Mo Xu

H3K36 Methylation Antagonizes PRC2-mediated H3K27 Methylation

H3K27 methylation mediated by the histone methyltransferase complex PRC2 is critical for transcriptional regulation, Polycomb silencing, Drosophila segmentation, mammalian X chromosome inactivation, and cancer. PRC2-mediated H3K27 methylation can spread along the chromatin and propagate the repressive chromatin environment; thus, chromatin components that antagonize the activity of PRC2 are important for restraining Polycomb silencing. Here we report that in HeLa cells, H3 histones unmethylated at Lys-36 are mostly methylated at Lys-27, with the exception of newly synthesized H3. In addition, K27me3 rarely co-exists with K36me2 or K36me3 on the same histone H3 polypeptide. Moreover, PRC2 activity is greatly inhibited on nucleosomal substrates with preinstalled H3K36 methylation. These findings collectively identify H3K36 methylation as a chromatin component that restricts the PRC2-mediated spread of H3K27 methylation. Finally, we provide evidence that the controversial histone lysine methyltransferase Ash1, a known Trithorax group protein that antagonizes Polycomb silencing in vivo, is an H3K36-specific dimethylase, not an H3K4 methylase, further supporting the role of H3K36 methylation in antagonizing PRC2-mediated H3K27 methylation.

Partitioning of Histone H3-H4 Tetramers During DNA Replication-Dependent Chromatin Assembly

Histone Inheritance Chromatin, the packaging material for eukaryotic genomes, is a potential repository for epigenetic information. The core structure of chromatin is the nucleosome, which consists of an octamer of histone proteins, two dimers each of histones H2A and H2B, and histones 3 and 4. Histones 3 and 4, in particular, carry a series of covalent modifications presumed to be passed on through cell division. Using mass spectrometry of tagged and isotope labeled histones, Xu et al. (p. 94; see the Perspective byRay-Gallet and Almouzni) followed the inheritance of the histones themselves through mitosis. The H2A-H2B dimers were inherited randomly through cell division, correlating with their lack of major covalent marks. In comparison, replication-deposited H3.1-H4 dimers did not separate through cell division, implying that H3 and H4 histone modifications might be maintained by copying from neighboring preexisting histones. Intriguingly, up to one-quarter of the nonreplication-deposited H3.3-H4 dimers, which mark active chromatin, did split during cell division. Inheritance of histones H3 and H4 implies that epigenetic marks are copied between nucleosomes. Semiconservative DNA replication ensures the faithful duplication of genetic information during cell divisions. However, how epigenetic information carried by histone modifications propagates through mitotic divisions remains elusive. To address this question, the DNA replication–dependent nucleosome partition pattern must be clarified. Here, we report significant amounts of H3.3-H4 tetramers split in vivo, whereas most H3.1-H4 tetramers remained intact. Inhibiting DNA replication–dependent deposition greatly reduced the level of splitting events, which suggests that (i) the replication-independent H3.3 deposition pathway proceeds largely by cooperatively incorporating two new H3.3-H4 dimers and (ii) the majority of splitting events occurred during replication-dependent deposition. Our results support the idea that “silent” histone modifications within large heterochromatic regions are maintained by copying modifications from neighboring preexisting histones without the need for H3-H4 splitting events.

Dense Chromatin Activates Polycomb Repressive Complex 2 to Regulate H3 Lysine 27 Methylation

Maintaining Repression The Polycomb Repressive Complex 2 (PRC2) plays a critical role in gene silencing in metazoans, methylating histone H3 on lysine 27 (H3K27) to generate a repressive chromatin mark. The catalytic subunit E(z)/Ezh2 requires the presence of two other subunits—ESC/EED and Su(z)12—for enzyme activity. Yuan et al. (p. 971; see the Perspective by Pirrotta) show that both a fragment of the histone H3 N-terminal tail, and histone H1 stimulated PRC2 enzyme activity on poor, low-density chromatin substrates, indicating that that PRC2 is regulated by the density and compaction states of chromatin. The histone H3 fragment binds to the Su(z)12 subunit of PRC2 to stimulate E(z)/Ezh2. Local chromatin compaction preceded establishment of histone H3K27 methylation indicating how PRC2 might maintain the repressed state. The density and compaction state of chromatin directly regulates the activity of a transcription repressor protein complex. Polycomb repressive complex 2 (PRC2)–mediated histone H3 lysine 27 (H3K27) methylation is vital for Polycomb gene silencing, a classic epigenetic phenomenon that maintains transcriptional silencing throughout cell divisions. We report that PRC2 activity is regulated by the density of its substrate nucleosome arrays. Neighboring nucleosomes activate the PRC2 complex with a fragment of their H3 histones (Ala31 to Arg42). We also identified mutations on PRC2 subunit Su(z)12, which impair its binding and response to the activating peptide and its ability in establishing H3K27 trimethylation levels in vivo. In mouse embryonic stem cells, local chromatin compaction occurs before the formation of trimethylated H3K27 upon transcription cessation of the retinoic acid–regulated gene CYP26a1. We propose that PRC2 can sense the chromatin environment to exert its role in the maintenance of transcriptional states.

A model for mitotic inheritance of histone lysine methylation

Histone lysine methylation has been implicated in epigenetic regulation of transcription. Using stable‐isotope labelling and quantitative mass spectrometry, we analysed the dynamics of histone lysine methylation. Here we report that histone methylation levels are transiently reduced during S phase and are gradually re‐established during subsequent cell cycle stages. However, despite the recovery of overall methylation levels before the next S phase, the methylation levels of parental and newly incorporated histones differ significantly. In addition, histone methylation levels are maintained at steady states by both restriction of methyltransferase activity and the active turnover of methyl groups in cells undergoing an extended G1/S phase arrest. Finally, we propose a ‘buffer model’ that unifies the imprecise inheritance of histone methylation and the faithful maintenance of underlying gene silencing.

H3.3-H4 Tetramer Splitting Events Feature Cell-Type Specific Enhancers

Previously, we reported that little canonical (H3.1–H4)2 tetramers split to form “hybrid” tetramers consisted of old and new H3.1–H4 dimers, but approximately 10% of (H3.3–H4)2 tetramers split during each cell cycle. In this report, we mapped the H3.3 nucleosome occupancy, the H3.3 nucleosome turnover rate and H3.3 nucleosome splitting events at the genome-wide level. Interestingly, H3.3 nucleosome turnover rate at the transcription starting sites (TSS) of genes with different expression levels display a bimodal distribution rather than a linear correlation towards the transcriptional activity, suggesting genes are either active with high H3.3 nucleosome turnover or inactive with low H3.3 nucleosome turnover. H3.3 nucleosome splitting events are enriched at active genes, which are in fact better markers for active transcription than H3.3 nucleosome occupancy itself. Although both H3.3 nucleosome turnover and splitting events are enriched at active genes, these events only display a moderate positive correlation, suggesting H3.3 nucleosome splitting events are not the mere consequence of H3.3 nucleosome turnover. Surprisingly, H3.3 nucleosomes with high splitting index are remarkably enriched at enhancers in a cell-type specific manner. We propose that the H3.3 nucleosomes at enhancers may be split by an active mechanism to regulate cell-type specific transcription.

Brush and Spray: A High-Throughput Systemic Acquired Resistance Assay Suitable for Large-Scale Genetic Screening

Systemic acquired resistance (SAR) is a defense mechanism induced in the distal parts of plants after primary infection. It confers long-lasting protection against a broad spectrum of microbial pathogens. Lack of high-throughput assays has hampered the forward genetic analysis of SAR. Here, we report the development of an easy and efficient assay for SAR and its application in a forward genetic screen for SAR-deficient mutants in Arabidopsis (Arabidopsis thaliana). Using the new assay for SAR, we identified six flavin-dependent monooxygenase1, four AGD2-like defense response protein1, three salicylic acid induction-deficient2, one phytoalexin deficient4, and one avrPphB-susceptible3 alleles as well as a gain-of-function mutant of CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR3 designated camta3-3D. Like transgenic plants overexpressing CAMTA3, camta3-3D mutant plants exhibit compromised SAR and enhanced susceptibility to virulent pathogens, suggesting that CAMTA3 is a critical regulator of both basal resistance and SAR.

Epigenetic inheritance mediated by histone lysine methylation: maintaining transcriptional states without the precise restoration of marks?

‘Epigenetics’ has been defined as the study of ‘mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence’. Chromatin modifications are major carriers of epigenetic information that both reflect and affect the transcriptional states of underlying genes. Several histone modifications are key players that are responsible for classical epigenetic phenomena. However, the mechanisms by which cells pass their histone modifications to daughter cells through mitotic division remain to be unveiled. Here, we review recent progress in the field and conclude that epigenetic modifications are not precisely maintained at a near-mononucleosome level of precision. We also suggest that transcription repression may be maintained by a buffer system that can tolerate a certain degree of fluctuation in repressive histone modification levels. This buffer system protects the repressed genes from potential improper derepression triggered by chromatin modification-level fluctuation resulting from cellular events, such as the cell-cycle-dependent dilution of the chromatin modifications and local responses to environmental cues.

Symmetrical modification within a nucleosome is not required globally for histone lysine methylation

Two copies of each core histone exist in every nucleosome; however, it is not known whether both histones within a nucleosome are required to be symmetrically methylated at the same lysine residues. We report that for most lysine methylation states, wild‐type histones paired with mutant, unmethylatable histones in mononucleosomes have comparable methylation levels to bulk histones. Our results indicate that symmetrical histone methylation is not required on a global scale. However, wild‐type H4 histones paired with unmethylatable H4K20R histones showed reduced levels of H4K20me2 and H4K20me3, suggesting that some fractions of these modifications might exist symmetrically, and enzymes mediating these modifications might, to some extent, favour nucleosome substrates with premethylated H4K20.

Nucleosome assembly and epigenetic inheritance

In eukaryotic cells, histones are packaged into octameric core particles with DNA wrapping around to form nucleosomes, which are the basic units of chromatin (Kornberg and Thomas, 1974). Multicellular organisms utilise chromatin marks to translate one single genome into hundreds of epigenomes for their corresponding cell types. Inheritance of epigenetic status is critical for the maintenance of gene expression profile during mitotic cell divisions (Allis et al., 2006). During S phase, canonical histones are deposited onto DNA in a replication-coupled manner (Allis et al., 2006). To understand how dividing cells overcome the dilution of epigenetic marks after chromatin duplication, DNA replication coupled (RC) nucleosome assembly has been of great interest. In this review, we focus on the potential influence of RC nucleosome assembly processes on the maintenance of epigenetic status.

A systematic evaluation of the compatibility of histones containing methyl-lysine analogues with biochemical reactions.

A systematic evaluation of the compatibility of histones containing methyl-lysine analogues with biochemical reactions