Biology subjects

Kimura, H.

Publications and source records attributed to Kimura, H..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

Escaping from multiple visual threats: Modulation of escape responses in Pacific staghorn sculpin (Leptocottus armatus).

Fish perform rapid escape responses to avoid sudden predatory attacks. During escape responses, fish bend their bodies into a C-shape and quickly turn away from the predator and accelerate. The escape trajectory is determined by the initial turn (Stage 1) and a contralateral bend (Stage 2). Previous studies have used a single threat or model predator as a stimulus. In nature, however, multiple predators may attack from different directions simultaneously or in close succession. It is unknown whether fish are able to change the course of their escape response when startled by multiple stimuli at various time intervals. Pacific staghorn sculpin (Leptocottus armatus) were startled with a left and right visual stimulus in close succession. By varying the timing of the second stimulus, we were able to determine when and how a second stimulus could affect the escape response direction. Four treatments were used: a single visual stimulus (control); or two stimuli coming from opposite sides separated by a 0 ms (simultaneous treatment); a 33 ms; or a 83 ms time interval. The 33 ms and 83 ms time intervals were chosen to occur shortly before and after a predicted 60 ms visual escape latency (i.e. during Stage 1). The 0 ms and 33 ms treatments influenced both the escape trajectory and the Stage 1 turning angle, compared to a single stimulation, whereas the 83 ms treatment had no effect on the escape response. We conclude that Pacific staghorn sculpin can modulate their escape response only between stimulation and the onset of the response, but that escape responses are ballistic after the body motion has started. SUMMARY STATEMENTUsing double stimulation from opposite sides at different time intervals to simulate coordinated predatory attacks, Pacific staghorn sculpin escape away from the first stimulus, but were unable to turn away from the second stimulus while the escape response was in progress.

animal behavior and cognition↗

bioRxiv2021

Visualizing looping of two endogenous genomic loci using synthetic zinc-finger proteins with anti-FLAG and anti-HA frankenbodies in living cells

In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here we demonstrate a new method to visualize endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps. First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously visualize the dynamics of the two loci in single living cells. This reveals close association between the two loci in the majority of cells, but the loci markedly separate upon the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof-of-principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.

cell biology↗

bioRxiv2021

A Drosophila Toolkit for Imaging of HA-tagged Proteins Unveiled a Block in Autophagy Flux in the Last Instar Larval Fat Body

For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with POI harboring different tags are needed but generation of these strains is still labor-intensive work. To overcome this, we developed a versatile Drosophila toolkit with a genetically encoded single-chain variable fragment for the HA epitope tag: "HA Frankenbody". This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Autophagy was dispensable for the swelling of lysosomes, indicating that lysosomal activity is downregulated at this stage. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, we propose that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.

cell biology↗

bioRxiv2021

Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry

DNA replication is a key step in initiating cell proliferation. Loading hexameric complexes of minichromosome maintenance (MCM) helicase onto DNA replication origins during the G1 phase is essential for initiating DNA replication. Here, we examined MCM hexamer states during the cell cycle in human hTERT-RPE1 cells using multicolor immunofluorescence-based, single-cell plot analysis, and biochemical size fractionation. Experiments involving cell-cycle arrest at the G1 phase and release from the arrest revealed that a double MCM hexamer was formed via a single hexamer during G1 progression. A single MCM hexamer was recruited to chromatin in the early G1 phase. Another single hexamer was recruited to form a double hexamer in the late G1 phase. We further examined relationship between the MCM hexamer states and the methylation levels at lysine 20 of histone H4 (H4K20) and found that the double MCM hexamer state was correlated with di/trimethyl-H4K20 (H4K20me2/3). Inhibiting the conversion from monomethyl-H4K20 (H4K20me1) to H4K20me2/3 retained the cells in the single MCM hexamer state. Non-proliferative cells, including confluent cells or Cdk4/6 inhibitor-treated cells, also remained halted in the single MCM hexamer state. We propose that the single MCM hexamer state is a halting step in the determination of cell cycle progression.

cell biology↗

bioRxiv2021

Visualizing transcription sites in living cells using a genetically encoded probe specific for the elongating form of RNA polymerase II

In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2). How RNAP2 transcription is regulated in the nucleus is a key to understanding the genome and cell function. The largest subunit of RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5- Pro6-Ser7) at the C-terminal domain and Ser2 is phosphorylated on an elongation form of RNAP2. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody probe exhibited numerous foci, possibly representing transcription "factories" in living HeLa cells, and foci were diminished when cells were treated with triptolide to induce RNAP2 degradation and with flavopiridol to inhibit Ser2ph. An in vitro binding assay using phospho-peptides confirmed the Ser2ph-specific binding of the mintbody. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2, such as the CDK12 and Paf1 complex component, compared to factors involved in transcription activation around the transcription start sites, such as CDK9 and BRD4. Tracking analysis revealed that RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but was more mobile compared to euchromatin domains, suggesting that the elongating RNAP2 complexes are separated from the more confined initiating clusters. SummaryThe authors developed a genetically encoded probe to specifically detect the Ser2- phosphorylated, elongating form of RNA Polymerase II in living cells. The motion of Ser2- phosphorylated polymerase foci was more dynamic than chromatin domains, suggesting that the elongating complexes are separated from the more confined initiating clusters.

cell biology↗

bioRxiv2021

Euchromatin factors HULC and Set1C affect heterochromatin organization for mating-type switching in fission yeast Schizosaccharomyces pombe

Mating-type (P or M) of fission yeast Schizosaccharomyces pombe is determined by the transcriptionally active mat1 cassette and is switched by gene conversion using a donor, either mat2 or mat3, located in an adjacent heterochromatin region (mating-type switching; MTS). In the process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the action of two recombination enhancers, SRE2 promoting the use of mat2-P and SRE3 promoting the use of mat3-M, leading to replacement of the content of the expressed mat1 cassette. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1{Delta}. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant indicated that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in MTS in these mutants suggested that HULC and Set1C inhibit the use of the SRE3 recombination enhancer in M cells, thus favoring SRE2 and mat2-P. Consistently, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C might control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain.

genetics↗

bioRxiv2020

RNA polymerase II condensate formation and association with Cajal and histone locus bodies in living human cells

In eukaryotic nuclei, a number of phase-separated nuclear bodies (NBs) are present. RNA polymerase II (Pol II) is the main player in transcription and forms large condensates in addition to localizing at numerous transcription foci. Cajal bodies (CBs) and histone locus bodies (HLBs) are NBs that are involved in transcriptional and post-transcriptional regulation of small nuclear RNA and histone genes. By live-cell imaging using human HCT116 cells, we here show that Pol II condensates (PCs) nucleated near CBs and HLBs, and the number of PCs increased during S phase concomitantly with the activation period of histone genes. Ternary PC-CB- HLB associates were formed via three pathways: nucleation of PCs and HLBs near CBs, interaction between preformed PC-HLBs with CBs, and nucleation of PCs near preformed CB- HLBs. Coilin knockout increased the co-localization rate between PCs and HLBs, whereas the number, nucleation timing, and phosphorylation status of PCs remained unchanged. Depletion of PCs did not affect CBs and HLBs. Treatment with 1,6-hexanediol revealed that PCs were more liquid-like than CBs and HLBs. Thus, PCs are dynamic structures often nucleated following the activation of gene clusters associated with other NBs. (187 words)

cell biology↗

bioRxiv2020

Modeling population size independent tissue epigenomes by ChIL-seq with single-thin sections

Recent advances in omics studies have enabled analysis at the single-cell level; however, methods for analyzing the whole cell of large organs and tissues remain challenging. Here, we developed a method named tsChIL to understand the diverse cellular dynamics at the tissue level using high-depth epigenomic data. tsChIL allowed the analysis of a single tissue section and could reproducibly acquire epigenomic profiles from several types of tissues, based on the distribution of target epigenomic states, tissue morphology, and number of cells. The proposed method enabled the independent evaluation of changes in cell populations and gene activation of cells in regenerating skeletal muscle tissues, using a statistical model of RNA polymerase II distribution on gene loci. Thus, the integrative analysis by tsChIL can elucidate in vivo cell-type dynamics of tissues.

molecular biology↗

bioRxiv2020

Cryptic promoter activation occurs by at least two different mechanisms in the Arabidopsis genome

In gene-trap screening of plant genomes, promoterless reporter constructs are often expressed without trapping of annotated gene promoters. The molecular basis of this phenomenon, which has been interpreted as the trapping of cryptic promoters, is poorly understood. In this study, using Arabidopsis gene-trap lines in which a firefly luciferase (LUC) open reading frame (ORF) was expressed from intergenic regions, we found that cryptic promoter activation occurs by at least two different mechanisms: one is the capturing of pre-existing promoter-like chromatin marked by H3K4me3 and H2A.Z, and the other is the entirely new formation of promoter chromatin near the 5 end of the inserted LUC ORF. To discriminate between these, we denoted the former mechanism as "cryptic promoter capturing", and the latter one as "promoter de novo origination". The latter finding raises a question as to how inserted LUC ORF sequence is involved in this phenomenon. To examine this, we performed a model experiment with chimeric LUC genes in transgenic plants. Using Arabidopsis psaH1 promoter-LUC constructs, we found that the functional core promoter region, where transcription start sites (TSS) occur, cannot simply be determined by the upstream nor core promoter sequences; rather, its positioning proximal to the inserted LUC ORF sequence was more critical. This result suggests that the insertion of the LUC ORF sequence alters the local distribution of the TSS in the plant genome. The possible impact of the two types of cryptic promoter activation mechanisms on plant genome evolution and endosymbiotic gene transfer is discussed.

evolutionary biology↗

bioRxiv2020

Point Technology Illuminates The Processing Of Polymerase-Associated Intact Nascent Transcripts

Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly due to technical limitations in purifying authentic nascent transcripts. We present a new approach to purify and profile nascent RNA, called Polymerase Intact Nascent Transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano) and profiles whole transcription units (POINT-seq). In particular we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5P-RNA at polyadenylation sites. Furthermore POINT-nano reveals that splicing occurs either immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing. HIGHLIGHTSO_LIPOINT methodology dissects intact nascent RNA processing C_LIO_LISpecificity of Xrn2 exonuclease in co-transcriptional RNA degradation C_LIO_LISplicing suppresses Xrn2-dependent premature termination C_LIO_LIDifferent kinetic classes of co-transcriptional splicing in human genes C_LI

genomics↗

bioRxiv2020

Tracking H3K27me3 and H4K20me1 dynamics during XCI reveals similarities in recruitment mechanism

During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long-noncoding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2-dependent H3K27me3 and SETD8-dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the molecular mechanism allowing for H4K20me1 enrichment. To follow XCI dynamics in living cells, we developed a genetically-encoded, H3K27me3-specific intracellular antibody, or H3K27me3-mintbody. By combining it with live-imaging of H4K20me1, the X chromosome and Xist RNA we uncover similarities in the initial accumulation dynamics of H3K27me3 and H4K20me1. Further ChIP-seq analysis confirmed concurrent accumulation of both marks during XCI albeit with distinct genomic distributions. Using a Xist B and C repeat mutant, which can silence the X but does not allow for H3K27me3 deposition, we also found a lack of H4K20me1 enrichment. Thus, these two marks accumulate at the X thanks to the same region of Xist and H4K20me1 in particular may have a role in the chromatin compaction that characterises facultative heterochromatin.

developmental biology↗

bioRxiv2020

Epstein-Barr virus tegument protein BGLF2 in exosomes released from virus-producer cells assists de novo infection by enhancing viral gene expression

Viruses must adapt to the environment of their host cells to establish infection and persist. Diverse mammalian cells, including virus-infected cells, secrete extracellular vesicles such as exosomes containing proteins and miRNAs, and use these vesicles to mediate intercellular communications. However, the roles of exosomes in viral infection remain unclear. Here we screened viral proteins to identify those responsible for the exosome-mediated upregulation of Epstein-Barr virus (EBV) infection. We found BGLF2 protein encapsulated in exosomes, which were released from EBV-infected cells. BGLF2 protein is a tegument protein that exists the space between the envelope and the nucleocapsid, and it is released into the cytoplasm shortly after infection. BGLF2 protein-containing exosomes enhanced viral gene expression and repressed innate immunity, thereby assisting the EBV infection. In summary, the EBV tegument protein BGLF2 is encapsulated not only encapsulated in viral particles, but also in exosomes secreted from infected cells. Therefore, BGLF2 may play a crucial role in establishing EBV latent infection.

microbiology↗

bioRxiv2020

Geometrical model explains multiple preferred escape trajectories of fish

The escape trajectory (ET) of prey - measured as the angle relative to the predators approach path - plays a major role in avoiding predation. Previous geometric models predict a single ET; however, many species show highly variable ETs with multiple preferred directions. Although such a high ET variability may confer unpredictability to avoid predation, the reasons why animals prefer specific multiple ETs remain unclear. Here, we constructed a novel geometric model that incorporates the time required for prey to turn and the predators position at the end of its attack. The optimal ET was determined by maximizing the time difference of arrival at the edge of the safety zone between the prey and predator. By fitting the model to the experimental data of fish Pagrus major we show that the model can clearly explain the observed multiple preferred ETs. By using the same model, we were able to explain different patterns of ETs empirically observed in other species (e.g., insects and frogs): a single preferred ET and multiple preferred ETs at small (20-50{degrees}) and large (150-180{degrees}) angles from the predator. Our results open new avenues of investigation for understanding how animals choose their ETs from behavioral and neurosensory perspectives.

animal behavior and cognition↗

bioRxiv2020

C. elegans episodic swimming is driven by multifractal kinetics

Fractal scaling is a common property of temporal change in various modes of animal behavior. The molecular mechanisms of fractal scaling in animal behaviors remain largely unexplored. The nematode C. elegans alternates between swimming and resting states in a liquid solution. Here, we report that C. elegans episodic swimming is characterized by scale-free kinetics with long-range temporal correlation and local temporal clusterization, which is characterized as multifractal kinetics. Residence times in actively-moving and inactive states were distributed in a power law-based scale-free manner. Multifractal analysis showed that temporal correlation and temporal clusterization were distinct between the actively-moving state and the inactive state. These results indicate that C. elegans episodic swimming is driven by transition between two behavioral states, in which each of two transition kinetics follows distinct multifractal kinetics. We found that a conserved behavioral modulator, cyclic GMP dependent kinase (PKG) may regulate the multifractal kinetics underlying an animal behavior. Our combinatorial analysis approach involving molecular genetics and kinetics provides a platform for the molecular dissection of the fractal nature of physiological and behavioral phenomena.

animal behavior and cognition↗

bioRxiv2020

Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

The carboxyl-terminal domain of RNA polymerase II is dynamically phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to unphosphorylated and phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as clusters of Serine 5 phosphorylated RNAP2 that form around active genes and are separated in both space and time from nascent mRNA synthesis. Computational models fit to our data determine that 5 to 40 RNAP2 cluster around the promoter of a gene during typical transcriptional bursts. Rapid imaging demonstrates nearly all RNAP2 in the cluster acquire Serine 5 phosphorylation within 3-6 seconds of arrival. Transcription from the cluster appears to be highly efficient, with nearly half of the clustered RNAP2 ultimately escaping the promoter in 1.5 minutes on average to elongate a full-length mRNA in approximately five minutes. The highly dynamic and spatially organized concentrations of RNAP2 we observe support the notion of highly efficient transcription clusters that form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

molecular biology↗

bioRxiv2020

Direct evidence of abortive lytic infection-mediated establishment of Epstein-Barr virus latency during B-cell infection

Viral infection induces dynamic changes in transcriptional profiles. Virus-induced and anti-viral responses are intertwined during the infection. Epstein-Barr virus (EBV) is a human gammaherpesvirus that provides a model of herpesvirus latency. To measure the transcriptome changes during the establishment of EBV latency, EBV-negative Akata cells were infected with EBV-EGFP and observed by transcriptome sequencing (RNA-seq) at 0, 2, 4, 7, 10, and 14 days after infection. We found transient downregulation of mitotic division-related genes, reflecting reprograming of cell growth by EBV. Moreover, a burst of viral lytic gene expression was detected in the early phase of infection. Experimental and mathematical investigations demonstrated that infectious virions were not produced in the pre-latent phase, suggesting the presence of an abortive lytic infection. Finally, we conducted fate mapping using recombinant EBV, enabling the noninvasive, continuous observation of infected cells during EBV infection. Our tracking analysis provided direct evidence that the abortive lytic infection in the pre-latent phase converges to latent infection during EBV infection of B-cells, shedding light on novel roles of viral lytic gene(s) in establishing latency. Author summaryViral infection is a complex process that activates both virus-triggered and host anti-viral responses. This process has classically been studied by snapshot analysis such as microarray and RNA-seq at discrete time points as population averages. Snapshot data lead to invaluable findings in host-pathogen interactions. However, these "snapshot" omics, even from a single cell, lack temporal resolution. Because the behavior of infected cells is highly dynamic and heterogenous, continuous analysis is required for deciphering the fate of infected cells during viral infection. Here, we exploited fate mapping techniques with recombinant Epstein-Barr virus (EBV) to track the infected cells and recorded a log of lytic gene expression during EBV infection. Our continuous observation of infected cells revealed that EBV established latency in B-cells via an abortive lytic infection in the pre-latent phase.

microbiology↗

bioRxiv2020

Inhibition of KDM1A activity restores adult neurogenesis and improves hippocampal memory in a mouse model of Kabuki syndrome

Kabuki syndrome (KS) is a rare cause of intellectual disability primarily caused by loss of function mutations in lysine-specific methyltransferase 2D (KMT2D), which normally adds methyl marks to lysine 4 on histone 3. Previous studies have shown that a mouse model of KS (Kmt2d+/{beta}Geo) demonstrates disruption of adult neurogenesis and hippocampal memory. Proof-of-principle studies have shown postnatal rescue of neurological dysfunction following treatments that promote chromatin opening, however, these strategies are non-specific and do not directly address the primary defect of histone methylation. Since lysine-specific demethylase 1A (LSD1/KDM1A) normally removes the H3K4 methyl marks added by KMT2D, we hypothesize that inhibition of KDM1A demethylase activity may ameliorate molecular and phenotypic defects stemming from KMT2D loss. To test this hypothesis, we evaluated a recently developed KDM1A inhibitor (TAK-418) in Kmt2d+/{beta}Geo mice. We find that orally administered TAK-418 increases the numbers of newly born Doublecortin (DCX)+ cells and processes in hippocampus in a dose dependent manner. We also observe TAK-418-dependent rescue of histone modification defects in hippocampus both by Western blot and ChIP-Seq. Treatment rescues gene expression abnormalities including those of immediate early genes such as FBJ osteosarcoma oncogene (Fos) and FBJ osteosarcoma oncogene homolog B (Fosb). After 2 weeks of TAK-418, Kmt2d+/{beta}Geo mice demonstrate normalization of hippocampal memory defects. In summary, our data suggest that KDM1A inhibition is a plausible treatment strategy for KS and support the hypothesis that the epigenetic dysregulation secondary to KMT2D dysfunction plays a major role in the postnatal neurological disease phenotype in KS.

genetics↗

bioRxiv2020

Histone modification dynamics as revealed by a multicolor immunofluorescence-based single-cell analysis

Post-translational modifications on histones can be stable epigenetic marks and transient signals that can occur in response to internal and external stimuli. Levels of histone modifications fluctuate during the cell cycle and vary among different cell types. Here we describe a simple system to monitor the levels of multiple histone modifications in single cells by multicolor immunofluorescence using directly labeled modification-specific antibodies. We first analyzed histone H3 and H4 modifications during the cell cycle. Levels of active marks, such as acetylation and H3K4 methylation, were increased during the S phase, in association with chromatin duplication. By contrast, levels of some repressive modifications gradually increased during the G2 and the next G1 phases. We applied this method to validate the target modifications of various histone demethylases in cells using a transient overexpression system. We also screened chemical compounds in marine organism extracts that affect histone modifications and identified psammaplin A, which was previously reported to inhibit histone deacetylases. Thus, the method presented here is a powerful and convenient tool for analyzing the changes in histone modifications.

cell biology↗