Histone lysine methyltransferases MpDot1 and MpSet9 are involved in the production of lovastatin and MonAzPs by histone crosstalk modification.

Increasing data suggested that histone methylation modification plays an important role in regulating biosynthesis of secondary metabolites (SMs). Monascus spp. have been applied to produce hypolipidemic drug lovastatin (also called monacolin K, MK) and edible Monascus-type azaphilone pigments (MonAzPs). However, little is known about how histone methylation regulates MK and MonAzPs. In this study, we constructed H3K9 methyltransferase deletion strain ΔMpDot1 and H4K20 methyltransferase deletion strain ΔMpSet9 using Monascus pilosus MS-1 as the parent. The result showed that deletion of MpDot1 reduced the production of MK and MonAzPs, and deletion of MpSet9 increased MonAzPs production. Real-time quantitative PCR (RT-qPCR) showed inactivation of mpdot1 and mpset9 disturbed the expression of genes responsible for the biosynthesis of MK and MonAzPs. Western blot suggested that deletion of MpDot1 reduced H3K79me and H4K16ac, and deletion of MpSet9 decreased H4K20me3 and increased H4pan acetylation. Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) showed ΔMpDot1 strain and ΔMpSet9 strain reduced the enrichment of H3K79me2 and H4K20me3 in the promoter regions of key genes for MK and MonAzPs biosynthesis, respectively. These results suggested that MpDot1 and MpSet9 affected the synthesis of SMs by regulating gene transcription and histone crosstalk, providing alternative approach for regulation of lovastatin and MonAzPs.

Phosphoserine inhibits neighboring arginine methylation in the RKS motif of histone H3.

The effects of phosphorylation of histone H3 at serine 10 have been studied in the context of other posttranslational modifications such as lysine methylation. We set out to investigate the impact of phosphoserine-10 on arginine-8 methylation. We performed methylation reactions using peptides based on histone H3 that contain a phosphorylated serine and compared the extent of arginine methylation with unmodified peptides. Results obtained via fluorography indicate that peptides containing a phosphorylated serine-10 inhibit deposition of methyl groups to arginine-8 residues. To further explore the effects of phosphoserine on neighboring arginine residues, we physically characterized the non-covalent interactions between histone H3 phosphoserine-10 and arginine-8 using 31P NMR spectroscopy. A salt bridge was detected between the negatively charged phosphoserine-10 and the positively charged unmodified arginine-8 residue. This salt bridge was not detected when arginine-8 was symmetrically dimethylated. Finally, molecular simulations not only confirm the presence of a salt bridge but also identify a subset of electrostatic interactions present when arginine is replaced with alanine. Taken together, our work suggests that the negatively charged phosphoserine maximizes its interactions. By limiting its exposure and creating new contacts with neighboring residues, it will inhibit deposition of neighboring methyl groups, not through steric hindrance, but by forming intrapeptide interactions that may mask substrate recognition. Our work provides a mechanistic framework for understanding the role of phosphoserine on nearby amino acid residues and arginine methylation.

Symmetric dimethylation on histone H4R3 associates with histone deacetylation to maintain properly polarized cell growth.

Yeast Hsl7 is recognized as a homolog of human arginine methyltransferase 5 (PRMT5) and shows type II PRMT activity by forming symmetric dimethylarginine residues on histones. Previously, we reported that Hsl7 is responsible for in vivo symmetric dimethylation on histone H4 arginine 3 (H4R3me2s) in a transcriptionally repressed state, possibly in association with histone deacetylation by Rpd3. Here, we investigated the function of Hsl7 during cell cycle progression. We found that the accumulation of Hsl7-mediated H4R3me2s is maintained by the histone deacetylase Rpd3 during transcriptional repression and that the low level of H4R3me2s is required for proper asymmetric cell growth during cell division. Our results suggest that the hypoacetylated state of histones is connected to the function of Hsl7 in regulating properly polarized cell growth during cell division and provide new insight into the epigenetic modifications that are important for cell cycle morphogenesis checkpoint control based on the repressive histone crosstalk between symmetric arginine methylation of H4 and histone deacetylation.

Structural Basis of H2B Ubiquitination-Dependent H3K4 Methylation by COMPASS.

The COMPASS (complex of proteins associated with Set1) complex represents the prototype of the SET1/MLL family of methyltransferases that controls gene transcription by H3K4 methylation (H3K4me). Although H2B monoubiquitination (H2Bub) is well known as a prerequisite histone mark for COMPASS activity, how H2Bub activates COMPASS remains unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of an extended COMPASS catalytic module (CM) bound to the H2Bub and free nucleosome. The COMPASS CM clamps onto the nucleosome disk-face via an extensive interface to capture the flexible H3 N-terminal tail. The interface also sandwiches a critical Set1 arginine-rich motif (ARM) that autoinhibits COMPASS. Unexpectedly, without enhancing COMPASS-nucleosome interaction, H2Bub activates the enzymatic assembly by packing against Swd1 and alleviating the inhibitory effect of the Set1 ARM upon fastening it to the acidic patch. By delineating the spatial configuration of the COMPASS-H2Bub-nucleosome assembly, our studies establish the structural framework for understanding the long-studied H2Bub-H3K4me histone modification crosstalk.

Structural Basis of Dot1L Stimulation by Histone H2B Lysine 120 Ubiquitination.

The essential histone H3 lysine 79 methyltransferase Dot1L regulates transcription and genomic stability and is deregulated in leukemia. The activity of Dot1L is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120Ub); however, the detailed mechanism is not understood. We report cryo-EM structures of human Dot1L bound to (1) H2BK120Ub and (2) unmodified nucleosome substrates at 3.5 Å and 4.9 Å, respectively. Comparison of both structures, complemented with biochemical experiments, provides critical insights into the mechanism of Dot1L stimulation by H2BK120Ub. Both structures show Dot1L binding to the same extended surface of the histone octamer. In yeast, this surface is used by silencing proteins involved in heterochromatin formation, explaining the mechanism of their competition with Dot1. These results provide a strong foundation for understanding conserved crosstalk between histone modifications found at actively transcribed genes and offer a general model of how ubiquitin might regulate the activity of chromatin enzymes.

Interactive and noninteractive roles of histone H2B monoubiquitination and H3K36 methylation in the regulation of active gene transcription and control of plant growth and development.

Covalent modifications of histones are essential to control a wide range of processes during development and adaptation to environmental changes. With the establishment of reference epigenomes, patterns of histone modifications were correlated with transcriptionally active or silenced genes. These patterns imply the need for the precise and dynamic coordination of different histone-modifying enzymes to control transcription at a given gene. Classically, the influence of these enzymes on gene expression is examined separately and their interplays rarely established. In Arabidopsis, HISTONE MONOUBIQUITINATION2 (HUB2) mediates H2B monoubiquitination (H2Bub1), whereas SET DOMAIN GROUP8 (SDG8) catalyzes H3 lysine 36 trimethylation (H3K36me3). In this work, we crossed hub2 with sdg8 mutants to elucidate their functional relationships. Despite similar phenotypic defects, sdg8 and hub2 mutations broadly affect genome transcription and plant growth and development synergistically. Also, whereas H3K4 methylation appears largely dependent on H2Bub1, H3K36me3 and H2Bub1 modifications mutually reinforce each other at some flowering time genes. In addition, SDG8 and HUB2 jointly antagonize the increase of the H3K27me3 repressive mark. Collectively, our data provide an important insight into the interplay between histone marks and highlight their interactive complexity in regulating chromatin landscape which might be necessary to fine-tune transcription and ensure plant developmental plasticity.

Interplay of Histone Marks with Serine ADP-Ribosylation.

Serine ADP-ribosylation (Ser-ADPr) is a recently discovered protein modification that is catalyzed by PARP1 and PARP2 when in complex with the eponymous histone PARylation factor 1 (HPF1). In addition to numerous other targets, core histone tails are primary acceptors of Ser-ADPr in the DNA damage response. Here, we show that specific canonical histone marks interfere with Ser-ADPr of neighboring residues and vice versa. Most notably, acetylation, but not methylation of H3K9, is mutually exclusive with ADPr of H3S10 in vitro and in vivo. We also broaden the O-linked ADPr spectrum by providing evidence for tyrosine ADPr on HPF1 and other proteins. Finally, we facilitate wider investigations into the interplay of histone marks with Ser-ADPr by introducing a simple approach for profiling posttranslationally modified peptides. Our findings implicate Ser-ADPr as a dynamic addition to the complex interplay of modifications that shape the histone code.

A Novel Histone Crosstalk Pathway Important for Regulation of UV-Induced DNA Damage Repair in Saccharomyces cerevisiae.

Histone post-translational modifications play vital roles in a variety of nuclear processes, including DNA repair. It has been previously shown that histone H3K79 methylation is important for the cellular response to DNA damage caused by ultraviolet (UV) radiation, with evidence that specific methylation states play distinct roles in UV repair. Here, we report that H3K79 methylation is reduced in response to UV exposure in Saccharomyces cerevisiae This reduction is specific to the dimethylated state, as trimethylation levels are minimally altered by UV exposure. Inhibition of this reduction has a deleterious effect on UV-induced sister chromatid exchange, suggesting that H3K79 dimethylation levels play a regulatory role in UV repair. Further evidence implicates an additional role for H3K79 dimethylation levels in error-free translesion synthesis, but not in UV-induced G1/S checkpoint activation or double-stranded break repair. Additionally, we find that H3K79 dimethylation levels are influenced by acetylatable lysines on the histone H4 N-terminal tail, which are hyperacetylated in response to UV exposure. Preclusion of H4 acetylation prevents UV-induced reduction of H3K79 dimethylation, and similarly has a negative effect on UV-induced sister chromatid exchange. These results point to the existence of a novel histone crosstalk pathway that is important for the regulation of UV-induced DNA damage repair.

Nucleosome competition reveals processive acetylation by the SAGA HAT module.

The Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex hyperacetylates histone tails in vivo in a manner that depends upon histone 3 lysine 4 trimethylation (H3K4me3), a histone mark enriched at promoters of actively transcribed genes. SAGA contains a separable subcomplex known as the histone acetyltransferase (HAT) module that contains the HAT, Gcn5, bound to Sgf29, Ada2, and Ada3. Sgf29 contains a tandem Tudor domain that recognizes H3K4me3-containing peptides and is required for histone hyperacetylation in vivo. However, the mechanism by which H3K4me3 recognition leads to lysine hyperacetylation is unknown, as in vitro studies show no effect of the H3K4me3 modification on histone peptide acetylation by Gcn5. To determine how H3K4me3 binding by Sgf29 leads to histone hyperacetylation by Gcn5, we used differential fluorescent labeling of histones to monitor acetylation of individual subpopulations of methylated and unmodified nucleosomes in a mixture. We find that the SAGA HAT module preferentially acetylates H3K4me3 nucleosomes in a mixture containing excess unmodified nucleosomes and that this effect requires the Tudor domain of Sgf29. The H3K4me3 mark promotes processive, multisite acetylation of histone H3 by Gcn5 that can account for the different acetylation patterns established by SAGA at promoters versus coding regions. Our results establish a model for Sgf29 function at gene promoters and define a mechanism governing crosstalk between histone modifications.

Writing and reading H2B monoubiquitylation.

Monoubiquitylation of histone H2B (H2Bub1), catalyzed by the heterodimeric ubiquitin ligase complex RNF20/40, regulates multiple molecular and biological processes. The addition of a large ubiquitin moiety to the small H2B is believed to change the biochemical features of the chromatin. H2B monoubiquitylation alters nucleosome stability, nucleosome reassembly and higher order compaction of the chromatin. While these effects explain some of the direct roles of H2Bub1, there is growing evidence that H2Bub1 can also regulate multiple DNA-templated processes indirectly, by recruitment of specific factors ("readers") to the chromatin. H2Bub1 readers mediate much of the effect of H2Bub1 on histone crosstalk, transcriptional outcome and probably other chromatin-related activities. Here we summarize the current knowledge about H2Bub1-specific readers and their role in various biological processes. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.