Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q؊ syndrome, a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction, but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14, the ribosomal protein gene deleted in the 5q؊ syndrome, or RPS19, the most commonly mutated gene in DBA. Induction of p53 led to lineagespecific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect, whereas nutlin-3, a compound that activates p53 through inhibition of HDM2, selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome, we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53, providing a basis for the failure of erythropoiesis in the 5q؊ syndrome, DBA, and perhaps other bone marrow failure syndromes. (Blood. 2011;117(9):2567-2576) IntroductionHeterozygous deletions or mutations of ribosomal protein genes have been implicated in 2 human disorders, Diamond-Blackfan anemia (DBA) 1-3 and the 5qϪ syndrome, a subtype of myelodysplastic syndrome (MDS). 4,5 Both disorders are characterized by a severe macrocytic anemia. 2,6 In DBA, a congenital disorder, approximately 25% of patients have mutations in the RPS19 gene, 1 and mutations or deletions have been identified in at least 9 additional ribosomal protein genes. 3,7 In the 5qϪ syndrome, somatic deletion of one allele of chromosome 5q causes haploinsufficiency for the RPS14 gene. 5 A central outstanding question is how a defect in ribosomes, which are expressed in and are essential to all cells, causes a primarily erythroid phenotype.Multiple animal models have demonstrated the effects of ribosomal dysfunction on erythropoiesis and the role of p53, which is known to monitor ribosome function. 8 Morpholinos targeting RPS19 in zebrafish cause an accumulation of p53 and a block in erythropoiesis that is reversed in the absence of p53. 9 Mice with germline mutations in the RPS19 or RPS20 genes have hyperpigmented foot pads with p53 accumulation in the epidermis, a decreased hematocrit, and an increased erythrocyte mean cell volume. Crossing the RPS19 mutant mice with p53 null mice rescued the skin and hematopoietic phenotypes. 10 Finally, a conditional heterozygous deletion of the Cd74-Nid76 interval syntenic to human chromosome 5q, containing 6 genes, including RPS14, in mouse hematopoietic cells, causes a macrocytic anemia that is rescued by crossing to a p53 null background. 11Although p53 appears to play a critical role in these d...

Antibodies, which are produced by B-lineage cells, consist of immunoglobulin heavy (IgH) and light (IgL) chains that have amino-terminal variable regions and carboxy-terminal constant regions. In response to antigens, B cells undergo two types of genomic alterations to increase antibody diversity. Affinity for antigen can be increased by introduction of point mutations into IgH and IgL variable regions by somatic hypermutation. In addition, antibody effector functions can be altered by changing the expressed IgH constant region exons through IgH class switch recombination (CSR). Somatic hypermutation and CSR both require the B-cell-specific activation-induced cytidine deaminase protein (AID), which initiates these reactions through its single-stranded (ss)DNA-specific cytidine deaminase activity. In biochemical assays, replication protein A (RPA), a ssDNA-binding protein, associates with phosphorylated AID from activated B cells and enhances AID activity on transcribed double-stranded (ds)DNA containing somatic hypermutation or CSR target sequences. This AID-RPA association, which requires phosphorylation, may provide a mechanism for allowing AID to access dsDNA targets in activated B cells. Here we show that AID from B cells is phosphorylated on a consensus protein kinase A (PKA) site and that PKA is the physiological AID kinase. Thus, AID from non-lymphoid cells can be functionally phosphorylated by recombinant PKA to allow interaction with RPA and promote deamination of transcribed dsDNA substrates. Moreover, mutation of the major PKA phosphorylation site of AID preserves ssDNA deamination activity, but markedly reduces RPA-dependent dsDNA deamination activity and severely impairs the ability of AID to effect CSR in vivo. We conclude that PKA has a critical role in post-translational regulation of AID activity in B cells.

Summary Although the BBAP E3 ligase and its binding partner, BAL, are overexpressed in chemotherapy-resistant lymphomas, the role of these proteins in DNA damage responses remains undefined. Since BAL proteins modulate promoter-coupled transcription and contain structural motifs associated with chromatin remodeling and DNA repair, we reasoned that the BBAP E3 ligase might target nucleosomal proteins. Herein, we demonstrate that BBAP selectively monoubiquitylates histone H4 lysine 91 and protects cells exposed to DNA damaging agents. Disruption of BBAP-mediated monoubiquitylation of histone H4K91 is associated with the loss of chromatin-associated H4K20 methylase, mono- and dimethyl H4K20, and a delay in the kinetics of 53BP1 foci formation at sites of DNA damage. Since 53BP1 localizes to DNA damage sites, in part, via an interaction with dimethyl H4K20, these data directly implicate BBAP in the monoubiquitylation and additional post-translational modification of histone H4 and an associated DNA damage response.