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Final Report Summary - NUCLEAR ACTIN (Elucidating the enigmatic role of nuclear actin in genomic stability)

Project title: Elucidating the enigmatic role of nuclear actin in genomic stability
The aim of project 626708 – Nuclear Actin was to characterize the role of actin in genome stability and, if possible, to discriminate between the contributions of nuclear versus cytoplasmatic actin. The observation that actin, which function is mostly known in the cytoskeletal context, appears to play an important role in chromosome integrity was made in the laboratory of Prof. Susan Gasser a few years ago: A chemicogenic screen that tested a vast amount of small molecule drugs with yeast cells that experience replication stress due to a genetic mutation revealed that inhibition of the TORC2 complex synergistically leads to hypersensitivity of the yeast genome to Zeocin mediated damage. More specifically, inhibition of Ypk1 and Ypk2, which are downstream kinases of TORC2 that regulate actin dynamics lead to a fast, cell-cycle and apoptosis independent fragmentation of the yeast genome in the presence of Zeocin damage (Shimada et al. Mol Cell 2013). Interestingly, this phenomenon called Yeast Chromosome Shattering (YCS) could be phenocopied by Zeocin and the actin depolymerizing drug Latrunculin A, which clearly suggests that actin regulation plays an important, yet unexpected role in genomic stability.
A phosphoproteomic screen that compared phosphorylation levels of proteins under normal or chromosomal shattering (YCS) conditions indeed revealed that a majority of actin regulating proteins, such as Pan1 or the yeast WASP homologue Las17 are differentially phosphorylated upon chromosomal fragmentation. Consequently, depletion of these proteins also lead to a hypersensitivity of the yeast genome to Zeocin and this observation corroborates, that proper regulation of actin dynamics is required for genomic stability facing oxidative DNA damage.
Consistently, strong actin overexpression in the baker’s yeast itself is toxic. Overexpression of actin that is mutated for nuclear export signals overcomes the general toxicity of excessive actin levels, yet renders cells sensitive to DNA damage. It is the polymerization capacity of actin that leads to this phenotype as overexpression of non-polymerizable actin is well tolerated also in the presence of Zeocin, but an actin filament stabilizing mutant (actin S14C) gives rise to even higher DNA damage sensitivity.
The identification of a mutant actin allele in S. cerevisiae that impairs chromosome fragmentation further strengthens the body of evidence that actin itself is directly involved in the phenomenon of YCS mediated through TORC2 inhibition in presence of oxidative DNA damage. The actin mutant actin1-111 harbors three mutations (D222A, E224A, E226A) that are not directly involved in actin filament contacts and yeast cells containing only this mutant copy of actin are viable albeit slow growing. In an act1-111 genetic background, neither TORC2 inhibition nor actin depolymerization by Latrunculin A lead to chromosome fragmentation with Zeocin, but ectopic expression of plasmid encoded wild-type actin in this mutant background allows strong fragmentation. Therefore, specific point mutations on actin interfere with the synergy of actin regulation and Zeocin mediated DNA damage (Shimada, Gerhold et al., manuscript in preparation).
We were able to show that the base excision repair pathway (BER) is responsible for shattering upon actin misregulation. Depletion of this pathway impairs YCS, while deletion of enzymes important for other repair pathways, such as non-homologous end joining (NHEJ) or homologous recombination (HR) did not affect chromosomal fragmentation. Processing oxidative DNA lesions that are caused by Zeocin are processed by glycosylases and AP-endonucleases, which first remove the damaged base and then induce a single strand break to DNA. Induction of YCS through actin dynamics misregulation appears to interfere with BER either through stimulation of the endonucleolytic cleavage or through inhibition of subsequent repair steps, such as the ligation of the repaired lesion. Indeed, we could show that several BER enzymes interact with actin filaments but not with microtubules. Moreover, some of the BER enzymes are post-translationally modified upon TORC2 inhibition or LatA mediated actin depolymerization, further corroborating the link between TORC2 signaling, actin dynamics and base excision repair.
Actin-containing chromatin remodelers are also involved in YCS. A functional INO80 complex mediates shattering, while a point mutation that deletes the ATPase activity of Ino80 is sufficient to inhibit fragmentation. Along that line, deletions of INO80 subunits that regulate ATPase activity of the remodeler also strongly dampen YCS. Moreover, actin in chromatin modifiers is found in a heterodimer with actin-related protein 4 (Arp4), therefore an Arp4-actin fusion protein was constructed to selectively mutate actin in the context of chromatin remodeling. Deletion of the essential arp4 gene could be complemented by this Arp4-actin fusion protein, but already wild-type actin in the fusion strongly abrogated YCS. Therefore, the influence of actin mutants in the context of chromatin remodeling could not be sufficiently addressed by this approach.
Finally and in collaboration with the Christian Heinis group at EPFL, we developed bicyclic peptides with high affinity to actin that can also discriminate between monomeric and filamentous actin in vitro. Their characterization and usefulness as a new generation of actin probes is currently underway.
During the funding period of grant 626708 – Nuclear Actin, we were able to further characterize the relevant pathways that lead to yeast chromosome shattering and we could identify a direct role of actin in that process. As recent results from us and others suggest that actin regulation also plays an important role in mammalian repair pathways, this work contributes to the understanding of a conserved molecular mechanism that connects chromosomal stability with actin, which is one of the most abundant proteins of the cell. Its role in DNA repair is a currently emerging concept that may have profound consequences in cancer treatment. Indeed, actin depolymerization and Zeocin mediated DNA damage give rise to strong synergistic lethality in the colon cancer cell line HCT116.

Reported by

FRIEDRICH MIESCHER INSTITUTE FOR BIOMEDICAL RESEARCH
Switzerland
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