Analysis Of Histone Modifications Research Articles

Pyruvate metabolism controls chromatin remodeling during CD4+ T cell activation

Upon antigen-specific Tcell receptor (TCR) engagement, human CD4+ Tcells proliferate and differentiate, a process associated with rapid transcriptional changes and metabolic reprogramming. Here, we show that the generation of extramitochondrial pyruvate is an important step for acetyl-CoA production and subsequent H3K27ac-mediated remodeling of histone acetylation. Histone modification, transcriptomic, and carbon tracing analyses of pyruvate dehydrogenase (PDH)-deficient Tcells show PDH-dependent acetyl-CoA generation as a rate-limiting step during T activation. Furthermore, Tcell activation results in the nuclear translocation of PDH and its association with both the p300 acetyltransferase and histone H3K27ac. These data support the tight integration of metabolic and histone-modifying enzymes, allowing metabolic reprogramming to fuel CD4+ Tcell activation. Targeting this pathway may provide a therapeutic approach to specifically regulate antigen-driven Tcell activation.

A single-cell multiomic analysis of kidney organoid differentiation

Kidney organoids differentiated from pluripotent stem cells are powerful models of kidney development and disease but are characterized by cell immaturity and off-target cell fates. Comparing the cell-specific gene regulatory landscape during organoid differentiation with human adult kidney can serve to benchmark progress in differentiation at the epigenome and transcriptome level for individual organoid cell types. Using single-cell multiome and histone modification analysis, we report more broadly open chromatin in organoid cell types compared to the human adult kidney. We infer enhancer dynamics by cis-coaccessibility analysis and validate an enhancer driving transcription of HNF1B by CRISPR interference both in cultured proximal tubule cells and also during organoid differentiation. Our approach provides an experimental framework to judge the cell-specific maturation state of human kidney organoids and shows that kidney organoids can be used to validate individual gene regulatory networks that regulate differentiation.

Abstract 4718: Analyses of epigenetic marks and mechanisms in disease: Your guide to a successful CUT&RUN assay

Abstract Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets & Release Using Nuclease (CUT&RUN) is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell. The CUT&RUN assay can be combined with downstream qPCR or NG-seq to analyze histone modifications and binding of transcription factors, DNA replication factors, or DNA repair proteins at specific target genes or across the entire genome. CUT&RUN provides a rapid, robust, and true low cell number assay for detection of protein-DNA interactions in the cell. Unlike the ChIP assay, CUT&RUN is free from formaldehyde cross-linking, chromatin fragmentation, and immunoprecipitation. Previously, we have shown that, compared to ChIP, CUT&RUN requires fewer starting cells (100K), has a much faster protocol (one day from cells to DNA), generates lower background signal (requires less sequencing depth), and offers spike-in control DNA for effective normalization of signal between samples and between experiments. We recently updated the CST CUT&RUN Assay Kit for use with 5,000-20,000 cells and added protocols for fixed cells and tissue. I will discuss the basics of the CUT&RUN assay and important factors to consider when setting up your experiment. In addition, I will provide data showing the versatility of this assay for mapping various histone modifications, transcription factor, and transcription cofactor binding across multiple sample types. Finally, I will discuss how the general protocol is optimized for greater signal to noise ratio, reduced number of starting cells, and provide an alternative digestion method to prepare the input DNA as a critical control of the CUT&RUN experiment. Citation Format: Angela H. Guo, Christopher R. Comeau, Fang Chen, Christopher J. Fry. Analyses of epigenetic marks and mechanisms in disease: Your guide to a successful CUT&RUN assay. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4718.

Molecular genetics of early-onset colorectal cancer.

Early-onset colorectal cancer (EOCRC) has been rising in global prevalence and incidence over the past several decades. Environmental influences, including generational lifestyle changes and rising obesity, contribute to these increased rates. While the rise in EOCRC is best documented in western countries, it is seen throughout the world, although EOCRC may have distinct genetic mutations in patients of different ethnic backgrounds. Pathological and molecular characterizations show that EOCRC has a distinct presentation compared with later-onset colorectal cancer (LOCRC). Recent studies have identified DNA, RNA, and protein-level alterations unique to EOCRC, revealing much-needed biomarkers and potential novel therapeutic targets. Many molecular EOCRC studies have been performed with Caucasian and Asian EOCRC cohorts, however, studies of other ethnic backgrounds are limited. In addition, certain molecular characterizations that have been conducted for LOCRC have not yet been repeated in EOCRC, including high-throughput analyses of histone modifications, mRNA splicing, and proteomics on large cohorts. We propose that the complex relationship between cancer and aging should be considered when studying the molecular underpinnings of EOCRC. In this review, we summarize current EOCRC literature, focusing on sporadic molecular alterations in tumors, and their clinical implications. We conclude by discussing current challenges and future directions of EOCRC research efforts.

Genome-wide analysis of transcriptome and histone modifications in Brassica napus hybrid.

Although utilization of heterosis has largely improved the yield of many crops worldwide, the underlying molecular mechanism of heterosis, particularly for allopolyploids, remains unclear. Here, we compared epigenome and transcriptome data of an elite hybrid and its parental lines in three assessed tissues (seedling, flower bud, and silique) to explore their contribution to heterosis in allopolyploid B. napus. Transcriptome analysis illustrated that a small proportion of non-additive genes in the hybrid compared with its parents, as well as parental expression level dominance, might have a significant effect on heterosis. We identified histone modification (H3K4me3 and H3K27me3) variation between the parents and hybrid, most of which resulted from the differences between parents. H3K4me3 variations were positively correlated with gene expression differences among the hybrid and its parents. Furthermore, H3K4me3 and H3K27me3 were rather stable in hybridization and were mainly inherited additively in the B. napus hybrid. Together, our data revealed that transcriptome reprogramming and histone modification remodeling in the hybrid could serve as valuable resources for better understanding heterosis in allopolyploid crops.

Dysfunctional epigenetic protein-coding gene-related signature is associated with the prognosis of pancreatic cancer based on histone modification and transcriptome analysis

Emerging evidence suggests that epigenetic alterations are responsible for the oncogenesis and progression of cancer. However, the role of epigenetic reprogramming in pancreatic cancer is still not clear. In this study, we used the limma R package to identify differentially expressed protein-coding genes (PCGs) between pancreatic cancer tissues and normal control tissues. The cell-type identification by the estimating relative subsets of RNA transcripts (CIBERSORT) package was used to quantify relative cell fractions in tumors. Prognostic molecular clusters were constructed using ConsensusClusterPlus analysis. Furthermore, the least absolute shrinkage and selection operator and stepAIC methods were used to construct a risk model. We identified 2351 differentially expressed PCGs between pancreatic cancer and normal control tissues in The cancer genome atlas dataset. Combined with histone modification data, we identified 363 epigenetic PCGs (epi-PCGs) and 19,010 non-epi-PCGs. Based on the epi-PCGs, we constructed three molecular clusters characterized by different expression levels of chemokines and immune checkpoint genes and distinct abundances of various immune cells. Furthermore, we generated a 9-gene model based on dysfunctional epi-PCGs. Additionally, we found that patients with high risk scores showed poorer prognoses than patients with low risk scores (p < 0.0001). Further analysis showed that the risk score was significantly related to survival and was an independent risk factor for pancreatic cancer patients. In conclusion, we constructed a 9-gene prognostic risk model based on epi-PCGs that might serve as an effective classifier to predict overall survival and the response to immunotherapy in pancreatic cancer patients.

Abstract 2078: Multi-omic analysis of metabolites, proteins, and histone modifications during human macrophage polarization

Abstract 2078: Multi-omic analysis of metabolites, proteins, and histone modifications during human macrophage polarization

Analysis of Chromatin Accessibility, Histone Modifications, and Transcriptional States in Specific Cell Types Using Flow Cytometry.

The past two decades in biomedical research have experienced an explosion of cell type-specific and single-cell studies, especially concerning the concomitant dissection of regulatory and transcriptional landscapes of those under investigation. Additionally, leveraging next-generation sequencing (NGS) platforms effortshave been undertaken to evaluate the effects of chromatin accessibility, histone modifications, or even transcription factor binding sites. We have shown that Fluorescence-Activated Nuclear Sorting (FANS) is an effective means to characterize the transcriptomes of nuclei from different tissues. In light of our own technical and experimental developments, we extend this effort to combine FACS/FANS with Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), Chromatin Immunoprecipitation sequencing (ChIP-seq), and RNA sequencing (RNA-seq) for profiling individual cell types according to their chromatin and transcriptional states.

Abstract B004: Analysis of epigenetic marks and mechanisms in disease: Your guide to a successful assay

Abstract Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets &amp; Release Using Nuclease is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell. The assay can be combined with downstream qPCR or NG-seq to analyze histone modifications and binding of transcription factors, DNA replication factors, or DNA repair proteins at specific target genes or across the entire genome. It provides a rapid, robust, and true low cell number assay for detection of protein-DNA interactions in the cell. Unlike the ChIP assay, it is free from formaldehyde cross-linking, chromatin fragmentation, and immunoprecipitation. Previously, we have shown that, compared to ChIP, it requires fewer starting cells (100K), has a much faster protocol (one day from cells to DNA), generates lower background signal (requires less sequencing depth), and offers spike-in control DNA for effective normalization of signal between samples and between experiments. We recently updated the CST Assay Kit for use with 5,000-20,000 cells and added protocols for fixed cells and tissue. I will discuss the basics of the assay and important factors to consider when setting up your experiment. In addition, I will provide data showing the versatility of this assay for mapping various histone modifications, transcription factor, and transcription cofactor binding across multiple sample types. Finally, I will discuss how the general protocol is optimized for greater signal to noise ratio, reduced number of starting cells, and provide an alternative digestion method to prepare the input DNA as a critical control of the experiment. Citation Format: Christopher C. Comeau, Angela H. Guo, Fang Chen, Christopher J. Fry. Analysis of epigenetic marks and mechanisms in disease: Your guide to a successful assay. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B004.

Whole-Genome Analysis of Histone Modifications Reveals New Insights into the Biology and Clinical Behavior of Mantle Cell Lymphoma Subtypes

Introduction: Mantle cell lymphoma (MCL) is a mature B-cell neoplasm characterized by the t(11;14)(q13;q32) leading to cyclin D1 overexpression. Two major clinico-biological subtypes of MCL have been recognized, conventional (cMCL) and leukemic non-nodal MCL (nnMCL), being the former clinically more aggressive than the latter. Although these subtypes have been well characterized at the genomic level, the genome-wide landscape of histone modifications is widely unknown. Aim: To explore whether an integrative multiomic analysis of chromatin marks and gene expression may help us to better understand the biology and clinical behavior of cMCL and nnMCL. Methods: We carried out 2 profiling strategies. On the one hand, we performed ChIP-seq for H3K27ac, which marks active regulatory elements, in a series of 52 MCL cases, and we also profiled chromatin accessibility by ATAC-seq in 28 of these cases. On the other hand, we performed ChIP-seq for 6 histone modifications with complementary functions (H3K4me1, H3K4me3, H3K27ac, H3K36me3, H3K27me3 and H3K9me3) in purified neoplastic cells of 3 cMCL and 3 nnMCL, and we mined ChIP-seq data from normal B cell subpopulations. Additionally, we analyzed previously published expression data from 44 MCL profiled by Affymetrix U219 microarrays. Clinical data were available for most cases. Data were analyzed using a battery of computational and statistical approaches. Results: An initial unsupervised principal component analyses of the H3K27ac and ATAC-seq data revealed 2 main clusters corresponding to cMCL and nnMCL, indicating that the chromatin landscape is markedly different between the 2 MCL subtypes. A supervised differential analysis of H3K27ac data between cMCL and nnMCL showed extensive differences, mostly associated with increased H3K27ac in cMCL. Putting the data in the context of normal B cell subpopulations, we identified 746 de novo active regions in cMCL and 126 in nnMCL (LFC>1, FDR ≤0.05). Interestingly, more than 60% of these regions were enhancers according to the chromatin states analysis (derived from the integration of 6 histone modifications), and the majority of them presented an open chromatin configuration. This chromatin activation patterns correlated with transcriptional differences between cMCL and nnMCL. Analyzing the target genes of these regions, we identified 167 and 20 upregulated genes in cMCL and nnMCL, respectively. Upregulated genes in cMCL were linked to cellular processes related to cell cycle and cell proliferation according to gene ontology analysis. Analyzing the transcription factor binding motifs present in the ATAC-seq peaks among the de novo active H3K27ac regions in cMCL, we observed an enrichment in specific transcription factor families such as Krüppel-like family (KLF) and E2F. However, there was no significant enrichment in any transcription factor family among nnMCL de novo active regions. Moreover, although the cMCL-associated de novo H3K27ac signature was common to all cMCL cases, we did observe varying H3K27ac levels among cases. We then explored whether H3K27ac levels could be associated with clinical features in cMCL. In fact, we identified 100 specific regions whose increased H3K27ac levels were significantly associated with shorter overall survival, leading to the identification of two subgroups of cMCL with markedly different clinical behavior. Conclusions: The results obtained with this multilayer chromatin analysis confirm that MCL is a heterogeneous disease showing widespread differences between the 2 subtypes. cMCL present a clear de novo chromatin activation associated with upregulation of key genes involved in cMCL pathogenesis, while nnMCL cases seem to be characterized by the absence of this signature and an overall chromatin profile more similar to normal B cells. Moreover, the level of activation in regulatory regions in cMCL seems to be related with the clinical outcome of the patients, with more activation associated with shorter overall survival.

Abstract 14259: Novel Long Noncoding RNA, Ppp1r1b-LncRNA , Epigenetically Promotes Myogenic Differentiation Program in Cardiac and Skeletal Myocytes

Introduction: Emerged as critical regulators of the transcriptome, the lncRNAs play important roles in cardiac development and might be targeted to treat human cardiomyocyte dysfunction in congenital heart disease. In our work, we identified a novel lncRNA, Ppp1r1b-lncRNA , as an essential, functionally conserved, positive regulator of myogenic differentiation. Hypothesis: Ppp1r1b-lncRNA promotes myogenic differentiation program of cardiac and skeletal myocytes via epigenic mechanisms. Methods: Antisense oligonucleotides (GapmeR) were used to suppress Ppp1r1b-lncRNA . Chromatin immunoprecipitation (CHIP) and chromatin isolation by RNA purification (CHIRP) were used to analyze gene specific histone modification level and lncRNA: chromatin interaction, respectively. RNA pull-down and RNA immunoprecipitation (RIP) were used to identify Ppp1r1b-lncRNA : protein interactome. Results: By silencing Ppp1r1b-lncRNA, skeletal myoblasts and human-induced pluripotent stem cell derived cardiomyocytes (hiPSCs-CMs) failed to develop fully differentiated myotubes. Analysis of GapmeR injected neonatal mouse heart further confirmed the role of Ppp1r1b-lncRNA in normal myogenesis. Members of muscle specific transcription factors, including MyoD1 and TBX5, were not induced during myogenic differentiation in the Ppp1r1b-lncRNA depleted cardiac and skeletal muscle precursor cells. Histone modification analysis revealed enrichment of H3K27 tri-methylation at MyoD1 and TBX5 promoters in GapmeR treated skeletal myoblasts. Furthermore, Ppp1r1b-lncRNA was found to bind to Ezh2, and ChIRP analysis revealed enriched interaction of Ppp1r1b-lncRNA with Myod1 and Tbx5 promoters, suggesting that Ppp1r1b-lncRNA induces transcription of myogenic regulatory factors by interacting with the polycomb repressive complex 2 (PRC2) at the chromatin interface. Correspondingly, the silencing of Ppp1r1b-lncRNA increased EZH2 binding at promoter regions of myogenic transcription factors. Conclusions: Ppp1r1b-lncRNA promotes myogenic differentiation by inhibiting histone methylation on the promotors of myogenic master regulators via competing for PRC2 binding with chromatin during heart and skeletal muscle development.

BESC from cloned embryos do not retain transcriptomic or epigenetic memory from somatic donor cells.

Epigenetic reprogramming after mammalian somatic cell nuclear transfer is often incomplete, resulting in low efficiency of cloning. However, gene expression and histone modification analysis indicated high similarities in transcriptome and epigenomes of bovine embryonic stem cells from in vitro fertilized and somatic cell nuclear transfer embryos. Embryonic stem cells (ESC) indefinitely maintain the pluripotent state of the blastocyst epiblast. Stem cells are invaluable for studying development and lineage commitment, and in livestock, they constitute a useful tool for genomic improvement and in vitro breeding programs. Although these cells have been recently derived from bovine blastocysts, a detailed characterization of their molecular state is lacking. Here, we apply cutting-edge technologies to analyze the transcriptomic and epigenomic landscape of bovine ESC (bESC) obtained from in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. bESC were efficiently derived from SCNT and IVF embryos and expressed pluripotency markers while retaining genome stability. Transcriptome analysis revealed that only 46 genes were differentially expressed between IVF- and SCNT-derived bESC, which did not reflect significant deviation in cellular function. Interrogating histone 3 lysine 4 trimethylation, histone 3 lysine 9 trimethylation, and histone 3 lysine 27 trimethylation with cleavage under targets and tagmentation, we found that the epigenomes of both bESC groups were virtually indistinguishable. Minor epigenetic differences were randomly distributed throughout the genome and were not associated with differentially expressed or developmentally important genes. Finally, the categorization of genomic regions according to their combined histone mark signal demonstrated that all bESC shared the same epigenomic signatures, especially at gene promoters. Overall, we conclude that bESC derived from SCNT and IVF embryos are transcriptomically and epigenetically analogous, allowing for the production of an unlimited source of pluripotent cells from high genetic merit organisms without resorting to transgene-based techniques.

Genome-wide analysis of bivalent histone modifications during Drosophila embryogenesis.

In eukaryotes, histone modifications are key epigenetic regulators that are associated with distinct chromatin features. Bivalent histone modifications describe a situation where a subset of promoters have with both activating (H3K4me3) and repressive (H3K27me3) markers in pluripotent cells (e.g., ESCs). However, it remains to be understood whether bivalent histone modifications are stable throughout developmental stages. Here, by systematically analyzing ChIP-seq data of H3K4me3 and H3K27me3, we provided the first panoramic view of bivalent histone modifications in Drosophila from embryonic 0-4 to 20-24 hr. In our study, we found that bivalent histone modifications occur at other locations in the genome in addition to the promoter region. Additionally, the different genomic regions occupied by bivalent histone modifications exhibit spatiotemporal specificity at each stage. Furthermore, gene ontology and motif analysis reflected continuous and gradual changes of target genes during different developmental process. In summary, we suggest that bivalent histone modifications have potential regulatory functions throughout Drosophila embryonic stage.

Histone Modification Analysis of Low-Mappability Regions.

Posttranslational modifications of histone are intimately related to chromatin/chromosome-mediated cellular events. Among all, the roles of histone modifications including acetylation, methylation, ubiquitination, and SUMOylation of lysine or arginine residue of nucleosome core histones in gene expression have been intensively studied. Genome-wide profiles of histone modification marks revealed their combinatorial organization in the functional features of chromatin. Analysis of histone modification by chromatin immunoprecipitation (ChIP) is one of the standard assays to examine chromatin states. Although high-throughput sequencing analysis (ChIP-seq) is now widely conducted, classical ChIP-qPCR analysis has advantages in investigation of multiple histone modification marks at a target site simply through theuse ofrelatively small numbers of cells. Since ChIP-qPCR is devoid of biases caused by overamplification and inaccurate mapping of sequencing reads, it is a more reliable quantification method than genome-wide ChIP-seq especially for analyses of the low-mappability regions, which harbor many repetitive sequences and/or highly homologous segmental multiplications as found in gene clusters. We have recently analyzed histone H3 and H4 modifications of the Zscan4 family gene loci in an 880kb gene cluster and found that the atypical enhancer-like structure is formed upon derepression of Zscan4. In this chapter, we describe the detailed protocols for histone modification ChIP-assay of repeat-enriched gene cluster regions. The protocol here we applied to mouse ES cells, but the protocol is perfectly applicableto humanculturedcellsand specimens.

SOC-I-05 Histone modification, DNA methylation, and mRNA expression analysis of murine liver repeatedly exposure to a chemical

SOC-I-05 Histone modification, DNA methylation, and mRNA expression analysis of murine liver repeatedly exposure to a chemical

The impact of cholesterol lowering drugs on metabolism and epigenetics

Background and Aims : Cardiovascular diseases (CVDs) are the leading cause of death globally. In patients with CVDs, treatment with cholesterol lowering drugs has been shown to reduce cardiovascular events. Bempedoic acid is a novel drug for lowering low-density lipoprotein cholesterol that has recently been approved for clinical use. Bempedoic acid specifically inhibits ATP citrate lyase (ACLY) in the liver, an enzyme that converts citrate to acetyl-CoA and acts at the interface of carbohydrate and lipid metabolism. ACLY is not only cytoplasmic, but is also found in the nucleus, where it regulates histone acetylation levels. Yet, it is unknown if and how bempedoic acid impacts ACLYs regulatory function in the nucleus.Methods: By combining chromatin immunoprecipitation sequencing, transcriptomics, tracer metabolomics and mass-spectrometry based histone post-translational modification analysis in primary human hepatocytes, we systematically investigate the impact of bempedoic acid on ACLY at different regulatory levels.Results: Our data from mouse liver tissue revealed binding of ACLY to promoter regions of metabolic genes. Consistently, gene expression analyses in primary human hepatocytes showed reduced expression of ACLY bound metabolic genes in response to bempedoic acid. Furthermore, histone modification analysis via mass spectrometry and ChIP-seq profiles displayed a highly specific loss of H3K9ac, a promoter-based histone modification known to stimulate gene transcription, upon bempedoic acid treatment. Metabolite profiling revealed reduction in lipid metabolism following bempedoic acid administration.Conclusions: Taken together, these data suggest that bempedoic acid's mechanism of action involves reprogramming of the epigenetic and transcriptional landscape of ACLY associated genes, together with a reduction in lipid production in hepatocytes. Background and Aims : Cardiovascular diseases (CVDs) are the leading cause of death globally. In patients with CVDs, treatment with cholesterol lowering drugs has been shown to reduce cardiovascular events. Bempedoic acid is a novel drug for lowering low-density lipoprotein cholesterol that has recently been approved for clinical use. Bempedoic acid specifically inhibits ATP citrate lyase (ACLY) in the liver, an enzyme that converts citrate to acetyl-CoA and acts at the interface of carbohydrate and lipid metabolism. ACLY is not only cytoplasmic, but is also found in the nucleus, where it regulates histone acetylation levels. Yet, it is unknown if and how bempedoic acid impacts ACLYs regulatory function in the nucleus. Methods: By combining chromatin immunoprecipitation sequencing, transcriptomics, tracer metabolomics and mass-spectrometry based histone post-translational modification analysis in primary human hepatocytes, we systematically investigate the impact of bempedoic acid on ACLY at different regulatory levels. Results: Our data from mouse liver tissue revealed binding of ACLY to promoter regions of metabolic genes. Consistently, gene expression analyses in primary human hepatocytes showed reduced expression of ACLY bound metabolic genes in response to bempedoic acid. Furthermore, histone modification analysis via mass spectrometry and ChIP-seq profiles displayed a highly specific loss of H3K9ac, a promoter-based histone modification known to stimulate gene transcription, upon bempedoic acid treatment. Metabolite profiling revealed reduction in lipid metabolism following bempedoic acid administration. Conclusions: Taken together, these data suggest that bempedoic acid's mechanism of action involves reprogramming of the epigenetic and transcriptional landscape of ACLY associated genes, together with a reduction in lipid production in hepatocytes.

Dysfunction of histone demethylase IBM1 in Arabidopsis causes autoimmunity and reshapes the root microbiome

Root microbiota is important for plant growth and fitness. Little is known about whether and how the assembly of root microbiota may be controlled by epigenetic regulation, which is crucial for gene transcription and genome stability. Here we show that dysfunction of the histone demethylase IBM1 (INCREASE IN BONSAI METHYLATION 1) in Arabidopsis thaliana substantially reshaped the root microbiota, with the majority of the significant amplicon sequence variants (ASVs) being decreased. Transcriptome analyses of plants grown in soil and in sterile growth medium jointly disclosed salicylic acid (SA)-mediated autoimmunity and production of the defense metabolite camalexin in the ibm1 mutants. Analyses of genome-wide histone modifications and DNA methylation highlighted epigenetic modifications permissive for transcription at several important defense regulators. Consistently, ibm1 mutants showed increased resistance to the pathogen Pseudomonas syringae DC3000 with stronger immune responses. In addition, ibm1 showed substantially impaired plant growth promotion in response to beneficial bacteria; the impairment was partially mimicked by exogenous application of SA to wild-type plants, and by a null mutation of AGP19 that is important for cell expansion and that is repressed with DNA hypermethylation in ibm1. IBM1-dependent epigenetic regulation imposes strong and broad impacts on plant-microbe interactions and thereby shapes the assembly of root microbiota.

Genome-wide profiling of histone H3K4me3 and H3K27me3 modifications in individual blastocysts by CUT&Tag without a solid support (NON-TiE-UP CUT&Tag)

Individual analysis of the epigenome of preimplantation embryos is useful for characterizing each embryo and for investigating the effects of environmental factors on their epigenome. However, it is difficult to analyze genome-wide epigenetic modifications, especially histone modifications, in a large number of single embryos due to the small number of cells and the complexity of the analysis methods. To solve this problem, we further modified the CUT&Tag method, which can analyze histone modifications in a small number of cells, such that the embryo is handled as a cell mass in the reaction solutions in the absence of the solid-phase magnetic beads that are used for antibody and enzyme reactions in the conventional method (NON-TiE-UP CUT&Tag; NTU-CAT). By using bovine blastocysts as a model, we showed that genome-wide profiles of representative histone modifications, H3K4me3 and H3K27me3, could be obtained by NTU-CAT that are in overall agreement with the conventional chromatin immunoprecipitation-sequencing (ChIP-seq) method, even from single embryos. However, this new approach has limitations that require attention, including false positive and negative peaks and lower resolution for broad modifications. Despite these limitations, we consider NTU-CAT a promising replacement for ChIP-seq with the great advantage of being able to analyze individual embryos.

Cytomixis in wheat male meiosis: influence analysis of the substitution of chromosome 1A, 2D, 5A, or 5D

ABSTRACT Cytomixis is a process of intercellular nuclear migration (INM) often observable in plant male meiosis. The genes responsible for this phenomenon have not been discovered yet. In an attempt to identify a “cytomictic chromosome,” we studied the male meiosis of wheat–rye single-chromosome substitution lines. Markers of the chromatin state were analyzed to figure out the nature of INM in cereal plants: is it a rather normal process or a consequence of random injury/deviation? There were no obvious correlations between the loss of wheat chromosome 1A, 2D, 5A, or 5D by hybrid plants and the frequency and cytological picture of INM in their meiosis. Acquisition of rye chromosome 1 Rv, 2 R, or 5 R did not correlate with any changes in the manifestation of INM either. Some unusual cytological patterns of the substituted-chromosome behavior were observed during INM. Chromosomes 1Rv, 2 R, and 5 R, which are alien to the wheat genome, were found to migrate preferentially between meiocytes. Analysis of post-translational histone modifications and synaptonemal-complex formation revealed that the migrating chromatin does not differ from the intact one. It is not inactivated, is properly condensed, and forms bivalents. Our findings suggest that none of the studied chromosomes carry “cytomictic genes.” At the same time, INM in male meiosis of cereals appears to be a normal process that may be involved in hybrid-genome rearrangements.

Aberrant H3K4me3 modification of epiblast genes of extraembryonic tissue causes placental defects and implantation failure in mouse IVF embryos.

Assisted reproductive technology has been widely applied in the treatment of human infertility. However, accumulating evidence indicates that invitro fertilization (IVF) is associated with a low pregnancy rate, placental defects, and metabolic diseases in offspring. Here, we find that IVF manipulation notably disrupts extraembryonic tissue-specific gene expression, and 334 epiblast (Epi)-specific genes and 24 Epi-specific transcription factors are abnormally expressed in extraembryonic ectoderm (ExE) of IVF embryos at embryonic day 7.5. Combined histone modification analysis reveals that aberrant H3K4me3 modification at the Epi active promoters results in increased expression of these genes in ExE. Importantly, we demonstrate that knockdown of the H3K4me3-recruited regulator Kmt2e, which is highly expressed in IVF embryos, greatly improves the development of IVF embryos and reduces abnormal gene expression in ExE. Our study therefore identifies that abnormal H3K4me3 modification in extraembryonic tissue is a major cause of implantation failure and abnormal placental development of IVF embryos.