ALBERT

Description: V(D)J recombination, the process by which antigen receptor genes are assembled from discrete DNA segments during lymphoid development, is responsible for the generation of the primary immune repertoire. Errors in V(D)J recombination have been implicated in the pathogenesis of lymphoid malignancies, including follicular lymphoma, MALT lymphoma and mantle cell lymphoma. V(D)J recombination is initiated by a specialized transposase, RAG, consisting of RAG-1 and RAG-2 subunits. RAG mobilizes participating gene segments in a site-specific fashion by cleaving DNA at conserved recombination signal sequences. The accessibility of these sequences to RAG is subject to locus- and developmental stage-specific control by mechanisms that are as yet poorly understood. Elucidation of these mechanisms is fundamental to our understanding of the off-target events linking RAG activity to tumorigenesis. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including trimethylation of histone H3 at lysine 4 (H3K4me3). RAG-2 contains a plant homeodomain (PHD) finger that binds specifically to H3K4me3. Disruption of this PHD finger impairs V(D)J recombination in vivo. Peptides bearing H3K4me3 stimulate substrate binding and catalysis of DNA cleavage by RAG. This stimulation is dependent on an intact PHD finger, suggesting that H3K4me3 is an allosteric activator of the V(D)J recombinase. Indeed, binding of H3K4me3 to the RAG-2 PHD induces dynamic conformational changes in RAG-1. Because substrate binding and catalysis are functions of RAG-1, information regarding occupancy of the RAG-2 PHD must be transmitted to the RAG-1 subunit. To understand how the recognition of active chromatin is coupled to the binding and cleavage of recombination signal sequences, we sought to trace the path of allostery from the RAG-2 PHD finger to RAG-1. Our strategy has been: (1) to generate chimeric RAG-2 proteins in which the mouse PHD finger is replaced by the PHD finger of a phylogenetically distant RAG-2; (2) to identify chimeric RAG-2 proteins that are capable of binding H3K4me3 but incapable of allosteric activation; (3) to systematically back-mutate residues in the foreign PHD to the mouse sequence; and (4) to identify back-mutations that rescue allosteric activation. A chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the bamboo shark, C. punctatum, fails to support V(D)J recombination in vivo. This chimeric protein retains the ability to bind H3K4me3 but engagement of H3K4me3 does not result in allosteric activation, suggesting that the allosteric interface of the PHD finger is disrupted. The amino acid sequence differences between mouse and C. punctatum form several clusters, located on the opposite side of the PHD from the H3K4me3 binding site. Each of these clusters in the C. punctatum PHD finger was mutated to the mouse sequence and the corresponding back-mutated chimeric RAG-2 proteins were tested for their ability to support V(D)J recombination. Strikingly, mutation of one such cluster, corresponding to residues 425 - 429, 431 and 433 of mouse RAG-2, was sufficient to rescue recombination activity to the level of wild-type. Taken together, our observations indicate that the binding of H3K4me3 by RAG-2 is itself insufficient to support recombination; rather, information regarding the engagement of H3K4me3 must be transmitted allosterically. Moreover, our mutational analysis has identified a putative allosteric surface within the PHD finger and distinct from the H3K4me3 binding site that is responsible for transmitting the allosteric signal. The requirement for allosteric activation by H3K4me3 may play a role in defining patterns of RAG-mediated DNA cleavage during normal development and in the generation of lymphoid malignancies. Disclosures Desiderio: Genentech: Consultancy; AbbVie: Consultancy.

Description: V(D)J recombination, the process by which antigen receptor genes are assembled, is implicated in the pathogenesis of lymphoid malignancies. Rearrangement is initiated by the RAG recombinase, a heterotetrameric complex consisting of two subunits each of RAG-1 and RAG-2, which cleaves participating DNA segments at conserved signal sequences. The susceptibility of gene segments to undergo recombination is positively correlated with active chromatin marks, such as tri-methylation of histone H3 at lysine 4 (H3K4me3). RAG-2 interacts directly with H3K4me3 through a plant homeodomain (PHD), and this interaction is required for efficient V(D)J recombination in vivo. Genetic and biochemical evidence suggest that H3K4me3 stimulates substrate binding and DNA cleavage by RAG through an allosteric mechanism. This predicted that H3K4me3 binding induces conformational changes in RAG-2 that are transmitted to RAG-1. To determine whether the conformation of the RAG complex is altered by H3K4me3, we mapped changes in solvent accessibility of cysteine thiols using pulse-alkylation mass spectrometry. Binding of H3K4me3 to the RAG-2 PHD induces conformational changes in RAG-1 within the DNA-binding domains and in the ZnH2 domain, which comprises part of the scaffold for the catalytic center. Our results demonstrate that engagement of the RAG-2 PHD induces dynamic conformational changes in the RAG-1 catalytic subunit, consistent with a model of allosteric control of RAG by active chromatin. Disclosures No relevant conflicts of interest to declare.

Description: We have generated an FLT3/ITD knock-in mouse model in which mice with an FLT3/ITD mutation develop myeloproliferative disease (MPD) and a block in early B-lymphocyte development. To elucidate the role of FLT3/ITD signaling in B-cell development, we studied VDJ recombination in the pro-B cells of FLT3/ITD mice and discovered an increased frequency of DNA double strand breaks (DSBs) introduced by the VDJ recombinase. Early pro-B cells from FLT3/ITD mice were found to have a lower efficiency and decreased accuracy of DSB repair by nonhomologous end joining (NHEJ), which is required for rejoining DSBs during VDJ recombination. Reduced NHEJ repair probably results from reduced expression of Ku86, a key component of the classic DNA-PK-dependent NHEJ pathway. In compensation, early pro-B cells from FLT3/ITD cells mice show increased levels of the alternative, and highly error-prone, NHEJ pathway protein PARP1, explaining the increase in repair errors. These data suggest that, in early pro-B cells from FLT3/ITD mice, impairment of classic NHEJ decreases the ability of cells to complete postcleavage DSB ligation, resulting in failure to complete VDJ recombination and subsequent block of B-lymphocyte maturation. These findings might explain the poor prognosis of leukemia patients with constitutive activation of FLT3 signaling.

Description: Abstract 184 Constitutive activation of FLT3 by internal tandem duplication (ITD) mutations is one of the most common molecular alterations found in up to 35% of acute myeloid leukemia (AML) and 3% of acute lymphoid leukemia (ALL). Its roles in leukemic transformation of normal hematopoietic stem/ progenitor cells (HSPCs) are not yet fully understood. We have generated a FLT3/ITD knock-in mouse model in which mice heterozygous for a FLT3/ITD mutation develop myeloid proliferation and a block in B-lymphocyte development at the Pro-B stage. To investigate the mechanisms for this block in lymphoid development, we studied V(D)J recombination in the B-lineage cells from the bone marrow (BM) of FLT3/ITD mice. The ligation-mediated PCR assay showed that Pro-B cells from FLT3/ITD mice accumulated high levels of signal-end recombination intermediates, generated by DNA cleavage at the DJH recombination signal sequences (RSSs), with a concomitant decrease in the frequency of completed DJH rearrangements when compared to wild-type littermate controls. These observations are consistent with decreased repair of double strand breaks (DSBs) introduced by the V(D)J recombinase in B cell progenitors from FLT3/ITD mice, resulting in impaired development beyond the Pro-B stage. The classical Ku and DNA-PK-dependent non-homologous end-joining (NHEJ) pathway is required for rejoining DSBs during V(D)J recombination. In vivo NHEJ assays demonstrate that BM cells, including Pro-B cells, with FLT3/ITD mutations have a lower efficiency and decreased accuracy of repair of DSBs, suggesting a defect in the classical NHEJ pathway. Quantitative RT-PCR and Western blotting analysis of Pro-B cells from FLT3/ITD mice demonstrate greatly reduced expression of Ku70/Ku86 and Ligase IV, key components of the the classical NHEJ pathway, confirming the idea that Classical NHEJ is defective. We and others have recently demonstrated that alternative (ALT) NHEJ pathway(s) compensate for deficiencies in classical NHEJ. Components of this pathway include DNA ligase III/XRCC1 and poly (ADP) ribose polymerase 1 (PARP1). Indeed, we show by Western blotting analysis that steady state levels of ALT NHEJ components DNA ligase III and PARP1 are increased in FLT3/ITD cells, compared with wild-type controls. These data suggest that during the process of DJH recombination in Pro-B cells from FLT3/ITD mice, impairment of the classical NHEJ pathway decreases the ability of cells to complete post-cleavage DSB ligation, resulting in failure to complete DJH recombination with a subsequent block of Pro-B cell maturation. With the classical NHEJ pathway impaired, BM cells from mice with FLT3/ITD mutations appear to repair DSBs through the highly error-prone ALT NHEJ pathway. The resultant increased genomic instability might underlie an important mechanism for the leukemic transformation of normal HSPCs by FLT3/ITD mutations and explain the poor prognosis of AML patients with this mutation. Disclosures: No relevant conflicts of interest to declare.

Description: The role of hedgehog (Hh) signaling in B lymphopoiesis has remained unclear. We observed that the proliferation of pro-B cells in stromal cocultures was impaired by interruption of Hh signaling, prompting us to investigate whether the target of Hh antagonism was intrinsic or extrinsic to the B-lymphoid compartment. In the present study, using conditional deletion of the pathway activator gene Smo, we found that cell-autonomous Hh signaling is dispensable for B-cell development, B-lymphoid repopulation of the BM, and humoral immune function. In contrast, depletion of the Smo protein from stromal cells was associated with impaired generation of B-lymphoid cells from hematopoietic stem progenitor cells, whereas reciprocal removal of Smo from these cells had no effect on the production of B-cell progenitors. Depletion of Smo from stromal cells was associated with coordinate down-regulation of genes for which expression is associated with osteoblastoid identity and B-lymphopoietic activity. The results of the present study suggest that activity of the Hh pathway within stromal cells promotes B lymphopoiesis in a non–cell-autonomous fashion.

Description: Key Points Hh signaling has been selectively extinguished in the mouse osteoblastoid lineage. Removal of Smo from osteoblasts results in a profound B-lymphopoietic defect.