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. 2023 Oct 18;51(20):11178–11196. doi: 10.1093/nar/gkad796

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Hydroxylation of SETDB1 at proline residues promotes VHL-mediated degradation. (A) Binding of Flag-tagged SETDB1 to PHD1-4 proteins in HEK293T cells analyzed by immunoprecipitation (IP) with anti-Flag antibodies followed by immunoblotting with the indicated antibodies. (B) Endogenous hydroxylated proline-containing SETDB1 in HeLa cells, captured by immunoprecipitation with an anti-OH antibody followed by immunoblotting with a SETDB1 antibody, compared with that in DMOG-treated (1 mM, 24 h) HeLa cells. (C) Interaction of SETDB1 with VHL, with or without DMOG treatment (1 mM, 12 h), assessed by immunoprecipitation with the indicated antibodies. (D) Alignment of hydroxylated proline residues of SETDB1 homologs in different organisms. Conserved proline residues are marked with red. (E) Effects of simultaneous mutation of conserved proline to alanine (3PA) in SETDB1 on physical associations between Flag-tagged SETDB1 (Flag-SETDB1) and HA-tagged VHL (HA-VHL) in HEK293T cells, assessed by immunoprecipitation with anti-Flag followed by immunoblotting with the indicated antibodies. (F) Hydroxylated proline residues of Flag-SETDB1 wild-type (WT) and 3PA mutant SETDB1 in HEK293T cells were captured via immunoprecipitation with the indicated antibodies followed by immunoblotting. (G) Effect of SETDB1 3PA mutation on SETDB1 protein in HEK293T cells under normoxia or hypoxia (1% O₂, 6 h) determined by immunoblotting with the indicated antibodies. (H) Effects of siRNA-mediated VHL depletion (siVHL) on polyubiquitination of Flag-tagged wild-type (WT) and 3PA mutant (3PA) SETDB1 in HEK293T cells determined with the ubiquitination assay as described in Supplementary Figure S1C. Non-targeting siRNA were used as a negative control. (I) Effect of 3PA mutation on polyubiquitination of Flag-tagged SETDB1 examined via an in vitro ubiquitination assay using recombinant histidine tagged-ubiquitin (His-Ub), E1, E2 and the CRL2^VHL complex, followed by immunoblotting with the indicated antibodies.