Figure 4.

Recruitment of HDAC3 to the rDNA Promoter by SETD5

(A) Immunoblot analysis of SETD5 and DDB1 (loading control) in control (WT) and Setd5 KO Neuro2a cells. Asterisks indicate nonspecific signals.

(B) RT-qPCR analysis of 47S and 18S rRNA in WT and Setd5 KO Neuro2a cells. Data are means ± SEM (n = 3 independent experiments). ∗p < 0.05, ∗∗p < 0.01 (Student's t test).

(C) Immunofluorescence analysis of SETD5 and UBF (nucleolar marker) in Neuro2a cells. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The boxed regions in the upper panels are shown at higher magnification in the lower panels. Scale bar, 10 μm.

(D) Immunoblot analysis of WT or Setd5 KO Neuro2a cells as well as of KO cells transfected with expression vectors for HA-tagged FL or N767 mutant forms of SETD5 (or with the empty vector, Vec). Tubulin served as a loading control.

(E) RT-qPCR analysis of 47S rRNA in Setd5 KO Neuro2a cells complemented (Comp) with FL or N767 mutant forms of SETD5 as in (D). Data are means ± SEM (n = 4 independent experiments). ∗p < 0.05 (one-way ANOVA followed by Tukey's test).

(F) ChIP-qPCR analysis of HA-SETD5 binding to the rDNA promoter in Setd5 KO Neuro2a cells complemented with FL or N767 forms of HA-SETD5 as in (D). ChIP was performed with antibodies to HA and with control immunoglobulin G (IgG). Data are means ± SEM (n = 4 independent experiments). ∗∗p < 0.01 (one-way ANOVA followed by Tukey's test).

(G) Immunoblot analysis of WT or Setd5 KO Neuro2a cells as well as of WT or KO cells modified by knockin of the DNA sequence encoding the 3×FLAG tag into the Hdac3 locus (HDAC-3×FLAG KI).

(H) ChIP-qPCR analysis of endogenous HDAC3-3×FLAG binding to the rDNA promoter in WT or Setd5 KO Neuro2a cells engineered as in (G). ChIP was performed with antibodies to FLAG and with control IgG. Data are means ± SEM (n = 3 independent experiments). ∗p < 0.05 (one-way ANOVA followed by Tukey's test).

See also Figures S7 and S10.