Ubiquitination of Lysine 867 of the Human SETDB1 Protein Upregulates Its Histone H3 Lysine 9 (H3K9) Methyltransferase Activity

Abstract

Posttranslational modifications (PTMs) of proteins play a crucial role in regulating protein-protein interactions, enzyme activity, subcellular localization, and stability of the protein. SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that regulates the methylation of histone H3 on lysine 9 (H3K9), gene silencing, and transcriptional repression. The C-terminal region of SETDB1 is a key site for PTMs, and is essential for its enzyme activity in mammalian and insect cells. In this study, we aimed to evaluate more precisely the effect of PTMs on the H3K9 methyltransferase activity of SETDB1. Using mass spectrometry analysis, we show that the C-terminal region of human SETDB1 purified from insect cells is ubiquitinated. We also demonstrate that the ubiquitination of lysine 867 of the human SETDB1 is necessary for full H3K9 methyltransferase activity in mammalian cells. Finally, we show that SETDB1 ubiquitination regulates the expression of its target gene, serpin peptidase inhibitor, clade E, member 1 (SERPINE1) by methylating H3K9. These results suggest that the ubiquitination of SETDB1 at lysine 867 controls the expression of its target gene by activating its H3K9 methyltransferase activity.

ATF7IP regulates SETDB1 nuclear localization and increases its ubiquitination

“…In addition, a slight increase of total SETDB1 amount by proteasome inhibition in ATF7IP KO HEK293T cells suggests a role of ATF7IP in SETDB1 stability, which is consistent with the previous finding [25]. Based on the findings of this study and previous findings [26,27], we illustrated a model for ATF7IP-mediated SETDB1 regulation ( Fig 6). Multiple types of regulations of SETDB1 by ATF7IP may explain loss-of-function phenotypes for ATF7IP, which are similar to the phenotypes observed after the loss of SETDB1 [25].…”

“…Under the similar conditions as in the co-transfection experiments ( Fig 4A), we examined the expression levels of exogenous SETDB1 and ATF7IP in HEK293T cells by Western blot analysis and found a change in the doublet banding pattern of V5-SETDB1: transfection of V5-SETDB1 alone resulted in a doublet band, whereas co-transfection with 3xFLAG-ATF7IP was mostly associated with the appearance of the upper band of V5-SETDB1 ( Fig 5A, lane 1 vs. 2). SETDB1 has been shown to be subjected to mono-ubiquitination and the upper band corresponded to its ubiquitinated form [26,27]. Anti-Ub antibody analysis of the immunoprecipitated (IPed) V5-SETDB1 samples confirmed that the upper band, the intensity of which was increased upon co-transfection with 3xF-ATF7IP, was indeed the ubiquitinated form ( Fig 5A, lane 1 vs. 2).…”

Essential roles of Windei and nuclear monoubiquitination of Eggless/ SETDB 1 in transposon silencing

“…The domain structures and predicted Egg post‐translational modification sites are summarized in Fig C. We first immunoisolated Egg from OSCs and probed with anti‐ubiquitin (Ub) antibodies.…”

“…The domain structures [4,11,31] and predicted Egg post-translational modification sites [27][28][29]36] are summarized in Fig 1C. We first immunoisolated Egg from OSCs and probed with antiubiquitin (Ub) antibodies. The upper two of the four Egg bands were positive for ubiquitin ( Fig 1D).…”

“…Proteomic analyses predict that Egg is phosphorylated at Ser215 and Thr217 . Results observed for SETDB1 suggest that Egg might also be monoubiquitinated. However, the actual post‐translational modification(s) of Egg and their functional significance have not been elucidated.…”