Genome-wide histone modification patterns in Kluyveromyces Lactis reveal evolutionary adaptation of a heterochromatin-associated mark [preprint]

The packaging of eukaryotic genomes into nucleosomes plays critical roles in all DNA-templated processes, and chromatin structure has been implicated as a key factor in the evolution of gene regulatory programs. While the functions of many histone modifications appear to be highly conserved throughout evolution, some well-studied modifications such as H3K9 and H3K27 methylation are not found in major model organisms such as Saccharomyces cerevisiae, while other modifications gain/lose regulatory functions during evolution. To study such a transition we focused on H3K9 methylation, a heterochromatin mark found in metazoans and in the fission yeast S. pombe, but which has been lost in the lineage leading to the model budding yeast S. cerevisiae. We show that this mark is present in the relatively understudied yeast Kluyveromyces lactis, a Hemiascomycete that diverged from S. cerevisiae prior to the whole-genome duplication event that played a key role in the evolution of a primarily fermentative lifestyle. We mapped genome-wide patterns of H3K9 methylation as well as several conserved modifications. We find that well-studied modifications such as H3K4me3, H3K36me3, and H3S10ph exhibit generally conserved localization patterns. Interestingly, we show H3K9 methylation in K. lactis primarily occurs over highly-transcribed regions, including both Pol2 and Pol3 transcription units. We identified the H3K9 methylase as the ortholog of Set6, whose function in S. cerevisiae is obscure. Functionally, we show that deletion of KlSet6 does not affect highly H3K9me3-marked genes, providing another example of a major disconnect between histone mark localization and function. Together, these results shed light on surprising plasticity in the function of a widespread chromatin mark

Posttranslational modifications of proteins in the pathobiology of medically relevant fungi

Peer reviewedPublisher PD

The role of multiple marks in epigenetic silencing and the emergence of a stable bivalent chromatin state

We introduce and analyze a minimal model of epigenetic silencing in budding yeast, built upon known biomolecular interactions in the system. Doing so, we identify the epigenetic marks essential for the bistability of epigenetic states. The model explicitly incorporates two key chromatin marks, namely H4K16 acetylation and H3K79 methylation, and explores whether the presence of multiple marks lead to a qualitatively different systems behavior. We find that having both modifications is important for the robustness of epigenetic silencing. Besides the silenced and transcriptionally active fate of chromatin, our model leads to a novel state with bivalent (i.e., both active and silencing) marks under certain perturbations (knock-out mutations, inhibition or enhancement of enzymatic activity). The bivalent state appears under several perturbations and is shown to result in patchy silencing. We also show that the titration effect, owing to a limited supply of silencing proteins, can result in counter-intuitive responses. The design principles of the silencing system is systematically investigated and disparate experimental observations are assessed within a single theoretical framework. Specifically, we discuss the behavior of Sir protein recruitment, spreading and stability of silenced regions in commonly-studied mutants (e.g., sas2, dot1) illuminating the controversial role of Dot1 in the systems biology of yeast silencing.Comment: Supplementary Material, 14 page

Toward a molecular understanding of yeast silent chromatin : roles for H4K16 acetylation and the Sir3 C-terminus

Discrete regions of the eukaryotic genome assume a heritable chromatin structure that is refractory to gene expression. In budding yeast, silent chromatin is characterized by the loading of the Silent Information Regulatory (Sir) proteins (Sir2, Sir3 and Sir4) onto unmodified nucleosomes. This requires the deacetylase activity of Sir2, extensive contacts between Sir3 and the nucleosome, as well as interactions between Sir proteins forming the Sir2-3-4 complex. During my PhD thesis I sought to advance our understanding of these phenomena from a molecular perspective. Previous studies of Sir-chromatin interactions made use of histone peptides and recombinant Sir protein fragments. This gave us an idea of possible interactions, but could not elucidate the role of histone modifications in the assembly of silent chromatin. This required that we examine nucleosomal arrays exposed to full length Sir proteins or the holo Sir complex. In Chapter 2, I made use of an in vitro reconstitution system, that allows the loading of Sir proteins (Sir3, Sir2-4 or Sir2-3-4) onto arrays of regularly spaced nucleosomes, to examine the impact of specific histone modifications (methylation of H3K79, acetylation of H3K56 and H4K16) on Sir protein binding and linker DNA accessibility. The “active” H4K16ac mark is thought to limit the loading of the Sir proteins to silent domain thus favoring the formation of silent regions indirectly by increasing Sir concentration locally. Strikingly, I found that the Sir2-4 subcomplex, unlike Sir3, has a slight higher affinity for H4K16ac-containing chromatin in vitro, consistent with H4K16ac being a substrate for Sir2. In addition the NAD-dependent deacetylation of H4K16ac promotes the binding of the holo Sir complex to chromatin beyond generating hypoacetylated histone tails. We conclude that the Sir2-dependent turnover of the “active” H4K16ac mark directly helps to seed repression. The tight association of the holo Sir complex within silent domains relies on the ability of Sir3 to bind unmodified nucleosomes. In addition, Sir3 dimerization is thought to reinforce and propagate silent domains. However, no Sir3 mutants that fail to dimerize were characterized to date. It was unclear which domain of Sir3 mediates dimerization in vivo. In Chapter 3, we present the X-ray crystal structure of the Sir3 extreme C-terminus (aa 840-978), which folds into a variant winged helix-turn-helix (Sir3 wH) and forms a stable homodimer through a large hydrophobic interface. Loss of wH homodimerization impairs holo Sir3 dimerization in vitro showing that the Sir3 wH module is key to Sir3-Sir3 interaction. Homodimerization mediated by the wH domain can be fully recapitulated by an unrelated bacterial homodimerization domain and is essential for stable association of the Sir2-3-4 complex with chromatin and the formation of silent chromatin in vivo

Genome-wide analysis of the effect of histone modifications on the coexpression of neighboring genes in Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Neighboring gene pairs in the genome of <it>Saccharomyces cerevisiae </it>have a tendency to be expressed at the same time. The distribution of histone modifications along chromatin fibers is suggested to be an important mechanism responsible for such coexpression. However, the extent of the contribution of histone modifications to the coexpression of neighboring genes is unclear.</p> <p>Results</p> <p>We investigated the similarity of histone modification between neighboring genes using autocorrelation analysis and composite profiles. Our analysis showed that neighboring genes had similar levels or changes of histone modifications, especially those transcribed in the same direction. The similarities, however, were restricted to 1 or 2 neighboring genes. Moreover, the expression of a gene was significantly correlated with histone modification of its neighboring gene(s), but this was limited to only 1 or 2 neighbors. Using a hidden Markov model (HMM), we found more than 2000 chromatin domains with similar acetylation changes as the cultures changed and a considerable number of these domains covered 2-4 genes. Gene pairs within domains exhibited a higher level of coexpression than random pairs and shared similar functions.</p> <p>Conclusions</p> <p>The results of this study suggest that similar histone modifications occur within only a small local chromatin region in yeast. The modifications generally have an effect on coexpression with only 1 or 2 neighboring genes. Some blocking mechanism(s) might strictly restrain the distribution of histone modifications in yeast.</p

Candida albicans repetitive elements display epigenetic diversity and plasticity

Transcriptionally silent heterochromatin is associated with repetitive DNA. It is poorly understood whether and how heterochromatin differs between different organisms and whether its structure can be remodelled in response to environmental signals. Here, we address this question by analysing the chromatin state associated with DNA repeats in the human fungal pathogen Candida albicans. Our analyses indicate that, contrary to model systems, each type of repetitive element is assembled into a distinct chromatin state. Classical Sir2-dependent hypoacetylated and hypomethylated chromatin is associated with the rDNA locus while telomeric regions are assembled into a weak heterochromatin that is only mildly hypoacetylated and hypomethylated. Major Repeat Sequences, a class of tandem repeats, are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Marker gene silencing assays and genome-wide RNA sequencing reveals that C. albicans heterochromatin represses expression of repeat-associated coding and non-coding RNAs. We find that telomeric heterochromatin is dynamic and remodelled upon an environmental change. Weak heterochromatin is associated with telomeres at 30?°C, while robust heterochromatin is assembled over these regions at 39?°C, a temperature mimicking moderate fever in the host. Thus in C. albicans, differential chromatin states controls gene expression and epigenetic plasticity is linked to adaptation

Displacement of Histones at Promoters of \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e Heat Shock Genes Is Differentially Associated with Histone H3 Acetylation

Chromatin remodeling at promoters of activated genes spans from mild histone modifications to outright displacement of nucleosomes in trans. Factors affecting these events are not always clear. Our results indicate that histone H3 acetylation associated with histone displacement differs drastically even between promoters of such closely related heat shock genes as HSP12, SSA4, and HSP82. The HSP12 promoter, with the highest level of histone displacement, showed the highest level of H3 acetylation, while the SSA4 promoter, with a lower histone displacement, showed only modest H3 acetylation. Moreover, for the HSP12 promoter, the level of acetylated H3 is temporarily increased prior to nucleosome departure. Individual promoters in strains expressing truncated versions of heat shock factor (HSF) showed that deletion of either one of two activating regions in HSF led to the diminished histone displacement and correspondingly lower H3 acetylation. The deletion of both regions simultaneously severely decreased histone displacement for all promoters tested, showing the dependence of these processes on HSF. The level of histone H3 acetylation at individual promoters in strains expressing truncated HSF also correlated with the extent of histone displacement. The beginning of chromatin remodeling coincides with the polymerase II loading on heat shock gene promoters and is regulated either by HSF binding or activation of preloaded HSF

Non-mammalian model organisms in epigenetic research : an overview

Recent advances in sequencing technology and genome editing tools had an indisputably enormous impact on our understanding of complex biological pathways and their genetic and epigenetic regulation. Unlike genetics, a study of phenotype development as a result of genotypic diversity, epigenetics studies the emergence of (possibly heritable) phenotypic assortment from one DNA sequence. Epigenetic modifications (i.e., DNA methylation, histone tail modifications, noncoding RNA interference, and many others) are diverse and can bring an additional layer of complexity to phenotype development and it's inheritance. Still, today, detailed mechanisms behind the development of epigenetic marks, their interaction, and their role in transgenerational inheritance of phenotypes are not fully understood. Therefore, chromatin biology and epigenetic research have a rich history of chasing discoveries in a variety of model organisms, including yeast, worms, flies, fish, and plants. Use of these models has opened numerous new avenues for investigation in the field. In the coming future, model organisms will continue to serve as an inseparable part of studies related to interpreting complex genomic and epigenomic data, gene–protein functional relationship, various diseases pathways, aging, and many others. Use of the model organism will provide insights not only into novel genetic players but also the profound impact of epigenetics on phenotype development. Here, we present a brief overview of the most commonly used nonmammalian model organism (i.e., fruit fly, nematode worm, zebrafish, and yeast) as potential experimental systems for epigenetic studies

Histone modifications influence the action of Snf2 family remodelling enzymes by different mechanisms

AbstractAlteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes