Subtelomeric Chromatin in the Fission Yeast S. pombe

Telomeres play important roles in safeguarding the genome. The specialized repressive chromatin that assembles at telomeres and subtelomeric domains is key to this protective role. However, in many organisms, the repetitive nature of telomeric and subtelomeric sequences has hindered research efforts. The fission yeast S. pombe has provided an important model system for dissection of chromatin biology due to the relative ease of genetic manipulation and strong conservation of important regulatory proteins with higher eukaryotes. Telomeres and the telomere-binding shelterin complex are highly conserved with mammals, as is the assembly of constitutive heterochromatin at subtelomeres. In this review, we seek to summarize recent work detailing the assembly of distinct chromatin structures within subtelomeric domains in fission yeast. These include the heterochromatic SH subtelomeric domains, the telomere-associated sequences (TAS), and ST chromatin domains that assemble highly condensed chromatin clusters called knobs. Specifically, we review new insights into the sequence of subtelomeric domains, the distinct types of chromatin that assemble on these sequences and how histone H3 K36 modifications influence these chromatin structures. We address the interplay between the subdomains of chromatin structure and how subtelomeric chromatin is influenced by both the telomere-bound shelterin complexes and by euchromatic chromatin regulators internal to the subtelomeric domain. Finally, we demonstrate that telomere clustering, which is mediated via the condensed ST chromatin knob domains, does not depend on knob assembly within these domains but on Set2, which mediates H3K36 methylation.

Chromatin accessibility profiling in Neurospora crassa reveals molecular features associated with accessible and inaccessible chromatin

Background Regulation of chromatin accessibility and transcription are tightly coordinated processes. Studies in yeast and higher eukaryotes have described accessible chromatin regions, but little work has been done in filamentous fungi. Results Here we present a genome-scale characterization of accessible chromatin regions in Neurospora crassa, which revealed characteristic molecular features of accessible and inaccessible chromatin. We present experimental evidence of inaccessibility within heterochromatin regions in Neurospora, and we examine features of both accessible and inaccessible chromatin, including the presence of histone modifications, types of transcription, transcription factor binding, and relative nucleosome turnover rates. Chromatin accessibility is not strictly correlated with expression level. Accessible chromatin regions in the model filamentous fungus Neurospora are characterized the presence of H3K27 acetylation and commonly associated with pervasive non-coding transcription. Conversely, methylation of H3 lysine-36 catalyzed by ASH1 is correlated with inaccessible chromatin within promoter regions. Conclusions: In N. crassa, H3K27 acetylation is the most predictive histone modification for open chromatin. Conversely, our data show that H3K36 methylation is a key marker of inaccessible chromatin in gene-rich regions of the genome. Our data are consistent with an expanded role for H3K36 methylation in intergenic regions of filamentous fungi compared to the model yeasts, S. cerevisiae and S. pombe, which lack homologs of the ASH1 methyltransferase.

The incorporation loci of H3.3K36M determine its preferential prevalence in chondroblastomas

The histone H3.3K36M mutation, identified in over 90% of chondroblastoma cases, reprograms the H3K36 methylation landscape and gene expression to promote tumorigenesis. However, it’s still unclear how the H3K36M mutation preferentially occurs in the histone H3 variant H3.3 in chondroblastomas. Here, we report that H3.3K36M-, but not H3.1K36M-, mutant cells showed increased colony formation ability and differentiation defects. H3K36 methylations and enhancers were reprogrammed to different status in H3.3K36M- and H3.1K36M-mutant cells. The reprogramming of H3K36 methylation and enhancers was depended on the specific loci at which H3.3K36M and H3.1K36M were incorporated. Moreover, targeting H3K36M-mutant proteins to the chromatin inhibited the H3K36 methylation locally. Taken together, these results highlight the roles of the chromatic localization of H3.3K36M-mutant protein in the reprogramming of the epigenome and the subsequent induction of tumorigenesis, and shed light on the molecular mechanisms by which the H3K36M mutation mainly occurs in histone H3.3 in chondroblastomas.

The role of H3K36 methylation and associated methyltransferases in chromosome-specific gene regulation

In Drosophila, two chromosomes require special mechanisms to balance their transcriptional output to the rest of the genome. These are the male-specific lethal complex targeting the male X-chromosome, and Painting of fourth targeting chromosome 4. The two systems are evolutionarily linked to dosage compensation of the X-chromosome and the chromosomes involved display specific chromatin structures. Here we explore the role of histone H3 tri-methylated at lysine 36 (H3K36me3) and the associated methyltransferases in these two chromosome-specific systems. We show that the loss of Set2 impairs the MSL complex mediated dosage compensation; however, the effect is not recapitulated by H3K36 replacement and indicates an alternative target of Set2. Unexpectedly, balanced transcriptional output from the 4th chromosome requires intact H3K36 and depends on the additive functions of NSD and the Trithorax group protein Ash1. We conclude that H3K36 methylation and the associated methyltransferases are important factors to balance transcriptional output of the male X-chromosome and the 4th chromosome. Furthermore, our study highlights the pleiotropic effects of these enzymes.

H3K36 methylation and DNA-binding are critical for Ioc4 recruitment and Isw1b remodeller function

The Isw1b chromatin-remodelling complex is specifically recruited to gene bodies to help retain pre-existing histones during transcription by RNA polymerase II. Recruitment is dependent on H3K36 methylation and the Isw1b subunit Ioc4, which contains an N-terminal PWWP domain. Here, we present the crystal structure of the Ioc4-PWWP domain including a detailed functional characterization of the domain on its own as well as in the context of full-length Ioc4 and the Isw1b remodeller. Ioc4-PWWP preferentially binds H3K36me3-containing nucleosomes. The ability of the PWWP domain to bind DNA is required for this interaction. It is also promoted by the unique insertion motif present in Ioc4-PWWP. The ability to bind H3K36me3 as well as DNA are also critical for full-length Ioc4 binding to nucleosomes in vitro as well as its recruitment to gene bodies in vivo. Furthermore, a fully functional Ioc4-PWWP domain is necessary for efficient remodelling by Isw1b and the maintenance of ordered chromatin in vivo, thereby preventing intragenic transcription initiation and the production of non-coding RNAs.

HP1γ regulates H3K36 methylation and pluripotency in embryonic stem cells

The heterochromatin protein 1 (HP1) family members are canonical effectors and propagators of gene repression mediated by histone H3 lysine 9 (H3K9) methylation. HP1γ exhibits an increased interaction with active transcription elongation-associated factors in embryonic stem cells (ESCs) compared to somatic cells. However, whether this association has a functional consequence remains elusive. Here we find that genic HP1γ colocalizes and enhances enrichment of transcription elongation-associated H3K36me3 rather than H3K9me3. Unexpectedly, sustained H3K36me3 deposition is dependent on HP1γ. HP1γ-deleted ESCs display reduced H3K36me3 enrichment, concomitant with decreased expression at shared genes which function to maintain cellular homeostasis. Both the H3K9me3-binding chromodomain and histone binding ability of HP1γ are dispensable for maintaining H3K36me3 levels. Instead, the chromoshadow together with the hinge domain of HP1γ that confer protein and nucleic acid-binding ability are sufficient because they retain the ability to interact with NSD1, an H3K36 methyltransferase. HP1γ-deleted ESCs have a slower self-renewal rate and an impaired ability to differentiate towards cardiac mesoderm. Our findings reveal a requirement for HP1γ in faithful establishment of transcription elongation in ESCs, which regulates pluripotency.