H3K9 methylation regulates heterochromatin silencing through incoherent feedforward loops.

Histone H3 lysine-9 methylation (H3K9me) is a hallmark of the condensed and transcriptionally silent heterochromatin. It remains unclear how H3K9me controls transcription silencing and how cells delimit H3K9me domains to avoid silencing essential genes. Here, using Arabidopsis genetic systems that induce H3K9me2 in genes and transposons de novo, we show that H3K9me2 accumulation paradoxically also causes the deposition of the euchromatic mark H3K36me3 by a SET domain methyltransferase, ASHH3. ASHH3-induced H3K36me3 confers anti-silencing by preventing the demethylation of H3K4me1 by LDL2, which mediates transcriptional silencing downstream of H3K9me2. These results demonstrate that H3K9me2 not only facilitates but orchestrates silencing by actuating antagonistic silencing and anti-silencing pathways, providing insights into the molecular basis underlying proper partitioning of chromatin domains and the creation of metastable epigenetic variation.

H3K4me1 recruits DNA repair proteins in plants.

DNA repair proteins can be recruited by their histone reader domains to specific epigenomic features, with consequences on intragenomic mutation rate variation. Here, we investigated H3K4me1-associated hypomutation in plants. We first examined 2 proteins which, in plants, contain Tudor histone reader domains: PRECOCIOUS DISSOCIATION OF SISTERS 5 (PDS5C), involved in homology-directed repair, and MUTS HOMOLOG 6 (MSH6), a mismatch repair protein. The MSH6 Tudor domain of Arabidopsis (Arabidopsis thaliana) binds to H3K4me1 as previously demonstrated for PDS5C, which localizes to H3K4me1-rich gene bodies and essential genes. Mutations revealed by ultradeep sequencing of wild-type and msh6 knockout lines in Arabidopsis show that functional MSH6 is critical for the reduced rate of single-base substitution (SBS) mutations in gene bodies and H3K4me1-rich regions. We explored the breadth of these mechanisms among plants by examining a large rice (Oryza sativa) mutation data set. H3K4me1-associated hypomutation is conserved in rice as are the H3K4me1-binding residues of MSH6 and PDS5C Tudor domains. Recruitment of DNA repair proteins by H3K4me1 in plants reveals convergent, but distinct, epigenome-recruited DNA repair mechanisms from those well described in humans. The emergent model of H3K4me1-recruited repair in plants is consistent with evolutionary theory regarding mutation modifier systems and offers mechanistic insight into intragenomic mutation rate variation in plants.

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 significantly attenuated in the ldl3 mutant, demonstrating the genome-wide impacts of the transcription-driven LDL3 pathway to control H3K4me2 in plants. Our findings implicate H3K4me2 demethylation in plants as chromatin records of transcriptional activity, which ensures robust gene control.

Identification of ecdysone receptor target genes in the worker honey bee brains during foraging behavior.

Ecdysone signaling plays central roles in morphogenesis and female ovarian development in holometabolous insects. In the European honey bee (Apis mellifera L.), however, ecdysone receptor (EcR) is expressed in the brains of adult workers, which have already undergone metamorphosis and are sterile with shrunken ovaries, during foraging behavior. Aiming at unveiling the significance of EcR signaling in the worker brain, we performed chromatin-immunoprecipitation sequencing of EcR to search for its target genes using the brains of nurse bees and foragers. The majority of the EcR targets were common between the nurse bee and forager brains and some of them were known ecdysone signaling-related genes. RNA-sequencing analysis revealed that some EcR target genes were upregulated in forager brains during foraging behavior and some were implicated in the repression of metabolic processes. Single-cell RNA-sequencing analysis revealed that EcR and its target genes were expressed mostly in neurons and partly in glial cells in the optic lobes of the forager brain. These findings suggest that in addition to its role during development, EcR transcriptionally represses metabolic processes during foraging behavior in the adult worker honey bee brain.

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 methyltransferase for H3K4me1 has been identified. Here, we identify three Arabidopsis methyltransferases that direct H3K4me1. Analyses of their genome-wide localization using ChIP-seq and machine learning reveal 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 methyltransferases 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.

Chromatin-based mechanisms to coordinate convergent overlapping transcription.

In eukaryotic genomes, the transcription units of genes often overlap with other protein-coding and/or noncoding transcription units1,2. In such intertwined genomes, the coordinated transcription of nearby or overlapping genes would be important to ensure the integrity of genome function3-6; however, the mechanisms underlying this coordination are largely unknown. Here, we show in Arabidopsis thaliana that genes with convergent orientation of transcription are major sources of antisense transcripts and that these genes transcribed on both strands are regulated by a putative Lysine-Specific Demethylase 1 family histone demethylase, FLOWERING LOCUS D (FLD)7,8. Our genome-wide chromatin profiling revealed that FLD, as well as its associating factor LUMINIDEPENDENS9, downregulates histone H3K4me1 in regions with convergent overlapping transcription. FLD localizes to actively transcribed genes, where it colocalizes with elongating RNA polymerase II phosphorylated at the Ser2 or Ser5 sites. Genome-wide transcription analyses suggest that FLD-mediated H3K4me1 removal negatively regulates the transcription of genes with high levels of antisense transcription. Furthermore, the effect of FLD on transcription dynamics is antagonized by DNA topoisomerase I. Our study reveals chromatin-based mechanisms to cope with overlapping transcription, which may occur by modulating DNA topology. This global mechanism to cope with overlapping transcription could be co-opted for specific epigenetic processes, such as cellular memory of responses to the environment10.

VISUAL-CC system uncovers the role of GSK3 as an orchestrator of vascular cell type ratio in plants.

The phloem transports photosynthetic assimilates and signalling molecules. It mainly consists of sieve elements (SEs), which act as "highways" for transport, and companion cells (CCs), which serve as "gates" to load/unload cargos. Though SEs and CCs function together, it remains unknown what determines the ratio of SE/CC in the phloem. Here we develop a new culture system for CC differentiation in Arabidopsis named VISUAL-CC, which almost mimics the process of the SE-CC complex formation. Comparative expression analysis in VISUAL-CC reveals that SE and CC differentiation tends to show negative correlation, while total phloem differentiation is unchanged. This varying SE/CC ratio is largely dependent on GSK3 kinase activity. Indeed, gsk3 hextuple mutants possess many more SEs and fewer CCs, whereas gsk3 gain-of-function mutants partially increase the CC number. Taken together, GSK3 activity appears to function as a cell-fate switch in the phloem, thereby balancing the SE/CC ratio.

Kenyon Cell Subtypes/Populations in the Honeybee Mushroom Bodies: Possible Function Based on Their Gene Expression Profiles, Differentiation, Possible Evolution, and Application of Genome Editing.

Mushroom bodies (MBs), a higher-order center in the honeybee brain, comprise some subtypes/populations of interneurons termed as Kenyon cells (KCs), which are distinguished by their cell body size and location in the MBs, as well as their gene expression profiles. Although the role of MBs in learning ability has been studied extensively in the honeybee, the roles of each KC subtype and their evolution in hymenopteran insects remain mostly unknown. This mini-review describes recent progress in the analysis of gene/protein expression profiles and possible functions of KC subtypes/populations in the honeybee. Especially, the discovery of novel KC subtypes/populations, the "middle-type KCs" and "KC population expressing FoxP," necessitated a redefinition of the KC subtype/population. Analysis of the effects of inhibiting gene function in a KC subtype-preferential manner revealed the function of the gene product as well as of the KC subtype where it is expressed. Genes expressed in a KC subtype/population-preferential manner can be used to trace the differentiation of KC subtypes during the honeybee ontogeny and the possible evolution of KC subtypes in hymenopteran insects. Current findings suggest that the three KC subtypes are unique characteristics to the aculeate hymenopteran insects. Finally, prospects regarding future application of genome editing for the study of KC subtype functions in the honeybee are described. Genes expressed in a KC subtype-preferential manner can be good candidate target genes for genome editing, because they are likely related to highly advanced brain functions and some of them are dispensable for normal development and sexual maturation in honeybees.

Increased complexity of mushroom body Kenyon cell subtypes in the brain is associated with behavioral evolution in hymenopteran insects.

In insect brains, the mushroom bodies (MBs) are a higher-order center for sensory integration and memory. Honeybee (Apis mellifera L.) MBs comprise four Kenyon cell (KC) subtypes: class I large-, middle-, and small-type, and class II KCs, which are distinguished by the size and location of somata, and gene expression profiles. Although these subtypes have only been reported in the honeybee, the time of their acquisition during evolution remains unknown. Here we performed in situ hybridization of tachykinin-related peptide, which is differentially expressed among KC subtypes in the honeybee MBs, in four hymenopteran species to analyze whether the complexity of KC subtypes is associated with their behavioral traits. Three class I KC subtypes were detected in the MBs of the eusocial hornet Vespa mandarinia and the nidificating scoliid wasp Campsomeris prismatica, like in A. mellifera, whereas only two class I KC subtypes were detected in the parasitic wasp Ascogaster reticulata. In contrast, we were unable to detect class I KC subtype in the primitive and phytophagous sawfly Arge similis. Our findings suggest that the number of class I KC subtypes increased at least twice - first with the evolution of the parasitic lifestyle and then with the evolution of nidification.