SPIN4 Is a Principal Endogenous Substrate of the E3 Ubiquitin Ligase DCAF16

DCAF16
is a substrate recognition component of Cullin-RING E3 ubiquitin
ligases that can be targeted by electrophilic PROTACs (proteolysis
targeting chimeras) to promote the nuclear-restricted degradation
of proteins. The endogenous protein substates of DCAF16 remain unknown.
In this study, we compared the protein content of DCAF16-wild type
and DCAF16-knockout (KO) cells by untargeted mass spectrometry-based
proteomics, identifying the Tudor domain-containing protein Spindlin-4
(SPIN4) as a protein with a level that was substantially increased
in cells lacking DCAF16. Very few other proteomic changes were found
in DCAF16-KO cells, pointing to a specific relationship between DCAF16
and SPIN4. Consistent with this hypothesis, we found that DCAF16 interacts
with and ubiquitinates SPIN4, but not other related SPIN proteins,
and identified a conserved lysine residue unique to SPIN4 that is
involved in DCAF16 binding. Finally, we provide evidence that SPIN4
preferentially binds trimethylated histone H3K4 over other modified
histone modifications. These results, taken together, indicate that
DCAF16 and SPIN4 form a dedicated E3 ligase–substrate complex
that regulates the turnover and presumed functions of SPIN4 in human
cells.