Ludovic Mousseron, Despoina Chousianiti, Françis Poulat, Bérangère Legois, Reiner A. Veitia, Anne‐Laure Todeschini
FOXL2 is a forkhead transcription factor (TF) essential for granulosa-cell identity and function, yet how post-translational modifications tune its activity remains incompletely understood. Here, we show that protein kinase C (PKC) phosphorylates FOXL2 in vitro. Two of the four phosphorylation sites, notably Ser101 and Ser107, map to the forkhead DNA-recognition helix. Phosphomimetic substitutions (S → D) at these positions (S101D/S107D) abolish binding to a consensus DNA sequence recognized by FOXL2 and luciferase reporter activation, whereas alanine substitutions are rather neutral. In HeLa cells, the S101D mutant and, to a lesser extent, S107A/S107D, relocalize at least partially to nucleoli and exhibit increased mobility consistent with reduced DNA engagement. This pattern was recapitulated in stably transduced KGN granulosa cells. RNA-seq of such KGN cells revealed that S101D and a C-terminal truncation (ΔC) induce a massive loss-of-function (LOF) relative to wild-type (WT) FOXL2. The LOF affects sets of genes involved in pathways central to granulosa physiology including ECM organization, cell migration/adhesion, and MAPK cascades, whereas S101A is largely WT-like. An analysis of the FOXL2 interactome in the transduced cells by mass spectrometry (MS) showed that S101D loses numerous interactions with TFs and chromatin remodelers, and Pol I/III regulators such as UBTF and TFIIIC components, while it gains other partners. By contrast, ΔC retains many of the protein-protein contacts of WT and preferentially loses ribosomal/TFIII interactions. Together, these data allow us to hypothesize that PKC-dependent phosphorylation within the FOXL2 DNA-recognition helix would underlie a rapid, reversible switch, weakening DNA binding, redirecting subnuclear partitioning, and rewiring protein-protein interactions, thereby reshaping FOXL2-dependent gene regulation in granulosa cells.