2). In addition, mitotic aberrations such as anaphase bridges were more frequently observed in the livers of TRRAP-CKO mice than in the controls (Fig. 3B,C), suggesting that cell cycle defects might be responsible for impaired hepatocyte proliferation and liver regeneration in the absence Ixazomib concentration of TRRAP. To assess whether deregulation of critical cell cycle players may be responsible for the observed decrease in hepatocyte proliferation in livers of TRRAP-CKO mice, we measured the steady-state levels of early cyclins

D and A (reliable markers of liver regeneration), cyclin-dependent kinases (cdk2 and cdk4), and CDC25A (a member of the CDC25 family of phosphatases), as well as c-Myc, a TRRAP-interacting transcription factor involved in cell cycle control. Although cyclin D1 and D2 levels in control livers increased and reached a peak between 36 and 48 hours after CCl4 treatment (Fig. 4A-C), consistent with Kinase Inhibitor Library concentration a synchronous exit of quiescent hepatocytes from G0 and entry into the cell cycle (Fig. 2), their levels were dramatically lower in TRRAP-CKO livers after CCl4 treatment, which is in agreement with impaired cell cycle reentry. Similarly, an increase in cyclins

E and cyclin A levels were also strongly counteracted in TRRAP-CKO livers after CCl4 treatment (Fig. 4A,D,E). Protein levels of other cell cycle regulators investigated (c-Myc, cdc25A, cdk2, and cdk4) were similar in both TRRAP-CKO and control livers after CCl4 treatment (Fig. 4A; Supporting Fig. 1), suggesting that expression of these cell cycle genes in regenerating livers is not controlled by TRRAP. These results show that TRRAP may be important for expression of cyclins D, A, and E and that it is dispensable for expression of cdk2 and cdk4, cdc25A, and c-Myc during liver regeneration. To elucidate the mechanism by which TRRAP regulates the expression of cell cycle regulators in liver regeneration, we next used ChIP assay to examine the status of histone acetylation and transcription factor binding within the cyclin A gene promoters after CCl4 treatment. Epothilone B (EPO906, Patupilone) Although

in TRRAP-containing livers histone H3 acetylation levels were significantly increased at different timepoints after CCl4 treatment, TRRAP-deficient cells failed to show an increase in histone H3 acetylation at later timepoints (Fig. 5A,B). Similar to histone H3, analysis of histone H4 acetylation status revealed that loss of TRRAP also compromised the increase in acetylation of histone H4 at the cyclin A promoter after CCl4 treatment (Fig. 5C). These results indicate that TRRAP is needed for the hyperacetylation of histone H3 and histone H4 associated with an increased expression of the cyclin gene in regenerating liver (Figs. 2, 4). We next examined the binding of c-Myc and E2F1 (the transcription factors known to bind TRRAP) to chromatin at the promoter of the cyclin A, a downstream target of these transcription factors.