Regulated nuclear entry of over-expressed Setdb1

Abstract

Setdb1 is a histone H3-lysine 9 (H3K9)-specific methyltransferase that interacts with various transcriptional regulators to induce local heterochromatin formation and participates as an indispensable component in building promyelocytic leukemia nuclear body (PML-NB), which is involved in various biological processes. We studied the effects of Setdb1 over-expression. We unexpectedly observed that exogenously expressed GFP-Setdb1 was retained in the cytoplasm, whereas endogenous Setdb1 showed a punctate nuclear signal. Leptomycin B (LMB) treatment, which blocks protein export from the nucleus, showed that entry of GFP-Setdb1 to the nucleus was regulated and that GFP-Setdb1 in the nucleus could localize at PML-NB as endogenous Setdb1. An analysis of Setdb1 deletion constructs showed that the N-terminal region was related to the nuclear export of Setdb1; supporting this, we detected two nuclear export signal motifs in this region. This N-terminal region had a SUMO interaction motif (SIM) whose mutation greatly reduced the ability of Setdb1 to associate with PML-NB and thus resulted in the disaggregation of PML-NB structure. We therefore presume that the cytoplasmic retention of over-expressed Setdb1 occurs as part of a regulatory mechanism to set a tight limit on the nuclear activity of Setdb1, whose excess activity might result in random and haphazard chromatin modifications that cause globally aberrant gene expression.