Figure 4

YY2 K247 methylation regulates YY2 DNA-binding activity. (a–d) DNA EMSA assay was performed by incubating biotinylated oligonucleotide containing YY1 consensus-binding site (a) or YY1-binding site in histone H3.2 gene-coding region (H3.2α; b), adeno-associated virus (AAV p5–60; c) or Cdc6 (Cdc6p) gene promoter region (d) with or without whole-cell lysates prepared from HeLa cells transfected with control vector or vectors expressing Flag-tagged YY2 (wt), YY2 (K247R) or YY2 (K139R). Intensity of shifted band was quantified by using Image J and was shown as indicated. (e) The expression of YY2 (wt), YY2 (K247R) and YY2 (K139R) in a–d was examined through IB with anti-Flag antibody. (f) DNA EMSA assay was performed by incubating biotinylated oligonucleotide containing YY1 consensus-binding site with whole-cell lysates prepared from control (wt), SET7/9 or LSD1 KO HeLa cells transfected with control vector or Flag-tagged YY2. (g) DNA EMSA assay was performed by incubating biotinylated oligonucleotide containing YY1 consensus-binding site with in vitro-purified bacterially expressed YY2 (wt) or YY2 (K247R) in the presence or absence of SET7/9. (h) The expression of YY2 (wt) and YY2 (K247R) in g was examined through C.B.S. (i) YY1 consensus-binding site was cloned into pGL2-luciferase vector (pGL2-YY1-luc). YY2 binding to the consensus-binding site can be examined through ChIP followed by qPCR using a primer set (P1 and P2) flanking the multiple cloning sites in pGL2 vector. (j) HeLa cells were co-transfected with pGL2-YY1-luc vector and vectors expressing HA-tagged YY2 (wt) or YY2 (K247R), followed by ChIP with anti-HA antibody and qPCR with primer set (P1+P2) as described in i. ChIP signals were presented as fold induction over wt after being normalized to input (±s.e.m., ***P<0.001). (k) The expression of YY2 (wt) and YY2 (K247R) in j was examined through IB. (l) Control (wt), SET7/9 or LSD1 KO HeLa cells were transfected with pGL2-YY1-luc vector in the presence of vector expressing HA-tagged YY2, followed by ChIP with anti-HA antibody and qPCR with primer set (P1+P2) as described in g. ChIP signals were presented as fold induction over wt cells after being normalized to input (±s.e.m., *P<0.05, ***P<0.001). (m) The expression of YY2 in l was examined through IB. (n) HeLa cells were transfected with pGL2-YY1-luc and control vector (CTL) or HA-tagged YY2 in the presence or absence of SET7/9 (wt), SET7/9 (m), LSD1 (wt) or LSD1 (m), followed by ChIP with anti-HA antibody and qPCR with primer set (P1+P2) as described in I. ChIP signals were presented as fold induction over CTL after being normalized to input (±s.e.m., **P<0.01, ***P<0.001, NS, nonsignificant). (o) The expression of YY2, SET7/9 (wt), SET7/9 (m), LSD1 (wt) and LSD1 (m) as described in n was examined by IB using antibodies as indicated.