Thalidomide and its metabolite 5‐hydroxythalidomide induce teratogenicity via the cereblon neosubstrate PLZF

Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)‐containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide‐ and 5‐hydroxythalidomide‐induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell‐free protein synthesis system, PLZF was identified as a thalidomide‐dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4 CRBN in complex with thalidomide, its derivatives or 5‐hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5‐hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide‐dependent CRBN substrate SALL4, was induced by thalidomide or 5‐hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide‐induced phenotypes. Our findings implicate PLZF as an important thalidomide‐induced CRBN neosubstrate involved in thalidomide teratogenicity. SYNOPSIS Thalidomide and its derivatives induce protein degradation via the ubiquitin ligase cereblon (CRBN), and leading to teratogenic phenotypes during embryogenesis. Here, the limb development regulator PLZF is identified as a CRBN neo‐substrate mediating teratogenicity of thalidomide as well as of its primary metabolite 5‐hydroxythalidomide. CRBN binds PLZF and induces its proteasomal degradation in the presence of thalidomide or its derivatives. 5‐hydroxythalidomide induces degradation of PLZF and the known CRBN neo‐substrate SALL4. PLZF downregulation causes defects in chicken limb development. In chicken embryos, thalidomide and 5‐hydroxythalidomide induce protein degradation of PLZF but not SALL4. Graphical Abstract The vertebrate transcription factor PLZF/ZBTB16 emerges as potential key mediator of limb defects caused by CRBN ubiquitin ligase modulators, providing insight into the teratogen