Dissecting the functions of the novel factors Pdp3 and Lem2 in heterochromatin regulation
Biochemistry
Evolutionary Cell and Developmental Biology (Zoology)
Final Report Abstract
The genome of eukaryotic cells is organized into distinct chromatin domains, referred to as euchromatin (transcriptionally active) and heterochromatin (transcriptionally repressed). This functional compartmentalization is reflected by the topological organization of these domains and is crucial for cellular functions and genome stability. Significant advances have been made in identifying enzymatic activities and structural components that are involved in establishing these chromatin states. However, the regulatory mechanisms that coordinate these factors are still poorly understood. Through unbiased genetic screens in the model system Schizosaccharomyces pombe (fission yeast), we isolated several new candidates that affect heterochromatic silencing. In this work, we studied how two of these new factors, Pdp3 and Lem2, control the spatial regulation of chromatin domains. Pdp3 is part of the conserved histone acetyltransferase (HAT) complex Mst2C (homologous to S. cerevisiae NuA3) and contains a putative methyl-binding PWWP domain. In collaboration with the lab of Marc Bühler (Friedrich Miescher Institute, Basel), we demonstrated that the PWWP domain of Pdp3 mediates recruitment of Mst2 to chromatin via trimethylated histone H3-Lys36 (H3K36me3), a mark associated with actively transcribed chromatin. Absence of Pdp3, or the methyltransferase Set2 that establishes H3K36me3, causes relocalization of Mst2C into heterochromatin. Promiscuous binding of Mst2C triggers a silent defect that can be suppressed by eliminating Mst2, the active subunit of the HAT complex. Vice versa, anchoring of Mst2C is also important for protecting euchromatin from the ectopic initiation of heterochromatin. Surprisingly, this function of Mst2C is mediated by acetylating a non-histone target, the ubiquitin ligase Brl1 involved in ubiquitylating the histone H2B (H2Bub). Acetylation of Brl1 increases its activity, thereby leading to increased levels of H2Bub and, as a consequence, H3K36me3. Thus, our work revealed a positive feedback loop that ensures the maintenance of distinct states of chromatin. These findings further indicate that controlling the specificity of chromatin modifiers is not only achieved by their recruitment to specific sites, but also by preventing promiscuous binding to other chromatin domains. In the second project, we studied the role of Lem2. This inner nuclear envelope protein is homologous to metazoan lamin-associated proteins (LAPs) that contain the well-conserved LEM domain. We found that Lem2 contributes to silencing of all major heterochromatic domains by acting redundantly with other factors at the nuclear periphery. Synthetic defects in silencing also correlate with mislocalization of perinuclear chromatin. Surprisingly, both functions of Lem2, repression and localization of heterochromatin, can be mechanistically separated. Whereas centromere binding and tethering to the nuclear periphery is mediated by the N-terminal part of Lem2 that includes the LEM domain, heterochromatin silencing requires exclusively the C- terminal MSC domain. Interestingly, this domain also contributes to the peripheral anchoring of telomeres, indicating that tethering of centromeres and telomeres are controlled through different mechanisms. On the level of gene silencing, Lem2 controls the proper balance between the repressor complex SHREC and the anti-silencing JmjC protein Epe1. Moreover, transcriptome analysis revealed that Lem2 also contributes to the repression of other loci that are independent of SHREC, suggesting that the MSC domain engages in multiple interactions. We propose that Lem2 coordinates the repression of various repressive pathways, revealing a more complex function for this type of LEM-containing protein than previously anticipated. In conclusion, our work revealed two complex mechanisms by which chromatin domains are spatially regulated in S. pombe. Given the strong conservation of the players involved (H3K36me3, PWWP-containing HAT complexes; LAPs), the underlying mechanisms may reflect basic principles that can likely be transferred to metazoans and humans.
Publications
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(2016). Beyond Tethering and the LEM domain: MSCellaneous functions of the inner nuclear membrane Lem2. Nucleus 7(6): 523-531
Braun S and Barrales RR
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(2016). Chromatin binding and silencing: Two roles of the same protein Lem2. Microbial Cell 3(4): 185-188
Barrales RR, Braun S
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(2016). Control of heterochromatin localization and silencing by the nuclear membrane protein Lem2. Genes Dev. 30(2): 133-48
Barrales RR, Forn M, Georgescu P, Sarkadi Z, Braun S
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(2017). The histone acetyltransferase Mst2 protects active chromatin from epigenetic silencing by acetylating the ubiquitin ligase Brl1. Mol Cell. 67(2):294-307
Flury V, Georgescu PR, Iesmantavicius V, Shimada Y, Kuzdere T, Braun S, and Bühler M
