Figure 3. Creation of IDKO mice. (A) A Western blot using nuclear extracts prepared from ventricles of the indicated genotypes and demonstrating reduc- tions in Rb protein levels after TAM treatment. (B) Expression of fetal cardiac-specific genes is up-regulated specifically in IDKO hearts. Semiquantitative RT-PCR was performed on total RNA extracted from purified ACMs with the indicated genotypes. For quantitation of changes in gene expression, see Fig. S3 A. CaA, cardiac actin; SkA, skeletal actin. (C) Hematoxylin and eosin staining of myocardial sections from mice with the indicated genotypes 1 wk after TAM treatment. Bars, 40 µm. Depletion of Rb and p130 in ACMs derepresses G2/M and cytokinesis genes and disrupts heterochromatin aggregation To determine whether Rb and p130 are involved in cell cycle gene silencing in postmitotic ACMs, expression of E2F-dependent genes was examined in IDKO mice. There was a 7–27-fold increase in expression of cell cycle genes in IDKO hearts com- pared with control mice, including genes involved in regulating G2/M and cytokinesis, which were stably silenced in control ACMs (Figs. 4 A and S4 A). Phosphorylation of H3S10, which is characteristic of G2/M, was also increased in IDKO hearts, but there was no change in global levels of H3K9me3 and H3K27me3 (Figs. 4 B and S4 [B and C]). Levels of H3K9me2, as well as G9a, the H3k9me2-specific methyltransferase, were decreased in ACMs from single Rb-deficient or IDKO mice, correlating with the up-regulation of G1/S cell cycle genes (cyclin E1 and E2F), which were regulated with hypertrophic stimuli. However, Rb-deficient ACMs do not reenter the cell cycle after growth stimuli, indicating that H3K9me2 is not suf- ficient for permanent cell cycle arrest. This reexpression of proliferation-promoting genes was associated with a marked reduction in the number of ACMs displaying heterochromatin aggregation (90 ± 1% in control ACMs vs. 14 ± 2% in IDKO ACMs, P < 0.01; Fig. 4, C and D). Consistent with the loss of heterochromatin, H3K9me3 immunostaining was diffuse in IDKO myocyte nuclei (Fig. S4 B). To determine whether depleting Rb and p130 also altered H3K9me3 or H3K27me3 at E2F-dependent promoters, isolated ACMs from IDKO and control hearts were cross-linked, and H3K9me3- and H3K27me3-associated promoter sequences were determined by ChIP. As shown, H3K9me3 and H3K27me3 levels at these E2F-dependent pro- moters remained unchanged (Fig. 4, E and F), suggesting that Rb and p130 are dispensable for maintaining these two types of histone modifications in ACMs. 412 JCB • VOLUME 194 • NUMBER 3 • 2011