FIG 7.

RP58 interacts with the PRC2 complex in the developing neocortex. (A) GSEA analysis showing the significant correlation of the genes upregulated in the Rp58^−/−neuronal cells with those genes targeted by the PRC2 complex and/or identified by H3K27m3 histone mark. (B) HEK293T cells were transfected with plasmids expressing tagged proteins (hemagglutinin [HA]-EED, HA-EZH2, or HA-SUZ12) and RP58, and then cultured for 48 h posttransfection. Protein extracts were used to immunoprecipitate HA-tagged proteins from transfected cells. (Left) Twenty percent of total protein extracts were probed with an HA tag or RP58 antibodies. (Right) Fifty percent of total immunoprecipitation was used for Western blot analysis. When EZH2 and SUZ12 are cotransfected with RP58, we observe an immunoreactive band for RP58 but not for EED-RP58 cotransfection, suggesting the specificity of the interaction. Arrow indicates IgG heavy chain on the HA blot detecting the EED protein. (C) Endogenous RP58 was immunoprecipitated from proteins lysate of E18.5 cortical tissue from Rp58^+/+ and Rp58^−/− brains. Ten percent of proteins lysate was used for input lanes. RP58 forms a complex with endogenous EZH2, SUZ12, and HDAC2 in Rp58^+/+ lysate but not in Rp58^−/− lysate used as a negative control. (D) Rp58^−/− E18.5 cortices show increased H3K9/K14Ace and decreased H3K27me3. Histone modifications were normalized to total H3, and statistical analysis was done using an unpaired t test. Analysis was done on n = 3 embryos from two different litters (P < 0.01).