Dynamic Competition of Polycomb and Trithorax in Transcriptional Programming.

Predicting regulatory potential from primary DNA sequences or transcription factor binding patterns is not possible. However, the annotation of the genome by chromatin proteins, histone modifications, and differential compaction is largely sufficient to reveal the locations of genes and their differential activity states. The Polycomb Group (PcG) and Trithorax Group (TrxG) proteins are the central players in this cell type-specific chromatin organization. PcG function was originally viewed as being solely repressive and irreversible, as observed at the homeotic loci in flies and mammals. However, it is now clear that modular and reversible PcG function is essential at most developmental genes. Focusing mainly on recent advances, we review evidence for how PcG and TrxG patterns change dynamically during cell type transitions. The ability to implement cell type-specific transcriptional programming with exquisite fidelity is essential for normal development.

A pair of readers of bivalent chromatin mediate formation of Polycomb-based "memory of cold" in plants.

The Polycomb-group chromatin modifiers play important roles to repress or switch off gene expression in plants and animals. How the active chromatin state is switched to a Polycomb-repressed state is unclear. In Arabidopsis, prolonged cold induces the switching of the highly active chromatin state at the potent floral repressor FLC to a Polycomb-repressed state, which is epigenetically maintained when temperature rises to confer "cold memory," enabling plants to flower in spring. We report that the cis-acting cold memory element (CME) region at FLC bears bivalent marks of active histone H3K4me3 and repressive H3K27me3 that are read and interpreted by an assembly of bivalent chromatin readers to drive cold-induced switching of the FLC chromatin state. In response to cold, the 47-bp CME and its associated bivalent chromatin feature drive the switching of active chromatin state at a recombinant gene to a Polycomb-repressed domain, conferring cold memory. We reveal a paradigm for environment-induced chromatin-state switching at bivalent loci in plants.

Tip60-mediated H2A.Z acetylation promotes neuronal fate specification and bivalent gene activation.

Cell lineage specification is accomplished by a concerted action of chromatin remodeling and tissue-specific transcription factors. However, the mechanisms that induce and maintain appropriate lineage-specific gene expression remain elusive. Here, we used an unbiased proteomics approach to characterize chromatin regulators that mediate the induction of neuronal cell fate. We found that Tip60 acetyltransferase is essential to establish neuronal cell identity partly via acetylation of the histone variant H2A.Z. Despite its tight correlation with gene expression and active chromatin, loss of H2A.Z acetylation had little effect on chromatin accessibility or transcription. Instead, loss of Tip60 and acetyl-H2A.Z interfered with H3K4me3 deposition and activation of a unique subset of silent, lineage-restricted genes characterized by a bivalent chromatin configuration at their promoters. Altogether, our results illuminate the mechanisms underlying bivalent chromatin activation and reveal that H2A.Z acetylation regulates neuronal fate specification by establishing epigenetic competence for bivalent gene activation and cell lineage transition.

Bivalent complexes of PRC1 with orthologs of BRD4 and MOZ/MORF target developmental genes in Drosophila.

Regulatory decisions in Drosophila require Polycomb group (PcG) proteins to maintain the silent state and Trithorax group (TrxG) proteins to oppose silencing. Since PcG and TrxG are ubiquitous and lack apparent sequence specificity, a long-standing model is that targeting occurs via protein interactions; for instance, between repressors and PcG proteins. Instead, we found that Pc-repressive complex 1 (PRC1) purifies with coactivators Fs(1)h [female sterile (1) homeotic] and Enok/Br140 during embryogenesis. Fs(1)h is a TrxG member and the ortholog of BRD4, a bromodomain protein that binds to acetylated histones and is a key transcriptional coactivator in mammals. Enok and Br140, another bromodomain protein, are orthologous to subunits of a mammalian MOZ/MORF acetyltransferase complex. Here we confirm PRC1-Br140 and PRC1-Fs(1)h interactions and identify their genomic binding sites. PRC1-Br140 bind developmental genes in fly embryos, with analogous co-occupancy of PRC1 and a Br140 ortholog, BRD1, at bivalent loci in human embryonic stem (ES) cells. We propose that identification of PRC1-Br140 "bivalent complexes" in fly embryos supports and extends the bivalency model posited in mammalian cells, in which the coexistence of H3K4me3 and H3K27me3 at developmental promoters represents a poised transcriptional state. We further speculate that local competition between acetylation and deacetylation may play a critical role in the resolution of bivalent protein complexes during development.

NFIB-MLL1 complex is required for the stemness and Dlx5-dependent osteogenic differentiation of C3H10T1/2 mesenchymal stem cells.

Despite significant progress in our understanding of the molecular mechanism of mesenchymal stem cell (MSC) differentiation, less is known about the factors maintaining the stemness and plasticity of MSCs. Here, we show that the NFIB-MLL1 complex plays key roles in osteogenic differentiation and stemness of C3H10T1/2 MSCs. We find that depletion of either NFIB or MLL1 results in a severely hampered osteogenic potential and failed activation of key osteogenic transcription factors, such as Dlx5, Runx2, and Osx, following osteogenic stimuli. In addition, the NFIB-MLL1 complex binds directly to the promoter of Dlx5, and exogenous expression of Myc-Dlx5, but not the activation of either the BMP- or the Wnt-signaling pathway, is sufficient to restore the osteogenic potential of cells depleted of NFIB or MLL1. Moreover, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis showed that the NFIB-MLL1 complex mediates the deposition of trimethylated histone H3K4 at both Dlx5 and Cebpa, key regulator genes that function at the early stages of osteogenic and adipogenic differentiation, respectively, in uncommitted C3H10T1/2 MSCs. Surprisingly, the depletion of either NFIB or MLL1 leads to decreased trimethylated histone H3K4 and results in elevated trimethylated histone H3K9 at those developmental genes. Furthermore, gene expression profiling and ChIP-sequencing analysis revealed lineage-specific changes in chromatin landscape and gene expression in response to osteogenic stimuli. Taken together, these data provide evidence for the hitherto unknown role of the NFIB-MLL1 complex in the maintenance and lineage-specific differentiation of C3H10T1/2 MSCs and support the epigenetic regulatory mechanism underlying the stemness and plasticity of MSCs.

Bivalent chromatin: a developmental balancing act tipped in cancer.

Bivalent chromatin is defined by the co-occurrence of otherwise opposing H3K4me3 and H3K27me3 modifications and is typically located at unmethylated promoters of lowly transcribed genes. In embryonic stem cells, bivalent chromatin has been proposed to poise developmental genes for future activation, silencing or stable repression upon lineage commitment. Normally, bivalent chromatin is kept in tight balance in cells, in part through the activity of the MLL/COMPASS-like and Polycomb repressive complexes that deposit the H3K4me3 and H3K27me3 modifications, respectively, but also emerging novel regulators including DPPA2/4, QSER1, BEND3, TET1 and METTL14. In cancers, both the deregulation of existing domains and the creation of de novo bivalent states is associated with either the activation or silencing of transcriptional programmes. This may facilitate diverse aspects of cancer pathology including epithelial-to-mesenchymal plasticity, chemoresistance and immune evasion. Here, we review current methods for detecting bivalent chromatin and discuss the factors involved in the formation and fine-tuning of bivalent domains. Finally, we examine how the deregulation of chromatin bivalency in the context of cancer could facilitate and/or reflect cancer cell adaptation. We propose a model in which bivalent chromatin represents a dynamic balance between otherwise opposing states, where the underlying DNA sequence is primed for the future activation or repression. Shifting this balance in any direction disrupts the tight equilibrium and tips cells into an altered epigenetic and phenotypic space, facilitating both developmental and cancer processes.

Bivalent chromatin accommodates survivin and BRG1/SWI complex to activate DNA damage response in CD4+ cells.

BACKGROUND: Bivalent regions of chromatin (BvCR) are characterized by trimethylated lysine 4 (H3K4me3) and lysine 27 on histone H3 (H3K27me3) deposition which aid gene expression control during cell differentiation. The role of BvCR in post-transcriptional DNA damage response remains unidentified. Oncoprotein survivin binds chromatin and mediates IFNγ effects in CD4+ cells. In this study, we explored the role of BvCR in DNA damage response of autoimmune CD4+ cells in rheumatoid arthritis (RA). METHODS: We performed deep sequencing of the chromatin bound to survivin, H3K4me3, H3K27me3, and H3K27ac, in human CD4+ cells and identified BvCR, which possessed all three histone H3 modifications. Protein partners of survivin on chromatin were predicted by integration of motif enrichment analysis, computational machine-learning, and structural modeling, and validated experimentally by mass spectrometry and peptide binding array. Survivin-dependent change in BvCR and transcription of genes controlled by the BvCR was studied in CD4+ cells treated with survivin inhibitor, which revealed survivin-dependent biological processes. Finally, the survivin-dependent processes were mapped to the transcriptome of CD4+ cells in blood and in synovial tissue of RA patients and the effect of modern immunomodulating drugs on these processes was explored. RESULTS: We identified that BvCR dominated by H3K4me3 (H3K4me3-BvCR) accommodated survivin within cis-regulatory elements of the genes controlling DNA damage. Inhibition of survivin or JAK-STAT signaling enhanced H3K4me3-BvCR dominance, which improved DNA damage recognition and arrested cell cycle progression in cultured CD4+ cells. Specifically, BvCR accommodating survivin aided sequence-specific anchoring of the BRG1/SWI chromatin-remodeling complex coordinating DNA damage response. Mapping survivin interactome to BRG1/SWI complex demonstrated interaction of survivin with the subunits anchoring the complex to chromatin. Co-expression of BRG1, survivin and IFNγ in CD4+ cells rendered complete deregulation of DNA damage response in RA. Such cells possessed strong ability of homing to RA joints. Immunomodulating drugs inhibited the anchoring subunits of BRG1/SWI complex, which affected arthritogenic profile of CD4+ cells. CONCLUSIONS: BvCR execute DNA damage control to maintain genome fidelity in IFN-activated CD4+ cells. Survivin anchors the BRG1/SWI complex to BvCR to repress DNA damage response. These results offer a platform for therapeutic interventions targeting survivin and BRG1/SWI complex in autoimmunity.

H3K4/H3K9me3 Bivalent Chromatin Domains Targeted by Lineage-Specific DNA Methylation Pauses Adipocyte Differentiation.

Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1, which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPß binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis.

Ubiquitination-dependent and -independent repression of target genes by SETDB1 reveal a context-dependent role for its methyltransferase activity during adipogenesis.

The lysine methyltransferase SETDB1, an enzyme responsible for methylation of histone H3 at lysine 9, plays a key role in H3K9 tri-methylation-dependent silencing of endogenous retroviruses and developmental genes. Recent studies have shown that ubiquitination of human SETDB1 complements its catalytic activity and the silencing of endogenous retroviruses in human embryonic stem cells. However, it is not known whether SETDB1 ubiquitination is essential for its other major role in epigenetic silencing of developmental gene programs. We previously showed that SETDB1 contributes to the formation of H3K4/H3K9me3 bivalent chromatin domains that keep adipogenic Cebpa and Pparg genes in a poised state for activation and restricts the differentiation potential of pre-adipocytes. Here, we show that ubiquitin-resistant K885A mutant of SETDB1 represses adipogenic genes and inhibits pre-adipocyte differentiation similar to wild-type SETDB1. We show this was due to a compensation mechanism for H3K9me3 chromatin modifications on the Cebpa locus by other H3K9 methyltransferases Suv39H1 and Suv39H2. In contrast, the K885A mutant did not repress other SETDB1 target genes such as Tril and Gas6 suggesting SETDB1 represses its target genes by two mechanisms; one that requires its ubiquitination and another that still requires SETDB1 but not its enzyme activity.

Asf1a resolves bivalent chromatin domains for the induction of lineage-specific genes during mouse embryonic stem cell differentiation.

Bivalent chromatin domains containing repressive H3K27me3 and active H3K4me3 modifications are barriers for the expression of lineage-specific genes in ES cells and must be resolved for the transcription induction of these genes during differentiation, a process that remains largely unknown. Here, we show that Asf1a, a histone chaperone involved in nucleosome assembly and disassembly, regulates the resolution of bivalent domains and activation of lineage-specific genes during mouse ES cell differentiation. Deletion of Asf1a does not affect the silencing of pluripotent genes, but compromises the expression of lineage-specific genes during ES cell differentiation. Mechanistically, the Asf1a-histone interaction, but not the role of Asf1a in nucleosome assembly, is required for gene transcription. Asf1a is recruited to several bivalent promoters, partially through association with transcription factors, and mediates nucleosome disassembly during differentiation. We suggest that Asf1a-mediated nucleosome disassembly provides a means for resolution of bivalent domain barriers for induction of lineage-specific genes during differentiation.

Intragenic CpG islands play important roles in bivalent chromatin assembly of developmental genes.

CpG, 5'-C-phosphate-G-3', islands (CGIs) have long been known for their association with enhancers, silencers, and promoters, and for their epigenetic signatures. They are maintained in embryonic stem cells (ESCs) in a poised but inactive state via the formation of bivalent chromatin containing both active and repressive marks. CGIs also occur within coding sequences, where their functional role has remained obscure. Intragenic CGIs (iCGIs) are largely absent from housekeeping genes, but they are found in all genes associated with organ development and cell lineage control. In this paper, we investigated the epigenetic status of iCGIs and found that they too reside in bivalent chromatin in ESCs. Cell type-specific DNA methylation of iCGIs in differentiated cells was linked to the loss of both the H3K4me3 and H3K27me3 marks, and disruption of physical interaction with promoter regions, resulting in transcriptional activation of key regulators of differentiation such as PAXs, HOXs, and WNTs. The differential epigenetic modification of iCGIs appears to be mediated by cell type-specific transcription factors distinct from those bound by promoter, and these transcription factors may be involved in the hypermethylation of iCGIs upon cell differentiation. iCGIs thus play a key role in the cell type-specific regulation of transcription.

Nickel exposure induces persistent mesenchymal phenotype in human lung epithelial cells through epigenetic activation of ZEB1.

Nickel (Ni) is an environmental and occupational carcinogen, and exposure to Ni is associated with lung and nasal cancers in humans. Furthermore, Ni exposure is implicated in several lung diseases including chronic inflammatory airway diseases, asthma, and fibrosis. However, the mutagenic potential of Ni is low and does not correlate with its potent toxicity and carcinogenicity. Therefore, mechanisms underlying Ni exposure-associated diseases remain poorly understood. Since the health risks of environmental exposures often continue post exposure, understanding the exposure effects that persist after the termination of exposure could provide mechanistic insights into diseases. By examining the persistent effects of Ni exposure, we report that Ni induces epithelial-mesenchymal transition (EMT) and that the mesenchymal phenotype remains irreversible even after the termination of exposure. Ni-induced EMT was dependent on the irreversible upregulation of ZEB1, an EMT master regulator, via resolution of its promoter bivalency. ZEB1, upon activation, downregulated its repressors as well as the cell-cell adhesion molecule, E-cadherin, resulting in the cells undergoing EMT and switching to persistent mesenchymal status. ZEB1 depletion in cells exposed to Ni attenuated Ni-induced EMT. Moreover, Ni exposure did not induce EMT in ZEB1-depleted cells. Activation of EMT, during which the epithelial cells lose cell-cell adhesion and become migratory and invasive, plays a major role in asthma, fibrosis, and cancer and metastasis, lung diseases associated with Ni exposure. Therefore, our finding of irreversible epigenetic activation of ZEB1 by Ni exposure and the acquisition of persistent mesenchymal phenotype would have important implications in understanding Ni-induced diseases.

A novel subtype classification and risk of breast cancer by histone modification profiling.

Breast cancer has been classified into several intrinsic molecular subtypes on the basis of genetic and epigenetic factors. However, knowledge about histone modifications that contribute to the classification and development of biologically distinct breast cancer subtypes remains limited. Here we compared the genome-wide binding patterns of H3K4me3 and H3K27me3 between human mammary epithelial cells and three breast cancer cell lines representing the luminal, HER2, and basal subtypes. We characterized thousands of unique binding events as well as bivalent chromatin signatures unique to each cancer subtype, which were involved in different epigenetic regulation programs and signaling pathways in breast cancer progression. Genes linked to the unique histone mark features exhibited subtype-specific expression patterns, both in cancer cell lines and primary tumors, some of which were confirmed by qPCR in our primary cancer samples. Finally, histone mark-based gene classifiers were significantly correlated with relapse-free survival outcomes in patients. In summary, we have provided a valuable resource for the identification of novel biomarkers of subtype classification and clinical prognosis evaluation in breast cancers.

LHP1 and INO80 cooperate with ethylene signaling for warm ambient temperature response by activating specific bivalent genes.

Ethylene signaling has been indicated as a potential positive regulator of plant warm ambient temperature response, but its underlying molecular mechanisms are largely unknown. Here, we show that LHP1 and INO80 cooperate with ethylene signaling for warm ambient temperature response by activating specific bivalent genes. We found that the presence of warm ambient temperature activates ethylene signaling through EIN2 and EIN3, leading to an interaction between LHP1 and accumulated EIN2-C to co-regulate a subset of LHP1-bound genes marked by H3K27me3 and H3K4me3 bivalency. Furthermore, we demonstrate that INO80 is recruited to bivalent genes by interacting with EIN2-C and EIN3, promoting H3K4me3 enrichment and facilitating transcriptional activation in response to a warm ambient temperature. Together, our findings illustrate a mechanism wherein ethylene signaling orchestrates LHP1 and INO80 to regulate warm ambient temperature response by activating specific bivalent genes in Arabidopsis.

A low-input high resolution sequential chromatin immunoprecipitation method captures genome-wide dynamics of bivalent chromatin.

BACKGROUND: Bivalent chromatin is an exemplar of epigenetic plasticity. This co-occurrence of active-associated H3K4me3 and inactive-associated H3K27me3 histone modifications on opposite tails of the same nucleosome occurs predominantly at promoters that are poised for future transcriptional upregulation or terminal silencing. We know little of the dynamics, resolution, and regulation of this chromatin state outside of embryonic stem cells where it was first described. This is partly due to the technical challenges distinguishing bone-fide bivalent chromatin, where both marks are on the same nucleosome, from allelic or sample heterogeneity where there is a mix of H3K4me3-only and H3K27me3-only mononucleosomes. RESULTS: Here, we present a robust and sensitive method to accurately map bivalent chromatin genome-wide, along with controls, from as little as 2 million cells. We optimized and refined the sequential ChIP protocol which uses two sequential overnight immunoprecipitation reactions to robustly purify nucleosomes that are truly bivalent and contain both H3K4me3 and H3K27me3 modifications. Our method generates high quality genome-wide maps with strong peak enrichment and low background, which can be analyzed using standard bioinformatic packages. Using this method, we detect 8,789 bivalent regions in mouse embryonic stem cells corresponding to 3,918 predominantly CpG rich and developmentally regulated gene promoters. Furthermore, profiling Dppa2/4 knockout mouse embryonic stem cells, which lose both H3K4me3 and H3K27me3 at approximately 10% of bivalent promoters, demonstrated the ability of our method to capture bivalent chromatin dynamics. CONCLUSIONS: Our optimized sequential reChIP method enables high-resolution genome-wide assessment of bivalent chromatin together with all required controls in as little as 2 million cells. We share a detailed protocol and guidelines that will enable bivalent chromatin landscapes to be generated in a range of cellular contexts, greatly enhancing our understanding of bivalent chromatin and epigenetic plasticity beyond embryonic stem cells.

Transcriptional Control of Subcutaneous Adipose Tissue by the Transcription Factor CTCF Modulates Heterogeneity in Fat Distribution in Women.

Determining the mechanism driving body fat distribution will provide insights into obesity-related health risks. We used functional genomics tools to profile the epigenomic landscape to help infer the differential transcriptional potential of apple- and pear-shaped women's subcutaneous adipose-derived stem cells (ADSCs). We found that CCCTC-binding factor (CTCF) expression and its chromatin binding were increased in ADSCs from pear donors compared to those from apple donors. Interestingly, the pear enriched CTCF binding sites were located predominantly at the active transcription start sites (TSSs) of genes with active histone marks and YY1 motifs and were also associated with pear enriched RNAPII binding. In contrast, apple enriched CTCF binding sites were mainly found at intergenic regions and when identified at TSS, they were enriched with the bivalent chromatin signatures. Altogether, we provide evidence that CTCF plays an important role in differential regulation of subcutaneous ADSCs gene expression and may influence the development of apple vs. pear body shape.

Slow RNAPII Transcription Elongation Rate, Low Levels of RNAPII Pausing, and Elevated Histone H1 Content at Promoters Associate with Higher m6A Deposition on Nascent mRNAs.

N6-methyladenosine modification (m6A) fine-tunes RNA fate in a variety of ways, thus regulating multiple fundamental biological processes. m6A writers bind to chromatin and interact with RNA polymerase II (RNAPII) during transcription. To evaluate how the dynamics of the transcription process impact m6A deposition, we studied RNAPII elongation rates in mouse embryonic stem cells with altered chromatin configurations, due to reductions in linker histone H1 content. We found that genes transcribed at slow speed are preferentially methylated and display unique signatures at their promoter region, namely high levels of histone H1, together with marks of bivalent chromatin and low RNAPII pausing. They are also highly susceptible to m6A loss upon histone H1 reduction. These results indicate that RNAPII velocity links chromatin structure and the deposition of m6A, highlighting the intricate relationship between different regulatory layers on nascent mRNA molecules.

Mcm2 promotes stem cell differentiation via its ability to bind H3-H4.

Mcm2, a subunit of the minichromosome maintenance proteins 2-7 (Mcm2-7) helicase best known for its role in DNA replication, contains a histone binding motif that facilitates the transfer of parental histones following DNA replication. Here, we show that Mcm2 is important for the differentiation of mouse embryonic stem (ES) cells. The Mcm2-2A mutation defective in histone binding shows defects in silencing of pluripotent genes and the induction of lineage-specific genes. The defects in the induction of lineage-specific genes in the mutant cells are likely, at least in part, due to reduced binding to Asf1a, a histone chaperone that binds Mcm2 and is important for nucleosome disassembly at bivalent chromatin domains containing repressive H3K27me3 and active H3K4me3 modifications during differentiation. Mcm2 localizes at transcription starting sites and the binding of Mcm2 at gene promoters is disrupted in both Mcm2-2A ES cells and neural precursor cells (NPCs). Reduced Mcm2 binding at bivalent chromatin domains in Mcm2-2A ES cells correlates with decreased chromatin accessibility at corresponding sites in NPCs. Together, our studies reveal a novel function of Mcm2 in ES cell differentiation, likely through manipulating chromatin landscapes at bivalent chromatin domains.

A novel bivalent chromatin associates with rapid induction of camalexin biosynthesis genes in response to a pathogen signal in Arabidopsis.

Temporal dynamics of gene expression underpin responses to internal and environmental stimuli. In eukaryotes, regulation of gene induction includes changing chromatin states at target genes and recruiting the transcriptional machinery that includes transcription factors. As one of the most potent defense compounds in Arabidopsis thaliana, camalexin can be rapidly induced by bacterial and fungal infections. Though several transcription factors controlling camalexin biosynthesis genes have been characterized, how the rapid activation of genes in this pathway upon a pathogen signal is enabled remains unknown. By combining publicly available epigenomic data with in vivo chromatin modification mapping, we found that camalexin biosynthesis genes are marked with two epigenetic modifications with opposite effects on gene expression, trimethylation of lysine 27 of histone 3 (H3K27me3) (repression) and acetylation of lysine 18 of histone 3 (H3K18ac) (activation), to form a previously uncharacterized type of bivalent chromatin. Mutants with reduced H3K27me3 or H3K18ac suggested that both modifications were required to determine the timing of gene expression and metabolite accumulation at an early stage of the stress response. Our study indicates that the H3K27me3-H3K18ac bivalent chromatin, which we name as kairostat, plays an important role in controlling the timely induction of gene expression upon stress stimuli in plants.

In the fight against harmful fungi and bacteria, plants have an arsenal of chemicals at their disposal. For instance, species in the crucifer family ­ which includes mustard, cabbages and the model plant Arabidopsis thaliana ­ can defend themselves with camalexin, a compound produced soon after the plant receives signals from its attacker. What controls this precise timing, however, is still unclear. For the genes that rule the production of camalexin to be 'read', interpreted, and ultimately converted into proteins, their DNA sequences first need to be physically accessible to the cell. This availability is controlled, in part, by adding or removing chemical groups onto histones, the spool-like structures which DNA wraps around. These precisely controlled modifications ultimately help to activate or repress a gene. Sometimes, activating and inhibiting chemical groups can be present in the same location, creating what is known as a bivalent chromatin domain. Zhao et al. investigated whether histone modifications regulate when A. thaliana produces camalexin in response to an attack. A combination of bioinformatics and experimental approaches highlighted two chemical modifications (one repressive, the other activating) which were present on the same histone, forming a previously unknown bivalent chromatin domain. Mutant plants which did not carry these modifications could not produce camalexin at the right time. Further experiments showed that under normal conditions, both histone modifications were present. However, when the plant was under attack, the level of repressive and activating modifications respectively decreased and increased, leading to gene activation. Together, the results by Zhao et al. suggest that both histone modifications are required for camalexin genes to respond appropriately to signals from a harmful agent. A deeper understanding of this new mechanism could, in turn, allow scientists to engineer crops that are better at resisting disease.

Stable DNMT3L overexpression in SH-SY5Y neurons recreates a facet of the genome-wide Down syndrome DNA methylation signature.

BACKGROUND: Down syndrome (DS) is characterized by a genome-wide profile of differential DNA methylation that is skewed towards hypermethylation in most tissues, including brain, and includes pan-tissue differential methylation. The molecular mechanisms involve the overexpression of genes related to DNA methylation on chromosome 21. Here, we stably overexpressed the chromosome 21 gene DNA methyltransferase 3L (DNMT3L) in the human SH-SY5Y neuroblastoma cell line and assayed DNA methylation at over 26 million CpGs by whole genome bisulfite sequencing (WGBS) at three different developmental phases (undifferentiated, differentiating, and differentiated). RESULTS: DNMT3L overexpression resulted in global CpG and CpG island hypermethylation as well as thousands of differentially methylated regions (DMRs). The DNMT3L DMRs were skewed towards hypermethylation and mapped to genes involved in neurodevelopment, cellular signaling, and gene regulation. Consensus DNMT3L DMRs showed that cell lines clustered by genotype and then differentiation phase, demonstrating sets of common genes affected across neuronal differentiation. The hypermethylated DNMT3L DMRs from all pairwise comparisons were enriched for regions of bivalent chromatin marked by H3K4me3 as well as differentially methylated sites from previous DS studies of diverse tissues. In contrast, the hypomethylated DNMT3L DMRs from all pairwise comparisons displayed a tissue-specific profile enriched for regions of heterochromatin marked by H3K9me3 during embryonic development. CONCLUSIONS: Taken together, these results support a mechanism whereby regions of bivalent chromatin that lose H3K4me3 during neuronal differentiation are targeted by excess DNMT3L and become hypermethylated. Overall, these findings demonstrate that DNMT3L overexpression during neurodevelopment recreates a facet of the genome-wide DS DNA methylation signature by targeting known genes and gene clusters that display pan-tissue differential methylation in DS.