Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation

Sumoylation controls expression of histone and growth control genes. (A) WI38 cells were infected with lentiviruses expressing control shCt or shUBC9 shRNAs. Five days post-selection, the expression of the indicated genes was analyzed by RT-qPCR. (B) Western blot analysis of WI38 cells expressing shCt or shUBC9 showing expression of histones H3, H2A, H2B, and H4. Tubulin and Ponceau were used as loading controls. Depletion of UBC9 and concomitant loss of global sumoylation and sumoylated SP100 are shown as controls for knockdown efficiency. (C) WI38 cells were transfected with a control siCt or siPIASY siRNAs, and the expression of the indicated genes was analyzed by RT-qPCR. (D) Affymetrix analysis of histone mRNA differential expression in retrovirally infected WI38 cells overexpressing PIASY or a control vector (WT). (E–G) As in A. (H) Forward scatter analysis (FSC) of Ubc9^+/+ (WT) and Ubc9^fl/-;T2 (KO) MEFs treated for 7 d by tamoxifen (Demarque et al. 2011) (four embryos/genotype); mean size (FSC units): 628 ± 17.5 (Ubc9^−/−) versus 594.3 ± 7.1 (Ubc9^+/+); P-value = 0.006; one representative example is shown. (I) Total protein levels as measured by OD normalized to cell number of Ubc9^+/+ (WT) and Ubc9^fl/-;T2 (KO) MEFs treated for 7 d by tamoxifen (four embryos/genotype); mean amount (μg/mL): 219.3 ± 32.9 (Ubc9^−/−) versus 193.5 ± 63.2 (Ubc9^+/+); P-value = 0.019. (J) As in A. For all RT-qPCR, experiments were carried out in triplicate and data are represented as mean ±SEM (n = 3).