Biology subjects

Kakutani, T.

Publications and source records attributed to Kakutani, T..

17 recordsLinked to original sources

bioRxiv2025

Antagonistic histone H2A variants and autonomous heterochromatin formation shape epigenomic patterns in Arabidopsis

Heterochromatin formation is pivotal in many eukaryotes with repetitive sequences, such as transposable elements (TEs). However, in plants, where the known de novo DNA methylation mechanism (RdDM) targets euchromatin, how heterochromatin is formed in a region-specific manner remains unclear. We previously reported an RdDM-independent de novo establishment of H3K9me and non-CpG methylation, both of which localize in heterochromatin. Here we show that the mutually exclusive histone H2A variants, H2A.W and H2A.Z, function as guides to initiate heterochromatin formation; H2A.W and H2A.Z promotes and inhibits heterochromatin establishment, respectively, especially in chromosomal arm regions with dispersed TEs. In contrast, pericentromeric TEs demonstrate autonomous heterochromatin formation, less dependently on these H2A variants. Furthermore, H2A.Z protects protein-coding genes from ectopic heterochromatin formation, possibly by preventing its spreading. We propose that the genome indexing mechanism driven by H2A variants, as well as the autonomous formation of pericentromeric heterochromatin, shapes proper epigenomic patterns in Arabidopsis.

genomics↗

bioRxiv2025

MBD8 is required for LDL2-mediated transcriptional repression downstream of H3K9me2 in Arabidopsis

Histone H3 lysine 9 methylation (H3K9me) is a conserved epigenetic mark for transcriptional gene repression. In Arabidopsis, H3K9 dimethylation (H3K9me2) promotes the removal of a transcription-associated mark, H3K4 monomethylation (H3K4me1), and this process within genes requires the histone demethylase LDL2. However, how LDL2 specifically functions in genes that accumulate H3K9me2 remains unknown. Here, we show that methyl-CpG-binding domain protein 8 (MBD8) binds to LDL2 and plays a role in H3K4me1 removal and transcriptional gene repression downstream of H3K9me2. MBD8 preferentially binds GC-poor DNA, regardless of its cytosine methylation status. Interestingly, LDL2 protein levels were significantly reduced in the mbd8 mutant background, suggesting that MBD8 stabilizes the LDL2 protein, facilitating H3K4me1 removal and the transcriptional repression of H3K9me-marked genes. The domain necessary for the MBD8-LDL2 interaction is conserved among plants. We propose that MBD proteins have diverse functions beyond their methylated DNA-binding capabilities.

plant biology↗

bioRxiv2025

H3K4me2 orchestrates H2A.Z and Polycomb repressive marks in Arabidopsis

The dimethylation of histone H3 lysine 4 (H3K4me2) plays an important role in developmental phase transitions in plants, such as regeneration from the callus and the initiation of flowering. H3K4me2 in plants is correlated with transcriptionally repressed states, which can be accounted for by transcription-coupled active H3K4me2 demethylation, but the converse molecular pathway by which H3K4me2 represses transcription remains largely unexplored. Here, we show that H3K4me2 colocalizes with the facultatively repressive marks H2A.Z, H2A ubiquitination (H2Aub), and H3K27me3 in the gene body. Our genetic analyses reveal that H3K4me2 functions upstream but not downstream of these repressive marks. Interestingly, in genes with diurnal H3K4me2 oscillation, H3K4me2 oscillates in antiphase with transcription but in phase with H2A.Z. In addition, the genetic manipulation of H3K4me2 affects the oscillating profiles of H2A.Z, suggesting the efficient relay from H3K4me2 to H2A.Z. Notably, the diurnal oscillation of H2Aub is much weaker than that of H2A.Z despite the overall similarity in their distributions. These results suggest that H3K4me2 orchestrates H2A.Z and H2Aub with distinct dynamics. Our findings reveal that plants employ H3K4me2 to transmit transcriptional cessation into repressive chromatin states.

genetics↗

bioRxiv2025

Natural variation modifies centromere proximal meiotic crossover frequency and segregation distortion in Arabidopsis thaliana

BackgroundCentromeres mediate chromosome segregation during cell division. In plants, centromeres are loaded with CENH3-variant nucleosomes, which direct kinetochore formation and spindle microtubule interaction. Plant centromeres are frequently composed of megabase-scale satellite repeat arrays, or retrotransposon nests. In monocentric genomes, such as Arabidopsis thaliana, extended regions of pericentromeric heterochromatin surround the CENH3-occupied satellite arrays. A zone of suppressed meiotic crossover recombination contains the centromere and extends into the pericentromeres. Here, we explore how Arabidopsis natural genetic variation influences centromere-proximal crossover frequency, and segregation distortion through meiosis, when homologous centromeres are structurally heterozygous. ResultsWe used fluorescent crossover reporters to survey the effect of natural variation on centromere-proximal recombination in twelve F1 hybrids, capturing Arabidopsis Eurasian and relict diversity. The majority of F1 hybrids showed either elevated or suppressed centromere-proximal crossovers (49 of 60), relative to inbreds. We relate hybrid crossover frequencies to patterns of centromeric structural variation, and in a subset of accessions, to epigenetic patterns of CENH3 loading and DNA methylation. The fluorescent reporters also allow segregation distortion through meiosis to be compared between inbred and hybrid strains. We observed a minority of hybrids (18 of 60) with distorted segregation through meiosis compared to inbreds, with and without simultaneous change to centromere-proximal crossover frequency. ConclusionsWe reveal a complex relationship between centromere structural variation, epigenetic state, crossover recombination, and segregation distortion. We propose a model for how Arabidopsis centromere structural heterozygosity may cause segregation distortion during female meiosis.

genetics↗

bioRxiv2023

H3K9me regulates heterochromatin silencing through incoherent feedforward loops

Histone H3 lysine-9 methylation (H3K9me) is associated with condensed and transcriptionally inactive heterochromatin1. Although it has long been known that H3K9me silences transcription to control a wide variety of biological phenomena in many eukaryotic species2,3, how the silencing is regulated under the control of H3K9me is still largely unclear. Moreover, how cells delimit regions with H3K9me to avoid silencing essential genes remains unexplored. Here, using Arabidopsis genetic systems that induce H3K9me2 and its associated non-CG DNA methylation (mCH) in the transcribed region of genes de novo, we show that the accumulation of H3K9me2/mCH paradoxically also leads to the deposition of the euchromatic mark H3K36me3. This induction of H3K36me3 depends on a SET domain methyltransferase, ASHH3, and brings about anti-silencing by preventing the demethylation of H3K4me1 by LDL2, which mediates transcriptional silencing downstream of H3K9me2/mCH4. H3K9me2/mCH-driven antagonistic actions of ASHH3-H3K36me3 and LDL2-H3K4me1-loss also regulate the de novo silencing of reactivated transposable elements (TEs). These results demonstrate that H3K9me2 both facilitates and impedes silencing, and the incoherent feedforward loops fine-tune the fate of genes and TEs. Our results illuminate a novel elaborate mechanism for partitioning chromatin domains and provide insights into the molecular basis underlying natural epigenetic variation.

genetics↗

bioRxiv2023

Retrotransposon addiction promotes centromere function via epigenetically activated small RNAs

Retrotransposons have invaded eukaryotic centromeres in cycles of repeat expansion and purging, but the function of centromeric retrotransposons, if any, has remained unclear. In Arabidopsis, centromeric ATHILA retrotransposons give rise to epigenetically activated short interfering RNAs (easiRNAs) in mutants in DECREASE IN DNA METHYLATION1 (DDM1), which promote histone H3 lysine-9 di-methylation (H3K9me2). Here, we show that mutants which lose both DDM1 and RNA dependent RNA polymerase (RdRP) have pleiotropic developmental defects and mis-segregation of chromosome 5 during mitosis. Fertility defects are epigenetically inherited with the centromeric region of chromosome 5, and can be rescued by directing artificial small RNAs to a single family of ATHILA5 retrotransposons specifically embedded within this centromeric region. easiRNAs and H3K9me2 promote pericentromeric condensation, chromosome cohesion and proper chromosome segregation in mitosis. Insertion of ATHILA silences transcription, while simultaneously making centromere function dependent on retrotransposon small RNAs, promoting the selfish survival and spread of centromeric retrotransposons. Parallels are made with the fission yeast S. pombe, where chromosome segregation depends on RNAi, and with humans, where chromosome segregation depends on both RNAi and HELLSDDM1.

molecular biology↗

bioRxiv2023

Molecular and structural basis of the heterochromatin-specific chromatin remodeling activity by Arabidopsis DDM1

The chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1) deposits the histone H2A variant H2A.W and silences transposons in Arabidopsis thaliana. However, the molecular mechanisms by which DDM1 specifically targets the nucleosome containing H2A.W and allows chromatin writers to access heterochromatin remained elusive. Here, we show that DDM1 promotes remodeling of the H2A.W nucleosome and requires interactions with the H2A.W-specific C-terminal tail. The cryo-EM structure of the DDM1-H2A.W nucleosome complex revealed that DDM1 binds to the N-terminal tail of H4 and the nucleosomal DNA. Comparison with the cryo-EM structure of the nucleosome containing H2A.W suggested that DDM1 increases the DNA end flexibility of nucleosomes. Based on these biochemical and structural results, we propose that the chromatin remodeling activity of DDM1 with the heterochromatin-specific H2A.W contributes to the maintenance of repressive epigenetic marks in heterochromatin by providing DNA methyltransferases with access to nucleosomal DNA.

molecular biology↗

bioRxiv2023

Cotranscriptional demethylation induces global loss of H3K4me2 from active genes in Arabidopsis

Based on studies of animals and yeasts, methylation of histone H3 lysine 4 (H3K4me1/2/3, for mono-, di-, and tri-methylation, respectively) is regarded as the key epigenetic modification of transcriptionally active genes. In plants, however, H3K4me2 correlates negatively with transcription, and the regulatory mechanisms of this counterintuitive H3K4me2 distribution in plants remain largely unexplored. A previous genetic screen for factors regulating plant regeneration identified Arabidopsis LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 3 (LDL3), which is a major H3K4me2 demethylase. Here, we show that LDL3-mediated H3K4me2 demethylation depends on the transcription elongation factor Paf1C and phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). In addition, LDL3 binds to phosphorylated RNAPII. These results suggest that LDL3 is recruited to transcribed genes by binding to elongating RNAPII and demethylates H3K4me2 cotranscriptionally. Importantly, the negative correlation between H3K4me2 and transcription is disrupted in the ldl3 mutant, demonstrating the genome-wide impacts of the transcription-driven LDL3 pathway to control H3K4me2 in plants. Our findings implicate H3K4me2 in plants as chromatin memory for transcriptionally repressive states, which ensures robust gene control.

plant biology↗

bioRxiv2022

The arms race in noncoding regions of transposable element: the evolution of anti-silencing and RNAi

Transposable elements (TEs) are among the most dynamic parts of genomes. Since TEs are potentially deleterious, eukaryotes silence them through epigenetic mechanisms such as DNA methylation and RNAi. We have previously reported that Arabidopsis TEs, called VANDALs, counteract epigenetic silencing through a group of sequence-specific anti-silencing proteins, VANCs. VANC proteins bind to noncoding regions of specific VANDAL copies and induce a loss of silent chromatin marks. Sequence-specific anti-silencing allows these TEs to proliferate with minimum host damage. Here, we show that RNAi efficiently targets noncoding regions of VANDAL TEs to silence them de novo. Target motifs of VANC, in turn, evolved to escape RNAi. Escaping RNAi could be the primary event leading to the differentiation of sequence-specific anti-silencing systems. We propose that this selfish behaviour of TEs paradoxically could make them less harmful to the host.

genetics↗

bioRxiv2022

Repetitive DNA promotes RNAi-mediated heterochromatin formation via an anti-silencing factor in fission yeast.

In most eukaryotes, constitutive heterochromatin, defined by histone H3 lysine 9 methylation (H3K9me), is enriched on repetitive DNA, such as pericentromeric repeats and transposons. Furthermore, repetitive transgenes also induce heterochromatin formation in diverse model organisms. However, the mechanisms that promote heterochromatin formation at repetitive DNA elements are still not clear. Here, using fission yeast, we show that tandemly repeated mRNA genes promote RNA interference (RNAi)-mediated heterochromatin formation in co-operation with an anti-silencing factor, Epe1. Although the presence of tandemly repeated genes itself does not cause heterochromatin formation, once complementary small RNAs are artificially supplied in trans, the RNAi machinery assembled on the repeated genes starts producing cognate small RNAs in cis to autonomously maintain heterochromatin at these sites. The establishment of this "repeat-induced RNAi" depends on the copy number of repeated genes and also requires Epe1, which is known to remove H3K9me and derepress the transcription of genes underlying heterochromatin. Analogous to repeated genes, the DNA sequence underlying constitutive heterochromatin encodes widespread transcription start sites (TSSs), from which Epe1 activates ncRNA transcription to promote RNAi-mediated heterochromatin formation. Our results suggest that, when repetitive transcription units underlie heterochromatin, Epe1 generates sufficient transcripts for the activation of RNAi without disruption of heterochromatin.

biochemistry↗

bioRxiv2022

The histone variant H2A.W cooperates with chromatin modifications and linker histone H1 to maintain transcriptional silencing of transposons in Arabidopsis

Transposable elements (TEs) are marked by a complex array of chromatin modifications, but a central unifying mechanism for how they are silenced remains elusive. Histone H3 Lysine 9 methylation (H3K9me) is an important component of heterochromatin in most eukaryotes, including plants. In flowering plants, the specialized histone variant H2A.W occupies nucleosomes found at TE sequences. This variant is deposited by the chromatin remodeler DDM1 and confers specific biophysical properties to the nucleosomes. Here we use genetic and genomic strategies to evaluate the role of H2A.W in transposon silencing in Arabidopsis. Compared with mutants lacking either H2A.W or H3K9me, the combined loss of both H2A.W and H3K9me causes a dramatic increase in both the number of expressed TEs and their expression levels. Synergistic effects are also observed when H2A.W is lost in combination with histone H1 or CH methylation. Collectively, these TEs are also upregulated in mutants lacking DDM1, which are impaired in H2A.W deposition and lose heterochromatic marks. We conclude that H2A.W acts in combination with different elements of heterochromatin to maintain silencing across a large spectrum of TEs present primarily in pericentric heterochromatin in Arabidopsis. In mammals, the DDM1 ortholog LSH deposits macroH2A to heterochromatin and silences TEs. We thus propose that specialized H2A variants localized to heterochromatin interact with a complex array of histone modifications to silence TEs in eukaryotes.

genomics↗

bioRxiv2021

Local and global crosstalk among heterochromatin marks drives epigenome patterning in Arabidopsis

Transposable elements (TEs) are robustly silenced by targeting of multiple epigenetic marks, but dynamics of crosstalk among these marks remains enigmatic. In Arabidopsis, TEs are silenced by cytosine methylation in both CpG and non-CpG contexts (mCG and mCH) and histone H3 lysine 9 methylation (H3K9me). While mCH and H3K9me are mutually dependent for their maintenance, mCG and mCH/H3K9me are independently maintained. Here we show that establishment, rather than maintenance, of mCH depends on mCG, accounting for the synergistic colocalization of these silent marks in TEs. When mCG is lost, establishment of mCH is abolished in TEs. mCG also guides mCH in active genes, although genic mCH/H3K9me is removed there. Unexpectedly, the targeting efficiency of mCH depends on relative, rather than absolute, levels of mCG, suggesting underlying global negative controls. We propose that the local positive feedback in heterochromatin dynamics, together with global negative feedback, drive robust and balanced epigenome patterning.

genomics↗

bioRxiv2021

Transcription-coupled and epigenome-encoded mechanisms direct H3K4 methylation

Mono-, di-, and trimethylation of histone H3 lysine 4 (H3K4me1/2/3) are associated with transcription, yet it remains controversial whether H3K4me1/2/3 promote or result from transcription. Our previous characterizations of Arabidopsis H3K4 demethylases suggest roles for H3K4me1 in transcription. However, the control of H3K4me1 remains unexplored in Arabidopsis, in which no methylase for H3K4me1 has been identified. Here, we identified three Arabidopsis methylases that direct H3K4me1. Analyses of their genome-wide localization using ChIP-seq and machine learning revealed that one of the enzymes cooperates with the transcription machinery, while the other two are associated with specific histone modifications and DNA sequences. Importantly, these two types of localization patterns are also found for the other H3K4 methylases in Arabidopsis and mice. These results suggest that H3K4me1/2/3 are established and maintained via interplay with transcription as well as inputs from other chromatin features, presumably enabling elaborate gene control.

molecular biology↗

bioRxiv2021

The genetic and epigenetic landscape of the Arabidopsis centromeres

Centromeres attach chromosomes to spindle microtubules during cell division and, despite this conserved role, show paradoxically rapid evolution and are typified by complex repeats. We used ultra-long-read sequencing to generate the Col-CEN Arabidopsis thaliana genome assembly that resolves all five centromeres. The centromeres consist of megabase-scale tandemly repeated satellite arrays, which support high CENH3 occupancy and are densely DNA methylated, with satellite variants private to each chromosome. CENH3 preferentially occupies satellites with least divergence and greatest higher-order repetition. The centromeres are invaded by ATHILA retrotransposons, which disrupt genetic and epigenetic organization of the centromeres. Crossover recombination is suppressed within the centromeres, yet low levels of meiotic DSBs occur that are regulated by DNA methylation. We propose that Arabidopsis centromeres are evolving via cycles of satellite homogenization and retrotransposon-driven diversification. One-sentence summaryLong read sequencing and assembly of the Arabidopsis centromeres reveals their genetic and epigenetic topography.

genomics↗

bioRxiv2020

Removal of repressive histone marks creates epigenetic memory of recurring heat in Arabidopsis

Acclimation to high temperature increases plants tolerance of subsequent lethal high temperatures1-3. Although epigenetic regulation of plant gene expression is well studied, how plants maintain a memory of environmental changes over time remains unclear. Here, we show that JUMONJI (JMJ) proteins4-8, demethylases involved in histone H3 lysine 27 trimethylation (H3K27me3), are necessary for Arabidopsis thaliana heat acclimation. Acclimation induces sustained H3K27me3 demethylation at key HEAT SHOCK PROTEIN (HSP) loci by JMJs, poising the HSP genes for subsequent activation. Upon sensing heat after a 3-day interval, JMJs directly reactivate HSP genes. Finally, jmj mutants fail to maintain heat memory under fluctuating field temperature conditions. Our findings of an epigenetic memory mechanism involving histone demethylases may have implications for environmental adaptation of field plants.

plant biology↗

bioRxiv2020

Chromatin-based mechanisms to coordinate convergent overlapping transcription

In eukaryotic genomes, transcription units of genes often overlap with other protein-coding and/or noncoding transcription units1,2. In such intertwined genomes, coordinated transcription of nearby or overlapping genes would be important to ensure integrity of genome function; however, the mechanisms underlying this coordination are largely unknown3-6. Here, we show in Arabidopsis thaliana that genes with convergent orientation of transcription are major sources of overlapping bidirectional transcripts and that these bidirectionally transcribed genes are regulated by a putative LSD1 family histone demethylase, FLD7,8. Our genome-wide chromatin profiling revealed that FLD downregulated histone H3K4me1 in regions with convergent overlapping transcription. FLD localizes to actively transcribed genes where it colocalizes with elongating RNA polymerase II phosphorylated at Ser-2 or Ser-5 sites. Genome-wide transcription analyses suggest that FLD-mediated H3K4me1 removal negatively regulates bidirectional transcription by retaining the elongating transcription machinery. Furthermore, this effect of FLD on transcription dynamics is mediated by DNA topoisomerase I. Our study has revealed chromatin-based mechanisms to cope with overlapping bidirectional transcription, likely by modulating DNA topology. This global mechanism to cope with bidirectional transcription could be co-opted for specific epigenetic processes, such as cellular memory of responses to environment9.

molecular biology↗

bioRxiv2019

Seasonal stability and dynamics of DNA methylation in plants in a natural environment

Organisms survive in naturally fluctuating environments by responding to long-term signals, such as seasonality, by filtering out short-term noise. DNA methylation has been considered a stable epigenetic mark but has also been reported to change in response to experimental manipulations of biotic and abiotic factors. However, it is unclear how they behave in natural environments. Here, we analyzed seasonal patterns of genome-wide DNA methylation at a single-base resolution using a single clone from a natural population of the perennial Arabidopsis halleri. The genome-wide pattern of DNA methylation was primarily stable, and most of the repetitive regions were methylated across the year. Although the proportion was small, we detected seasonally methylated cytosines (SeMCs) in the genome. SeMCs in the different contexts showed distinct seasonal patterns of methylation. SeMCs in CHH context were detected predominantly at repetitive sequences in intergenic regions. Additionally, we found that CHH methylation within AhgFLC locus showed a seasonal pattern that was negatively associated with changes in gene expression. Gene-body CG methylation (gbM) itself was generally stable across seasons, but the levels of gbM were positively associated with seasonal stability of RNA expression of the genes. These results suggest the existence of two distinct aspects of DNA methylation in natural environments: sources of epigenetic variation and epigenetic marks for stable gene expression.

plant biology↗