Figure 3

SET7/9-mediated YY1 methylation regulates YY1 DNA-binding activity.

(A) DNA EMSA assay was performed by incubating biotinylated oligonucleotide containing YY1 consensus binding site (wt) (top panel) or its mutant form (m) (middle panel) with or without whole cell lysates prepared from HEK293T cells transfected with control vector or vectors expressing Flag-tagged YY1(wt), YY1(K173R), YY1(K174R), YY1(K409R) or YY1(K411R). Unlabeled oligonucleotide was included as indicated to demonstrate the specificity of YY1 binding with its consensus binding site. The binding affinity (gel intensity) was quantified by using Image J, with the ratio of lane 3:4:5:6:7:8 being 1:0.15:0.94:0.62:0.85:0.2. The expression of YY1(wt), YY1(K173R), YY1(K174R), YY1(K409R) and YY1(K411R) was examined through IB with anti-Flag antibody. (B) YY1 consensus binding site or its mutant form was cloned into pGL2-luciferase vector (pGL2-YY1(wt)-luc or pGL2-YY1(m)-luc). YY1 binding to the consensus binding site or its mutant form can be examined through ChIP using a primer set specifically targeting to upstream (P1) or downstream (P2) of multiple cloning site in pGL2 vector. (C) HeLa cells were transfected with pGL2-YY1(wt)-luc or pGL2-YY1(m)-luc vector in the presence or absence of vectors expressing Flag-tagged YY1(wt), YY1(K173R) or YY1(K411R), followed by ChIP with anti-Flag antibody and then q-PCR with the primer set (P1 + P2) as described in (B). ChIP signals were presented as percentage of inputs (±s.e.m., **P < 0.01). Experiments were repeated three times and representative data was shown. (D) HeLa cells were transfected with pGL2-YY1(wt)-luc vector together with or without Flag-tagged YY1(wt), YY1(K173R) or YY1(K411R) in the presence or absence of SET7/9, followed by ChIP with anti-Flag antibody and q-PCR with the primer set (P1 + P2) as described in (B). ChIP signals were presented as percentage of inputs (±s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001). Experiments were repeated three times and representative data was shown.