Systematic yeast two-hybrid screening identifies novel functions for SET1C/COMPASS

ABSTRACT Set1 is the catalytic subunit of SET1C or COMPASS, which methylates histone H3K4 and serves as a scaffold for the association of seven tightly bound polypeptides. We have employed yeast two-hybrid screenings to determine the interactome of Set1 and each subunit, providing a unique resource for exploring known and novel roles of the complex. Our screenings identified a multitude of potential interactors involved in chromatin regulation, DNA replication, meiotic breaks, and Ty transposition, processes previously associated with SET1C. Consistent with Set1 being an RNA-binding protein, the screens link SET1C to multiple aspects of RNA biogenesis, including pre-mRNA splicing and polyadenylation. The results reveal that several importins are candidate interactors of Set1, along with RGG motif-containing proteins, providing insights into the mechanisms by which Set1 moves between cytoplasmic and nuclear compartments. We further reveal that reconstituted SET1C interacts with the AT hook domain of the chromatin remodeler Snf2 and methylates multiple arginines within this domain. In vivo, we report that the ARTSTRGR AT-hook motif is methylated in a Set1-dependent manner revealing new interplay between lysine and arginine methylation. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵¶ Joint authors Isabella E. Maudlin, Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK., Marion Dubarry, Univ Lyon, Université Claude Bernard Lyon 1, INSA-Lyon, CNRS, UMR5240, Microbiologie, Adaptation et Pathogénie, 10 rue Raphaël Dubois, F-69622, Villeurbanne, France, Carlos A. Niño, IFOM ETS, The AIRC Institute of Molecular Oncology, Milan, Italy. Vincent Géli, Institute for Research on Cancer and Aging, Nice (IRCAN), Nice, France We present a systematic SET1C interaction map that provides a structured resource for generating and testing new hypotheses on SET1C function. We emphasise that these interactions represent a hypothesis generating resource rather than a set of validated protein-protein interactions. To reflect this, the manuscript has been carefully revised to distinguish clearly between observation and interpretation, and to avoid overstatement of the data. Accordingly, we have revised the title and the abstract. Selected examples are explored further to illustrate how candidates from the dataset can be followed up, but the primary contribution of this work is to provide a structured framework and resource that can guide future mechanistic studies of SET1C function. We thank the reviewers for their thoughtful comments. We have followed their recommendations by modifying the structure of the manuscript, removing distracting results and relocating some figures to the supplementary materials to improve the readability of the manuscript. At the same time, the reviewers acknowledge that the dataset is extensive and that aspects of the validation work are valuable. Below, we provide point-by-point responses to the public reviews and referees recommendations.