H3K36-dependent anchoring of the KAT Mst2C is required to maintain the balance between euchromatic and heterochromatic domains in S. pombe

PWWP domains are highly conserved in eukaryotes and act in recruiting histone modifiers to chromatin that is decorated by methylation. In S. cerevisiae, the NuA3b subunit Pdp3 targets this H3K14-specific HAT complex histone H3 (di- and) trimethylated at K36, which promotes transcriptional elongation. However, in its S. pombe homolog Mst2C the function and target of Pdp3 have yet remained unknown. In this doctoral thesis, I provide evidence that Mst2C functions in euchromatic and heterochromatic transcription but through entirely different means. My research revealed that the deletion of pdp3+ in S. pombe results in perturbed silencing at pericentromeric and subtelomeric heterochromatin domains. However, this is suppressed in the absence of Mst2, a catalytic subunit of Mst2C, which is also required for the functional integrity of the complex. Based on this observation, and in cooperation with the Buhler group in Basel, I studied the distribution of Mst2 and Pdp3 on chromatin. We could show that pdp3+ deletion or mutation of its PWWP domain led to a loss of Mst2 binding and its encroachment on heterochromatin, thereby demonstrating that Mst2 localization to euchromatin is dependent on Pdp3. In addition, I could reveal that the PWWP domain of Pdp3 is able to discriminate between the different methylation states of H3K36. Both binding of Mst2 and of Pdp3 was abolished in a Set2 truncation mutant, which mediates mono- and dimethylation but not trimethylation of H3K36. Lastly, my collaborators could show that in addition to H3K14, euchromatic Mst2C acetylates the HULC subunit Brl1, thereby promoting transcription and preventing the initiation of ectopic silencing. Several studies have reported that loss of Set2 results in a silencing defect itself. Through studying heterochromatic transcription in set2∆ in conjunction with deletion mutants of pdp3+, mst2+, and nto1+ and ptf2+, which are essential for Mst2C integrity, I determined that, as in pdp3∆, the silencing defect of set2∆ is solely founded in the encroachment of Mst2C on heterochromatin. Intriguingly, the deletion of any of the three critical subunits resulted in suppression below the level found in wild-type strains, implying that Mst2C is required to maintain basal transcription in heterochromatin. Together with the previous observations, this suggests that loss of Pdp3 and Set2 leads to a silencing defect via the same pathway that promotes basal transcription. Surprisingly, I found that Mst2C promotes heterochromatin transcription via an entirely different Pdp3-independent mechanism than in euchromatin, as it functions neither through Brl1 nor H3K14ac, but a yet unknown target. In conclusion, this thesis has demonstrated that Pdp3-dependent anchoring of Mst2C to H3K36me3 has a dual purpose: (a) in euchromatin, it prevents the formation of ectopic heterochromatin at regions decorated with H3K36me3 and promoting transcription in a Brl1-dependent manner; (b) in heterochromatin, this sequestration protects Mst2C-mediated but Pdp3 and Brl1-independent basal transcription from becoming hyperactivated and interfering with maintenance of this region.