Domain Model Explains Propagation Dynamics and Stability of Histone H3K27 and H3K36 Methylation Landscapes

Chromatin states must be maintained during cell proliferation to uphold cellular identity and genome integrity. Inheritance of histone modifications is central in this process. However, the histone modification landscape is challenged by incorporation of new unmodified histones during each cell cycle, and the principles governing heritability remain unclear. We take a quantitative computational modeling approach to describe propagation of histone H3K27 and H3K36 methylation states. We measure combinatorial H3K27 and H3K36 methylation patterns by quantitative mass spectrometry on subsequent generations of histones. Using model comparison, we reject active global demethylation and invoke the existence of domains defined by distinct methylation endpoints. We find that H3K27me3 on pre-existing histones stimulates the rate of de novo H3K27me3 establishment, supporting a read-write mechanism in timely chromatin restoration. Finally, we provide a detailed quantitative picture of the mutual antagonism between H3K27 and H3K36 methylation and propose that it stabilizes epigenetic states across cell division

Epigenetic regulation of Epichloë festucae secondary metabolite biosynthesis and symbiotic interaction with Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand

Histone methylation is one of several epigenetic layers for transcriptional regulation. Most studies on the importance of this histone modification in regulating fungal secondary metabolite gene expression and pathogenicity have focussed on the role of histone methyltransferases, while few studies have focussed on the role of histone demethylases that catalyse the reversal of the modification. Epichloë festucae (Ascomycota) is an endophyte that forms a mutualistic interaction with perennial ryegrass. The fungus contributes to the symbiosis by the production of several classes of secondary metabolites, these have anti-insect and/or anti-mammalian activity. The EAS and LTM clusters in E. festucae are located subtelomerically and contain the biosynthetic genes for two of these important metabolites which are only synthesised in planta. Thus, in the host plant these genes are highly expressed, but they are tightly silenced in culture conditions. Previous study has shown that histone H3K9 and H3K27 methylation and their corresponding histone methyltransferases are important for this process. In this study, the role of histone lysine demethylases (KDMs) in regulating these genes and the symbiotic interaction is described. Eight candidate histone demethylases (Jmj1-Jmj8) were identified in E. festucae, among these proteins are homologues of mammalian KDM4, KDM5, KDM8, JMDJ7, and N. crassa Dmm-1. The genes for the proteins were overexpressed in E. festucae and histone methylation levels were determined in the strains. Overexpression of the genes was not observed to cause any change to the culture and symbiotic phenotypes of the fungus. Western blot analysis subsequently identified one of the proteins, KdmB, as the histone H3K4me3 demethylase. Further analysis by ChIP- and RT-qPCR showed that demethylation of H3K4me3 by KdmB at the eas/ltm genes is crucial for the activation of these genes in planta. The full expression of several other telomeric genes was similarly found to require KdmB. On the other hand, the COMPASS H3K4 methyltransferase complex subunit CclA that is required for H3K4 trimethylation in E. festucae represses the eas/ltm genes in culture conditions by maintaining H3K4me3 levels at the loci. Thus, these findings suggest a repressive role for H3K4me3 at these subtelomeric secondary metabolite loci and are consistent with the role of H3K4me3 in yeast telomeric silencing. Disruption of kdmB did not affect the symbiotic interaction of E. festucae with the host grass but severely reduced the levels of lolitrem B, an animal neurotoxin. At the same time, the levels of ergovaline, another animal toxin, and peramine, an insect feeding deterrent, were not affected. Therefore, disruption or inhibition of KdmB may also serve as a promising approach for future endophyte improvement programmes. The E. festucae homologue of KDM8 (an H3K36me2 demethylase), Jmj4, was further investigated in this study but no H3K6 demethylase activity was found for the protein. Both disruption and overexpression of the gene encoding Jmj4 similarly had no effect on the culture and symbiotic phenotypes of E. festucae. However, deletion of setB, encoding the homologue of yeast Set2 (H3K36 methyltransferase) specifically reduced histone H3K36me3 levels in E. festucae. This contrasts with deletion of Set2 in other fungi which affected H3K36 mono-, di- and trimethylation. The ΔsetB mutant was severely impeded in development, and was unable to establish infection of the host plant. Introduction of the wild-type setB gene reversed these phenotypes. This study shows that H3K4 trimethylation controlled by CclA and KdmB is an important regulator of subtelomeric secondary metabolite genes in E. festucae but not for the symbiotic interaction of the fungus with perennial ryegrass. On the other hand, the histone H3K36 methyltransferase SetB specifically controls H3K36 trimethylation in E. festucae and is required for normal vegetative growth and ability of the fungus to infect the host plant

Identification and Characterization of Histone H3K36 Demethylases in <i>Drosophila melanogaster</i>

Covalent modifications of histones, such as acetylation, methylation, phosphorylation and ubiquitination, have an important role in regulating gene expression. Histone methylation is implicated in both gene activation and repression depending on the methylation site and the state of methylation (Li et al., 2007a). Historically, histone methylation was considered to be a static modification. Recent discoveries of histone demethylases demonstrate that histone methylation is reversible. Numerous studies have shown that dynamic regulation of histone methylation plays an important role in many cellular processes (Cloos et al., 2008). However, mechanisms governing the targeting and regulation of histone demethylation remain elusive. In this thesis, I identified two Drosophila melanogaster JmjC domain-containing proteins, dKDM4A and dDKM4B, which are histone H3K36 demethylases. Affinity purification and mass spectrometry analysis revealed that Heterochromatin Protein 1a (HP1a) associates with dKDM4A. I found that the chromoshadow domain of HP1a and a HP1-interacting motif within dKDM4A are responsible for this interaction. HP1a stimulates the histone H3K36 demethylation activity of dKDM4A and this stimulation depends on HP1a binding to the H3K9me. Loss of HP1a leads to increased level of histone H3K36me3. By chromatin immunoprecipitation using an antibody against H3K36me3 in wild type and dKDM4A mutant embryos, I identified candidate target genes of dKDM4A. A subset of dKDM4A target genes are also shown to be bound by HP1a, suggesting dKDM4A-HP1a complex may function in regulating H3K36 levels at these genes

Conservation and Function of the Histone Methyltransferase Set2

Histone methylation is an important post-translational modification involved in the regulation of eukaryotic gene expression. While many methylation sites on histone proteins have been identified to play roles in both gene activation and repression, the enzymes mediating these modifications and their exact functions are just beginning to be discovered. In the budding yeast Saccharomyces cerevisiae, methylation of histone H3 at lysine 36 (H3K36) by the histone methyltransferase Set2 has been linked to the process of transcription elongation. Previous findings indicate that through an interaction with the elongating RNA polymerase, Set2 targets H3K36 for methylation in the coding region of genes. However, the exact functions for this enzyme and its modification were largely unknown. In these studies, I demonstrate that Set2 methylation of H3K36 is highly conserved and associated with elongating RNA polymerase II in organisms distinct from budding yeast. These results reveal that Set2 and H3K36 methylation have a conserved role in the transcription elongation process. Furthermore, I have contributed to the finding that Set2 regulates global histone acetylation patterns by recruiting a small Rpd3 deacetylase (Rpd3S) complex to the coding region of genes. This is among one of the first studies to identify a functional mechanism for Set2-mediated H3K36 methylation in transcription elongation. Finally, I have identified a novel and conserved modification on H3K36. Independent of being methylated, my studies reveal H3K36 is acetylated by the transcriptional co-activator Gcn5 at promoter regions. Collectively, these results suggest that distinct modifications on H3K36 play diverse roles in the transcription process

Emerging roles of telomeric chromatin alterations in cancer

Telomeres, the nucleoprotein structures that cap the ends of eukaryotic chromosomes, play important and multiple roles in tumorigenesis. Functional telomeres need the establishment of a protective chromatin structure based on the interplay between the specific complex named shelterin and a tight nucleosomal organization. Telomere shortening in duplicating somatic cells leads eventually to the destabilization of the telomere capping structure and to the activation of a DNA damage response (DDR) signaling. The final outcome of this process is cell replicative senescence, which constitute a protective barrier against unlimited proliferation. Cells that can bypass senescence checkpoint continue to divide until a second replicative checkpoint, crisis, characterized by chromosome fusions and rearrangements leading to massive cell death by apoptosis. During crisis telomere dysfunctions can either inhibit cell replication or favor tumorigenesis by the accumulation of chromosomal rearrangements and neoplastic mutations. The acquirement of a telomere maintenance mechanism allows fixing the aberrant phenotype, and gives the neoplastic cell unlimited replicative potential, one of the main hallmarks of cancer. Despite the crucial role that telomeres play in cancer development, little is known about the epigenetic alterations of telomeric chromatin that affect telomere protection and are associated with tumorigenesis. Here we discuss the current knowledge on the role of telomeric chromatin in neoplastic transformation, with a particular focus on H3.3 mutations in alternative lengthening of telomeres (ALT) cancers and sirtuin deacetylases dysfunctions

A chromatin modifying enzyme, SDG8, is involved in morphological, gene expression, and epigenetic responses to mechanical stimulation

Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzyme, SDG8/ASHH2, which can regulate the expression of many touch responsive genes identified in Arabidopsis. SDG8 is required for the permissive expression of touch induced genes; and the loss of function of sdg8 perturbs the maximum levels of induction on selected touch gene targets. SDG8 is required to maintain permissive H3K4 trimethylation marks surrounding the Arabidopsis touch-inducible gene TOUCH 3 (TCH3), which encodes a calmodulin-like protein (CML12). The gene neighboring was also slightly down regulated, revealing a new target for SDG8 mediated chromatin modification. Finally, sdg8 mutants show perturbed morphological response to wind-agitated mechanical stimuli, implicating an epigenetic memory-forming process in the acclimation response of thigmomorphogenesis

Identification and characterization of Smyd2: a split SET/MYND domain-containing histone H3 lysine 36-specific methyltransferase that interacts with the Sin3 histone deacetylase complex

BACKGROUND: Disrupting the balance of histone lysine methylation alters the expression of genes involved in tumorigenesis including proto-oncogenes and cell cycle regulators. Methylation of lysine residues is commonly catalyzed by a family of proteins that contain the SET domain. Here, we report the identification and characterization of the SET domain-containing protein, Smyd2. RESULTS: Smyd2 mRNA is most highly expressed in heart and brain tissue, as demonstrated by northern analysis and in situ hybridization. Over-expressed Smyd2 localizes to the cytoplasm and the nucleus in 293T cells. Although accumulating evidence suggests that methylation of histone 3, lysine 36 (H3K36) is associated with actively transcribed genes, we show that the SET domain of Smyd2 mediates H3K36 dimethylation and that Smyd2 represses transcription from an SV40-luciferase reporter. Smyd2 associates specifically with the Sin3A histone deacetylase complex, which was recently linked to H3K36 methylation within the coding regions of active genes in yeast. Finally, we report that exogenous expression of Smyd2 suppresses cell proliferation. CONCLUSION: We propose that Sin3A-mediated deacetylation within the coding regions of active genes is directly linked to the histone methyltransferase activity of Smyd2. Moreover, Smyd2 appears to restrain cell proliferation, likely through direct modulation of chromatin structure

Rpd3l contributes to the DNA damage sensitivity of saccharomyces cerevisiae checkpoint mutants

DNA replication forks that are stalled by DNA damage activate an S-phase checkpoint that prevents irreversible fork arrest and cell death. The increased cell death caused by DNA damage in budding yeast cells lacking the Rad53 checkpoint protein kinase is partially suppressed by deletion of the EXO1 gene. Using a whole-genome sequencing approach, we identified two additional genes, RXT2 and RPH1, whose mutation can also partially suppress this DNA damage sensitivity. We provide evidence that RXT2 and RPH1 act in a common pathway, which is distinct from the EXO1 pathway. Analysis of additional mutants indicates that suppression works through the loss of the Rpd3L histone deacetylase complex. Our results suggest that the loss or absence of histone acetylation, perhaps at stalled forks, may contribute to cell death in the absence of a functional checkpoint.Cancer Research UK FC001066UK Medical Research Council FC001066Wellcome Trust FC001066European Molecular Biology Organization ALTF 263–2011European Research Council Advanced 669424-CHROMORE

H3K56me3 is a novel, conserved heterochromatic mark that largely but not completely overlaps with H3K9me3 in both regulation and localization.

Histone lysine (K) methylation has been shown to play a fundamental role in modulating chromatin architecture and regulation of gene expression. Here we report on the identification of histone H3K56, located at the pivotal, nucleosome DNA entry/exit point, as a novel methylation site that is evolutionary conserved. We identify trimethylation of H3K56 (H3K56me3) as a modification that is present during all cell cycle phases, with the exception of S-phase, where it is underrepresented on chromatin. H3K56me3 is a novel heterochromatin mark, since it is enriched at pericentromeres but not telomeres and is thereby similar, but not identical, to the localization of H3K9me3 and H4K20me3. Possibly due to H3 sequence similarities, Suv39h enzymes, responsible for trimethylation of H3K9, also affect methylation of H3K56. Similarly, we demonstrate that trimethylation of H3K56 is removed by members of the JMJD2 family of demethylases that also target H3K9me3. Furthermore, we identify and characterize mouse mJmjd2E and its human homolog hKDM4L as novel, functionally active enzymes that catalyze the removal of two methyl groups from trimethylated H3K9 and K56. H3K56me3 is also found in C. elegans, where it co-localizes with H3K9me3 in most, but not all, tissues. Taken together, our findings raise interesting questions regarding how methylation of H3K9 and H3K56 is regulated in different organisms and their functional roles in heterochromatin formation and/or maintenance