Phosphoproteomics of aged insulin-resistant bone identifies P70S6K phosphorylation of AFF4 as a gene-specific transcriptional regulator

Insulin action on the skeleton is essential for bone development and whole-body energy metabolism, however a global view of signaling in this tissue is lacking. Furthermore, whether there are signaling differences that drive the gene-specific activation under insulin-resistant (IR) or ageing conditions is unknown. Here, we perform a phosphoproteomic analysis of insulin signaling in the bones of young, lean, insulin-sensitive versus old, obese, IR mice revealing a rewiring of phosphorylation. We target dysregulated phosphoproteins in a zebrafish functional genomic screen of bone development and mineralization revealing candidates important for skeletal formation. One of these is ALF Transcription Elongation Factor 4 (AFF4), the core scaffold of the Super Elongation Complex and we show that phosphorylation of S831 on AFF4 is an insulin-dependent substrate of P70S6K and attenuated in aged, IR bone. Phosphorylation of S831 is defective in IR osteoblasts and associated with reduced transcriptional elongation at discrete locations in the genome. Mechanistically, we show phosphorylation of S831 increases recruitment of chromatin remodelers, ENL/AF9 to crotonylated histone via the YEATS domain, and promotes gene-specific activation. Our analysis identifies regulators of insulin action on the skeleton, further uncovering a mechanism of IR via locus-specific changes in transcriptional elongation and gene activation.