Repression of p53-target gene Bbc3/PUMA by MYSM1 is essential for the survival of hematopoietic multipotent progenitors and contributes to stem cell maintenance.

p53 is a central mediator of cellular stress responses, and its precise regulation is essential for the normal progression of hematopoiesis. MYSM1 is an epigenetic regulator essential for the maintenance of hematopoietic stem cell (HSC) function, hematopoietic progenitor survival, and lymphocyte development. We recently demonstrated that all developmental and hematopoietic phenotypes of Mysm1 deficiency are p53-mediated and rescued in the Mysm1(-/-)p53(-/-) mouse model. However, the mechanisms triggering p53 activation in Mysm1(-/-) HSPCs, and the pathways downstream of p53 driving different aspects of the Mysm1(-/-) phenotype remain unknown. Here we show the transcriptional activation of p53 stress responses in Mysm1(-/-) HSPCs. Mechanistically, we find that the MYSM1 protein associates with p53 and colocalizes to promoters of classical p53-target genes Bbc3/PUMA (p53 upregulated modulator of apoptosis) and Cdkn1a/p21. Furthermore, it antagonizes their p53-driven expression by modulating local histone modifications (H3K27ac and H3K4me3) and p53 recruitment. Using double-knockout mouse models, we establish that PUMA, but not p21, is an important mediator of p53-driven Mysm1(-/-) hematopoietic dysfunction. Specifically, Mysm1(-/-)Puma(-/-) mice show full rescue of multipotent progenitor (MPP) viability, partial rescue of HSC quiescence and function, but persistent lymphopenia. Through transcriptome analysis of Mysm1(-/-)Puma(-/-) MPPs, we demonstrate strong upregulation of other p53-induced mediators of apoptosis and cell-cycle arrest. The full viability of Mysm1(-/-)Puma(-/-) MPPs, despite strong upregulation of many other pro-apoptotic mediators, establishes PUMA as the essential non-redundant effector of p53-induced MPP apoptosis. Furthermore, we identify potential mediators of p53-dependent but PUMA-independent Mysm1(-/-)hematopoietic deficiency phenotypes. Overall, our study provides novel insight into the cell-type-specific roles of p53 and its downstream effectors in hematopoiesis using unique models of p53 hyperactivity induced by endogenous stress. We conclude that MYSM1 is a critical negative regulator of p53 transcriptional programs in hematopoiesis, and that its repression of Bbc3/PUMA expression is essential for MPP survival, and partly contributes to maintaining HSC function.

Modifying chemotherapy response by targeted inhibition of eukaryotic initiation factor 4A.

Translation is regulated predominantly at the initiation phase by several signal transduction pathways that are often usurped in human cancers, including the PI3K/Akt/mTOR axis. mTOR exerts unique administration over translation by regulating assembly of eukaryotic initiation factor (eIF) 4F, a heterotrimeric complex responsible for recruiting 40S ribosomes (and associated factors) to mRNA 5' cap structures. Hence, there is much interest in targeted therapies that block eIF4F activity to assess the consequences on tumor cell growth and chemotherapy response. We report here that hippuristanol (Hipp), a translation initiation inhibitor that selectively inhibits the eIF4F RNA helicase subunit, eIF4A, resensitizes Eµ-Myc lymphomas to DNA damaging agents, including those that overexpress eIF4E-a modifier of rapamycin responsiveness. As Mcl-1 levels are significantly affected by Hipp, combining its use with the Bcl-2 family inhibitor, ABT-737, leads to a potent synergistic response in triggering cell death in mouse and human lymphoma and leukemia cells. Suppression of eIF4AI using RNA interference also synergized with ABT-737 in murine lymphomas, highlighting eIF4AI as a therapeutic target for modulating tumor cell response to chemotherapy.

Foot-and-mouth disease virus leader proteinase: involvement of C-terminal residues in self-processing and cleavage of eIF4GI.

The leader proteinase (L(pro)) of foot-and-mouth disease virus frees itself from the nascent polyprotein, cleaving between its own C terminus and the N terminus of VP4 at the sequence Lys-Leu-Lys- downward arrow-Gly-Ala-Gly. Subsequently, the L(pro) impairs protein synthesis from capped mRNAs in the infected cell by processing a host protein, eukaryotic initiation factor 4GI, at the sequence Asn-Leu-Gly- downward arrow-Arg-Thr-Thr. A rabbit reticulocyte lysate system was used to examine the substrate specificity of L(pro) and the relationship of the two cleavage reactions. We show that L(pro) requires a basic residue at one side of the scissile bond to carry out efficient self-processing. This reaction is abrogated when leucine and lysine prior to the cleavage site are substituted by serine and glutamine, respectively. However, the cleavage of eIF4GI is unaffected by the inhibition of self-processing. Removal of the 18-amino acid C-terminal extension of L(pro) slowed eIF4GI cleavage; replacement of the C-terminal extension by unrelated amino acid sequences further delayed this cleavage. Surprisingly, wild-type L(pro) and the C-terminal variants all processed the polyprotein cleavage site in an intermolecular reaction at the same rate. However, when the polyprotein cleavage site was part of the same polypeptide chain as the wild-type Lb(pro), the rate of processing was much more rapid. These experiments strongly suggest that self-processing is an intramolecular reaction.