The UBTF2 isoform mediates histone gene expression. (A) Total protein lysates from NIH3T3 cells transfected with sirEgfp, sirUbtf1/2#1, sirUbtf1/2#2, or sirUbtf1 for 48 h were analyzed by Western blotting (top panel). RNA was also extracted and 45S rRNA precursor levels were determined by reverse transcription qPCR using primers to the 5′ external transcribed region (ETS) (Supplemental Table 20). 45S rRNA levels were normalized to Gapdh mRNA and expressed as fold change relative to sirEgfp control (n = 3; Ave ± SEM). (*) P-value < 0.05 (bottom panel). (B) (Top panel) qChIP analysis of UBTF1/2 binding to histone H2a, H1, and H4 genes in NIH3T3 cells transfected with either sirEgfp, sirUbtf1/2#1, or sirUbtf1 for 48 h (n = 3; Ave ± SEM). (*) P-value < 0.05 compared to sirEgfp sample. qChIPs were performed as described in Figure 4C. (Bottom panel) G1 populations of NIH3T3 cells transfected as above were sorted as described in Figure 4A, and total RNA was extracted. Histone H2a, H1, and H4 mRNA levels were determined by reverse transcription qPCR. mRNA levels were normalized to B2m mRNA and expressed as fold change relative to the sirEgfp control (n = 4; Ave ± SEM). (*) P-value < 0.05 relative to corresponding sirEgfp samples.
