was used as a reference gene to normalize the gene transcription data.

RNA sequencing and data analysis

For the RNA-seq analysis, total RNA was extracted from 100 mg of 14-day-old seedlings using the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China). Three independent biological replicates were prepared per line. The strand-specific RNA-seq libraries were constructed according to the KAPA Stranded mRNA-seq Kit instructions (Illumina® Platforms, KR0960-v5.17, Kapa Biosystems, Wilmington, MA, USA). The libraries were sequenced on the Illumina Novaseq 6000 instrument at GENERGY BIO (Shanghai, China).

Reads containing adapters and low-quality reads (q < 20) were trimmed using Cutadapt (v3.5)⁵⁹. HISAT2 (v2.1.0) was used to align the trimmed reads to the rice reference genome (MSU7)⁶⁰. High-quality reads were obtained using SAMtools (v1.9)⁶¹. The number of reads for each gene was calculated using featureCounts (v2.0.1)⁶². The DESeq2 (v1.34.0) program⁶³ and the following criteria were used to identify differentially expressed genes: |log₂(fold-change)| ≥ log₂(1.5) and p ≤ 0.05. Differentially expressed TEs were identified using TEtranscripts (v2.2.3) and the following criteria: |log₂(fold-change)| ≥ log₂(1.5) and padj ≤ 0.05⁶⁴.

ChIP sequencing and data analysis

2 g of 14-day-old rice seedlings grown under short-day conditions were used for ChIP-seq assay. Plants were fixed in a buffer consisting of 0.4 M sucrose, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% v/v formaldehyde, and 1 mM PMSF. Nuclei were extracted using a buffer comprising 0.25 M sucrose, 15 mM PIPES (pH 6.8), 5 mM MgCl₂, 60 mM KCl, 15 mM NaCl, 1 mM CaCl₂, 0.9% v/v Triton X-100, and a protease inhibitor cocktail. Chromatin in lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1% w/v SDS, 0.1% w/v Na deoxycholate, 1% v/v Triton X-100, and a protease inhibitor cocktail was sonicated to generate DNA fragments (200–500 bp). The immunoprecipitation was performed using the following antibodies: anti-GFP (ab290, Abcam; 1:300 dilution), anti-H2A.Z²² (1:100 dilution), anti-H2A⁶⁵ (1:100 dilution), anti-H3K9me2 (ab1220, Abcam; 1:300 dilution), anti-H3K4me2 (07-030, Millipore; 1:200 dilution), anti-H3K4me3 (ab8580, Abcam; 1:300 dilution), anti-H3K27me3 (A22396, ABclonal; 1:50 dilution), anti-H2Aub (8240, Cell Signaling; 1:100 dilution), anti-H3K36me3 (ab9050, Abcam; 1:300 dilution), and anti-H3 (ab1791, Abcam; 1:300 dilution). The supernatant was incubated with Protein A beads at 4 °C for 1 h. The beads were sequentially washed using the following buffers: low-salt buffer [50 mM HEPES (pH 7.5), 1 mM EDTA, 150 mM NaCl, and 0.1% v/v Triton X-100], high-salt buffer [50 mM HEPES (pH 7.5), 1 mM EDTA, 500 mM NaCl, and 0.1% v/v Triton X-100], LiCl buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% v/v NP-40, and 0.25 M LiCl], and TE buffer [10 mM Tris-HCl (pH 8.0) and 1 mM EDTA]. The immunoprecipitated complex was then eluted twice using elution buffer (1% w/v SDS and 0.1 M NaHCO₃). The supernatant was incubated overnight at 65 °C (reverse cross-linking) and then treated with RNase A (TaKaRa) and proteinase K (Invitrogen) to degrade RNA and proteins. The obtained DNA fragments were then extracted to construct the ChIP-seq library using the VAHTSTM Universal DNA Library Prep Kit (Vazyme). The ChIP-seq libraries for OsINO80, H2A.Z, H2A, H3, H3K4me2, H3K4me3, H3K9me2, H3K27me3, H2Aub, and H3K36me3 were sequenced on the Illumina Novaseq 6000 or Illumina HiSeq 2000 instrument to generate 150-bp reads at GENERGY BIO (Shanghai, China). For OsINO80-YFP, WT-YFP, H3, H2A.Z, H3K4me2, H3K4me3, H3K36me3, and H2Aub ChIP-seq experiments, two replicates were performed along with DNA input as ChIP-seq control. For H3K27me3 and H3K9me2 ChIP-seq experiments, three replicates were performed. For pUBI::YFP ChIP-seq experiments, four replicates were performed.

Adapters and low-quality reads (q < 20) were removed using Cutadapt (v3.5)⁵⁹. For the ChIP-seq reads of OsINO80, H2A.Z, H2A, H3, H3K9me2, H3K4me2, H3K4me3, H3K27me3, H2Aub, and H3K36me3, we used Bowtie2 (v2.4.5)⁶⁶ to map the clean reads. SAMtools (v1.9)⁶¹ was used to sort the reads and remove potential PCR duplicates. SICER (v1.1)⁶⁷ was used to call peaks on the basis of a comparison of the IP and the input (FDR ≤ 0.05). Reads were normalized as reads per kilobase per million mapped reads (RPKM) in 10-bp windows using the bamCoverage tool of deepTools (v3.5.1)⁶⁸. The data in the bigWig format files were visualized using Integrative Genomics Viewer⁶⁹. The enrichment signals around the gene body, transcription start site (TSS), and transcription termination site (TTS) were detected using the computeMatrix tool of deepTools. The resulting matrix of the windowed coverage was used to generate a mean profile in R (v4.2.1) or a heatmap using plotHeatmap in deepTools, in which each row represents a single gene or peak. The genes corresponding to the detected peaks were annotated using ChIPpeakAnno (v3.28.1)⁷⁰. The distribution of the peaks was determined using ChIPseeker (v1.32.1)⁷¹. MAnorm (v.1.3.0)⁷² was used in quantitative comparison of ChlP-Seq data sets. Enrichment analysis was employed to investigate the correlation between changes in H2A.Z/histone modifications and changes in gene transcription by using a published procedure⁴³.

Whole-genome bisulfite sequencing and data analysis

Genomic DNA was extracted from 14-day-old rice seedlings using the DNeasy Plant Maxi kit (Qiagen). The sodium bisulfite treatment, library construction, and sequencing were completed by GENERGY BIO (Shanghai, China). For each genotype, four replicates were obtained and sequenced for BS-seq and two replicates were performed for DNA-seq.

Cutadapt (v3.5)⁵⁹ was used to trim the paired-end reads to obtain high-quality reads (q ≥ 20) with no adapters. Bismark (v0.22.1) was used to map the filtered reads to the rice reference genome (MSU7) and to identify the regions with methylated DNA on the basis of the filtered mapping results⁷³. The DNA methylation levels were determined and visualized using ViewBS (v0.1.9)⁷⁴.

Examination of transposable element insertion

Transposable elements were analyzed according to the procedure⁷⁵. Cutadapt (v.3.5)⁵⁹ was used to remove adapters and low-quality reads (q < 20). Bowtie2 (v.2.4.5)⁶⁶ was used to map the clean reads to the rice reference genome (MSU7) with the parameter “--very-sensitive”. Rice TEs were annotated using the default settings of RepeatMasker (http://www.repeatmasker.org/)⁴⁶. The TE insertion events were detected using SPLITREADER⁷⁶. The TE insertion events with 3–20 target site duplications were selected. The events that occurred only in the osino80-2 mutant were analyzed and further validated.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The raw and processed RNA-seq, ChIP-seq, and BS-seq data generated in this study have been deposited in the NCBI GEO database [https://www.ncbi.nlm.nih.gov/gds/] with accession numbers GSE225485, GSE225484, and GSE225483, respectively. DNA-seq data in this study have been deposited in the Sequence Read Archive (SRA) database [https://www.ncbi.nlm.nih.gov/sra/] and are available with the following accession code: PRJNA1093549. Source data are provided with this paper.

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Acknowledgements

We thank Drs Guodong Ren and Chengguo Duan and Jun Zhu for critically reading the manuscript. We thank Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the English text of a draft of this manuscript. This work was supported by the National Natural Science Foundation of China (grants NSFC31930017 and 32100453), China Postdoctoral Science Foundation (2023M732505), Postdoctoral Fellowship Program of CPSF (GZB20230498), and Sichuan Science and Technology Program (2024NSFSC0333).

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These authors contributed equally: Kangxi Du, Jiabing Wu, Jiachen Wang.

Authors and Affiliations

State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, PR China
Kangxi Du, Jiabing Wu, Jiachen Wang, Wenhao Xie, Liufan Yin, Xiang Li, Chao Li & Aiwu Dong
State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Rice Research Institute, Sichuan Agricultural University, Chengdu, PR China
Kangxi Du

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Kangxi Du
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Jiabing Wu
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A.D. conceived and designed the research. A.D. supervised the experiments and analysis. K.D., J. Wu, and C.L. performed the experiments. K.D., J. Wang, L.Y., W.X., and X.L. analyzed the data. K.D. and A.D. wrote the manuscript. All authors read, revised, and approved the manuscript.

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Correspondence to Aiwu Dong.

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Du, K., Wu, J., Wang, J. et al. The chromatin remodeling factor OsINO80 promotes H3K27me3 and H3K9me2 deposition and maintains TE silencing in rice. Nat Commun 15, 10919 (2024). https://doi.org/10.1038/s41467-024-55387-4

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Received: 23 March 2023
Accepted: 05 December 2024
Published: 30 December 2024
DOI: https://doi.org/10.1038/s41467-024-55387-4