Identifying the substrates and mechanisms of ubiquitin E3 ligases that shape the heterochromatin landscape in the fission yeast S. pombe
Final Report Abstract
The chromatin of eukaryotic cells is organized into transcriptionally active and repressed domains, referred to as euchromatin and heterochromatin. A hallmark of heterochromatin is methylation of histone H3 on Lysine 9 (H3K9me), which is mediated by the conserved Suvar3-9 family of histone methyltransferases. In the fission yeast Schizosaccharomyces pombe, the Suvar3-9 homolog is present in a complex known as CLRC, which also comprises a ubiquitin E3 ligase multimeric complex. All members of the E3 ligase complex are essential for the establishment of H3K9me, yet the substrate and function of this posttranslational modification remain elusive. In this project, we aimed to identify potential substrates of CLRC through a proteomics approach. Using the UBA trap method, which allows stable binding of the substrate via fusing the E3 to a ubiquitin binding domain (UBA), we identified Ccq1 and other members of the telomeric shelterin complex involved in capping and protecting telomeres. Ccq1 has previously been shown to recruit telomerase and another heterochromatic repressor complex called SHREC, a homolog of the mammalian NuRD complex. Surprisingly, while we found that shelterin members are ubiquitylated in vivo, their ubiquitylation and protein levels are not affected by the presence of CLRC, suggesting a different function for the interaction of CLCR and shelterin. We show that Ccq1 recruits CRLC to subtelomeric chromatin and is crucial for maintaining H3K9me levels, in agreement with two recent publications. Yet our study challenges the previous notion that H3K9me is controlled at the level of histone modification. Specifically, we demonstrate that the substrate of H3K9 methylation, histone H3, is the actual target of this regulation and that subtelomeres in general have an intrinsic low affinity for nucleosomes. This nucleosome instability is independent of the chromosomal position but linked to the DNA sequence of subtelomeres, which are rich of poly[A/T] tracts. Maintaining critical level of nucleosomes at subtelomeres requires Ccq1, which acts through recruiting the downstream factors CLRC and SHREC. Notably, SHREC contains a nuclear remodeler subunit and the remodeler activity is critical for maintaining nucleosome levels. Our data further indicate that transcriptional silencing is linked to nucleosome maintenance. Finally, our study provides evidence that the specific subtelomeric DNA sequence promotes recombination between chromosomal ends in the absence of telomerase, resembling the alternative lengthening of telomeres (ALT) maintenance mechanism in human cells. We propose that Ccq1 co-evolved with the appearance of fragile subtelomeric sequence to regulate telomere plasticity. In conclusion, while the ubiquitylation substrate of CLRC remains to be identified, our study reveals a novel regulatory mechanism that acts specifically at subtelomeric DNA sequences and controls nucleosome and genome stability. Whereas gene silencing at other heterochromatic regions is regulated through histone modifications, our findings provide evidence that repression of subtelomeric heterochromatin is predominantly regulated through nucleosome occupancy.
Publications
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(2019) Shelterin and subtelomeric DNA sequences control nucleosome maintenance and genome stability. EMBO reports 20 (1) e47181
van Emden, Thomas S.; Forn, Marta; Forné, Ignasi; Sarkadi, Zsuzsa; Capella, Matías; Martín Caballero, Lucía; Fischer-Burkart, Sabine; Brönner, Cornelia; Simonetta, Marco; Toczyski, David; Halic, Mario; Imhof, Axel; Braun, Sigurd
