R e s e a R c h a R t i c l e3 5 3 3 jci.orgVolume 125 Number 9 September 2015 levels decreased dramatically, suggesting important posttranscriptional regulation of PRMT5 expression in these cells. Since straight Prmt5-KO mice die before birth (12), we generated Prmt5-conditional KO mice by first crossing Prmt5 FLIP-OUT mice (obtained from the European Mutant Mouse Archive [EMMA]) with Flp recombinase-expressing transgenic (Tg) mice to generate Prmt5-floxed mice, whereby exon 7 of the Prmt5 time PCR (qPCR) ( Figure 1A) and Western blot analysis ( Figure 1B). Prmt5 mRNA and protein levels were readily detected in HSPCs, with little change in mRNA levels in the various stem and progenitor cell populations. However, when cells underwent myeloid, erythroid, or lymphoid differentiation, PRMT5 protein levels decreased to 5% to 24% of the levels seen in HSPCs. Although Prmt5 mRNA was maintained in differentiated B cells, its protein BM cells and found a complete loss of this modification 7 days after Cre induction ( Figure 1C), suggesting that PRMT5 is indeed the major type 2 PRMT in these cells. Deletion of PRMT5 during adult hematopoiesis leads to severe cytopenias. The deletion of PRMT5 in 2-month-old mice had a profound effect on hematopoiesis. Two weeks after the first poly(I:C) injection, the Mx1Cre + Prmt5 fl/fl mice developed severe pallor due to anemia, and most of these PRMT5-deleted mice were moribund 1 to 2 days later, requiring euthanasia. Analysis of the peripheral blood of these mice revealed severe pancytopenia, with a more than 10-fold decrease in wbc, a 5-fold decrease in rbc, and a 100-fold decrease in platelet counts 15 days after Cre induction ( Figure 1D). BM cellularity of the PRMT5-deleted mice was reduced by more than 50% on day 7 and by more than 95% on day 15 ( Figure 1E), consistent with the development of aplastic anemia ( Figure 1G). While PRMT5 loss had little effect on spleen weight (or cellularity) on day 15 (Supplemental Figure 2, A and B), the size and cellularity of the thymus were significantly reduced on day 15 gene was flanked by 2 LoxP sites (Supplemental Figure 1A; mice and Mx1Cre-negative littermate controls. To delete Prmt5 in hematopoietic cells, 2 i.p. injections of poly(I:C) (10 mg/kg on days 0 and 1) were given to both the Mx1Cre -and Mx1Cre + Prmt5-floxed mice. Prmt5 loss was confirmed by PCR analysis of genomic DNA (Supplemental Figure 1B), by quantitative qPCR to detect Prmt5 mRNA (Supplemental Figure 1C), and by Western blot analysis to detect PRMT5 protein (Supplemental Figure 1D). This injection strategy was used in all subsequent experiments. Interestingly, loss of PRMT5 triggered the loss of MEP50 protein (Supplemental Figure 1D), a cofactor that is required for the enzymatic activity of PRMT5 on histones, without changing Mep50 mRNA levels (data not shown). This suggests a probable posttranscriptional effect of PRMT5 on MEP50 protein levels. We also determined the overall level of symmetrically dimethylated arginine in PRMT5-deleted and Prmt5 Δ/Δ mice (n = 3). P...

The p53 tumor suppressor protein is a transcription factor that exerts its effects on the cell cycle via regulation of gene expression. Although the mechanism of p53-dependent transcriptional activation has been well-studied, the molecular basis for p53-mediated repression has been elusive. The E2F family of transcription factors has been implicated in regulation of cell cycle-related genes, with E2F6, E2F7, and E2F8 playing key roles in repression. In response to cellular DNA damage, E2F7, but not E2F6 or E2F8, is up-regulated in a p53-dependent manner, with p53 being sufficient to increase expression of E2F7. Indeed, p53 occupies the promoter of the E2F7 gene after genotoxic stress, consistent with E2F7 being a novel p53 target. Ablation of E2F7 expression abrogates p53-dependent repression of a subset of its targets, including E2F1 and DHFR, in response to DNA damage. Furthermore, E2F7 occupancy of the E2F1 and DHFR promoters is detected, and expression of E2F7 is sufficient to inhibit cell proliferation. Taken together, these results show that p53-dependent transcriptional up-regulation of its target, E2F7, leads to repression of relevant gene expression. In turn, this E2F7-dependent mechanism contributes to p53-dependent cell cycle arrest in response to DNA damage.

Protein arginine methyltransferase 5 (PRMT5) is overexpressed in many cancer types and is a promising therapeutic target for several of them, including leukemia and lymphoma. However, we and others have reported that PRMT5 is essential for normal physiology. This dependence may become dose limiting in a therapeutic setting, warranting the search for combinatorial approaches. Here, we report that PRMT5 depletion or inhibition impairs homologous recombination (HR) DNA repair, leading to DNA-damage accumulation, p53 activation, cell-cycle arrest, and cell death. PRMT5 symmetrically dimethylates histone and non-histone substrates, including several components of the RNA splicing machinery. We find that PRMT5 depletion or inhibition induces aberrant splicing of the multifunctional histone-modifying and DNA-repair factor TIP60/KAT5, which selectively affects its lysine acetyltransferase activity and leads to impaired HR. As HR deficiency sensitizes cells to PARP inhibitors, we demonstrate here that PRMT5 and PARP inhibitors have synergistic effects on acute myeloid leukemia cells.

The p53 tumor suppressor is a transcription factor that mediates varied cellular responses. The C terminus of p53 is subjected to multiple and diverse post-translational modifications. An attractive hypothesis is that differing sets of combinatorial modifications therein determine distinct cellular outcomes. To address this in vivo, a Trp53ΔCTD/ΔCTD mouse was generated in which the endogenous p53 is targeted and replaced with a truncated mutant lacking the C-terminal 24 amino acids. These Trp53ΔCTD/ΔCTD mice die within 2 wk post-partum with hematopoietic failure and impaired cerebellar development. Intriguingly, the C terminus acts via three distinct mechanisms to control p53-dependent gene expression depending on the tissue. First, in the bone marrow and thymus, the C terminus dampens p53 activity. Increased senescence in the Trp53ΔCTD/ΔCTD bone marrow is accompanied by up-regulation of Cdkn1 (p21). In the thymus, the C-terminal domain negatively regulates p53-dependent gene expression by inhibiting promoter occupancy. Here, the hyperactive p53ΔCTD induces apoptosis via enhanced expression of the proapoptotic Bbc3 (Puma) and Pmaip1 (Noxa). In the liver, a second mechanism prevails, since p53ΔCTD has wild-type DNA binding but impaired gene expression. Thus, the C terminus of p53 is needed in liver cells at a step subsequent to DNA binding. Finally, in the spleen, the C terminus controls p53 protein levels, with the overexpressed p53ΔCTD showing hyperactivity for gene expression. Thus, the C terminus of p53 regulates gene expression via multiple mechanisms depending on the tissue and target, and this leads to specific phenotypic effects in vivo.