An updated definition of V(D)J recombination signal sequences revealed by high-throughput recombination assays.

In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel assay to evaluate V(D)J recombination activity on thousands of RSSs where the 12-RSS heptamer and adjoining spacer region contained randomized sequences. While the consensus heptamer sequence (CACAGTG) was marginally preferred, V(D)J recombination was highly active on a wide range of non-consensus sequences. Select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site were generally preferred. In addition, while different coding flanks and nonamer sequences affected recombination efficiency, the relative dependency on the purine/pyrimidine motifs in the RSS heptamer remained unchanged. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions.

Full length RAG2 expression enhances the DNA damage response in pre-B cells.

V(D)J recombination by the RAG1 and RAG2 protein complex in developing lymphocytes includes DNA double strand break (DSB) intermediates. RAG2 undergoes export from the nucleus and enrichment at the centrosome minutes following production of DSBs by genotoxic stress, suggesting that RAG2 participates in cellular responses to DSBs such as those generated during V(D)J recombination. To determine the effect of RAG2 expression on cell viability following DSB generation, we measured pre-B cells that expressed either full length (FL) wild-type RAG2, or a T490A mutant of RAG2 that has increased stability and fails to undergo nuclear export following generation of DSBs. Each RAG2 construct was labeled with GFP at the N-terminus. Compared to the T490A mutant, cells expressing FL RAG2 exhibited elevated apoptosis by 24 h following irradiation, and this coincided with a greater amount of Caspase 3 cleavage measured in cell lysates. Pre-B cells expressing either RAG2 protein exhibited similar increases in phospho-p53 levels following irradiation. Interestingly, FL RAG2-expressing cells exhibited elevated division relative to the T490A clone beginning ~24 h following irradiation, as well as an increased percentage of cells proceeding through mitosis, suggesting an improved rate of recovery following the initial burst in apoptosis. Altogether, these data show that FL RAG2, but not its stable nuclear export-defective T490A mutant, participates in pre-B cell decisions between apoptosis versus DNA repair and cell cycle progression following DNA damage.

RAG2 localization and dynamics in the pre-B cell nucleus.

RAG2 of the V(D)J recombinase is essential for lymphocyte development. Within the RAG2 noncore region is a plant homeodomain (PHD) that interacts with the modified histone H3K4me3, and this interaction is important for relieving inhibition of the RAG recombinase for V(D)J recombination. However, the effect of the noncore region on RAG2 localization and dynamics in cell nuclei is poorly understood. Here, we used cell imaging to measure the effect of mutating the RAG2 noncore region on properties of the full length protein. We measured GFP-labeled full length RAG2 (FL), the RAG2 core region alone (Core), and a T490A mutant in the noncore region, which has unique regulatory properties. This showed that FL, T490A, and Core localized to nuclear domains that were adjacent to DAPI-rich heterochromatin, and that contained the active chromatin marker H3K4me3. Within the RAG2-enriched regions, T490A exhibited greater colocalization with H3K4me3 than either FL or Core. Furthermore, colocalization of H3K4me3 with FL and T490A, but not Core, increased in conditions that increased H3K4me3 levels. Superresolution imaging showed H3K4me3 was distributed as puncta that RAG2 abutted, and mobility measurements showed that T490A had a significantly lower rate of diffusion within the nucleus than either FL or Core proteins. Finally, mutating Trp453 of the T490A mutant (W453A,T490A), which blocks PHD-dependent interactions with H3K4me3, abolished the T490A-mediated increased colocalization with H3K4me3 and slower mobility compared to FL. Altogether, these data show that Thr490 in the noncore region modulates RAG2 localization and dynamics in the pre-B cell nucleus, such as by affecting RAG2 interactions with H3K4me3.

Identification of Apolipoprotein A-I as a Retinoic Acid-binding Protein in the Eye.

All-trans-retinoic acid may be an important molecular signal in the postnatal control of eye size. The goal of this study was to identify retinoic acid-binding proteins secreted by the choroid and sclera during visually guided ocular growth. Following photoaffinity labeling with all-trans-[11,12-(3)H]retinoic acid, the most abundant labeled protein detected in the conditioned medium of choroid or sclera had an apparent Mr of 27,000 Da. Following purification and mass spectrometry, the Mr 27,000 band was identified as apolipoprotein A-I. Affinity capture of the radioactive Mr 27,000 band by anti-chick apolipoprotein A-I antibodies confirmed its identity as apolipoprotein A-I. Photoaffinity labeling and fluorescence quenching experiments demonstrated that binding of retinoic acid to apolipoprotein A-I is 1) concentration-dependent, 2) selective for all-trans-retinoic acid, and 3) requires the presence of apolipoprotein A-I-associated lipids for retinoid binding. Expression of apolipoprotein A-I mRNA and protein synthesis were markedly up-regulated in choroids of chick eyes during the recovery from induced myopia, and apolipoprotein A-I mRNA was significantly increased in choroids following retinoic acid treatment. Together, these data suggest that apolipoprotein A-I may participate in a regulatory feedback mechanism with retinoic acid to control the action of retinoic acid on ocular targets during postnatal ocular growth.

Membrane-cytoskeleton interactions in cholesterol-dependent domain formation.

Since the inception of the fluid mosaic model, cell membranes have come to be recognized as heterogeneous structures composed of discrete protein and lipid domains of various dimensions and biological functions. The structural and biological properties of membrane domains are represented by CDM (cholesterol-dependent membrane) domains, frequently referred to as membrane 'rafts'. Biological functions attributed to CDMs include signal transduction. In T-cells, CDMs function in the regulation of the Src family kinase Lck (p56lck) by sequestering Lck from its activator CD45. Despite evidence of discrete CDM domains with specific functions, the mechanism by which they form and are maintained within a fluid and dynamic lipid bilayer is not completely understood. In the present chapter, we discuss recent advances showing that the actomyosin cytoskeleton has an integral role in the formation of CDM domains. Using Lck as a model, we also discuss recent findings regarding cytoskeleton-dependent CDM domain functions in protein regulation.

An interdomain boundary in RAG1 facilitates cooperative binding to RAG2 in formation of the V(D)J recombinase complex.

V(D)J recombination assembles functional antigen receptor genes during lymphocyte development. Formation of the recombination complex containing the recombination activating proteins, RAG1 and RAG2, is essential for the site-specific DNA cleavage steps in V(D)J recombination. However, little is known concerning how complex formation leads to a catalytically-active complex. Here, we combined limited proteolysis and mass spectrometry methods to identify regions of RAG1 that are sequestered upon association with RAG2. These results show that RAG2 bridges an interdomain boundary in the catalytic region of RAG1. In a second approach, mutation of RAG1 residues within the interdomain boundary were tested for disruption of RAG1:RAG2 complex formation using fluorescence-based pull down assays. The core RAG1 mutants demonstrated varying effects on complex formation with RAG2. Interestingly, two mutants showed opposing results for the ability to interact with core versus full length RAG2, indicating that the non-core region of RAG2 participates in binding to core RAG1. Significantly, all of the RAG1 interdomain mutants demonstrated altered stoichiometries of the RAG complexes, with an increased number of RAG2 per RAG1 subunit compared to the wild type complex. Based on our results, we propose that interaction of RAG2 with RAG1 induces cooperative interactions of multiple binding sites, induced through conformational changes at the RAG1 interdomain boundary, and resulting in formation of the DNA cleavage active site.

Spatio-temporal regulation of RAG2 following genotoxic stress.

V(D)J recombination of lymphocyte antigen receptor genes occurs via the formation of DNA double strand breaks (DSBs) through the activity of RAG1 and RAG2. The co-existence of RAG-independent DNA DSBs generated by genotoxic stressors potentially increases the risk of incorrect repair and chromosomal abnormalities. However, it is not known whether cellular responses to DSBs by genotoxic stressors affect the RAG complex. Using cellular imaging and subcellular fractionation approaches, we show that formation of DSBs by treating cells with DNA damaging agents causes export of nuclear RAG2. Within the cytoplasm, RAG2 exhibited substantial enrichment at the centrosome. Further, RAG2 export was sensitive to inhibition of ATM, and was reversed following DNA repair. The core region of RAG2 was sufficient for export, but not centrosome targeting, and RAG2 export was blocked by mutation of Thr(490). In summary, DNA damage triggers relocalization of RAG2 from the nucleus to centrosomes, suggesting a novel mechanism for modulating cellular responses to DSBs in developing lymphocytes.

CD28 sensitizes TCR Ca²âº signaling during Ag-independent polarization of plasma membrane rafts.

T cells become polarized during initial interactions with an APC to form an Ag-independent synapse (AIS) composed of membrane rafts, TCR, and TCR-proximal signaling molecules. AISs occur temporally before TCR triggering, but their role in downstream TCR signaling is not understood. Using both human and murine model systems, we studied the signals that activate AIS formation and the effect of these signals on TCR-dependent responses. We show that CD28 produces AISs detectable by spinning disc confocal microscopy seconds following initial interactions between the T cell and APC. AIS formation by CD28 coincided with costimulatory signaling, evidenced by a cholesterol-sensitive activation of the MAPK ERK that potentiated Ca²âº signaling in response to CD3 cross-linking. CD45 also enriched in AISs but to modulate Src kinase activity, because localization of CD45 at the cell interface reduced the activation of proximal Lck. In summary, we show that signaling by CD28 during first encounters between the T cell and APC both sensitizes TCR Ca²âº signaling by an Erk-dependent mechanism and drives formation of an AIS that modulates the early signaling until TCR triggering occurs. Thus, early Ag-independent encounters are an important window for optimizing T cell responses to Ag by CD28.

Early and dynamic polarization of T cell membrane rafts and constituents prior to TCR stop signals.

Polarization of membrane rafts and signaling proteins to form an immunological synapse is a hallmark of T cell stimulation. However, the kinetics of raft polarization and associated proteins in relation to the initial contact of the T cell with the APC are poorly defined. We addressed this question by measuring the distribution of membrane-targeted fluorescent protein markers during initial T cell interactions with B cell APCs. Experiments with unpulsed B cells lacking cognate Ag demonstrated an MHC class II-independent capping that was specific to membrane raft markers and required actin rearrangements and signals from Src kinases and PI3K. By live cell imaging experiments, we identified a similar specific polarization of membrane raft markers before TCR-dependent stop signals, and which occurred independently of cognate peptide-MHC class II. T cells conjugated to unpulsed B cells exhibited capping of CD4 and microclusters of the TCR zeta-chain, but only the CD4 enrichment was cholesterol dependent. Furthermore, raft association of CD4 was necessary for its efficient targeting to the Ag-independent caps. Interestingly, anergic Vbeta8(+) T cells isolated from staphylococcal enterotoxin B-injected mice did not exhibit Ag-independent capping of membrane rafts, showing that inhibition of these early, Ag-independent events is a property associated with tolerance. Altogether, these data show that membrane raft capping is one of the earliest events in T cell activation and represents one avenue for promoting and regulating downstream peptide-MHC-dependent signaling within the T cell.

Visualization of transfer of a fluorescently-labeled membrane raft protein to T cells using lentivirus.

Lentivirus vector systems have been developed for the safe delivery of foreign genes to target tissues. However, the use of these systems for delivering specific proteins to target cells has been largely unexplored. To test this concept, the lentivirus expression plasmid pLenti was utilized to overexpress in producer cells a YFP-fusion protein that is specifically targeted to glycolipid-enriched membrane rafts, which is the site of virus assembly. Our data show that virus generated in producer cells that expressed the YFP fusion protein were able to effectively label target cells by a 2-3 hr incubation with the virus. Labeling of the target cells was specific to the lentivirus, as it was blocked by pre-incubating the virus with antibody to the surface protein, and it was not affected by pre-treating the target cells with cyclohexamide. T cells that were labeled using the lentivirus underwent a robust stimulation following crosslinking the T cell receptor, thus showing that T cells labeled using lentivirus remained responsive to extracellular cues. Altogether, these results show that overexpression of foreign proteins in lentivirus producer cells can yield protein-loaded viruses, which can then function to deliver the protein to target cells. Thus, our findings suggest an avenue for targeting specific proteins to cells where foreign gene expression is not feasible.

Visualization of inositol phosphate-dependent mobility of Ku: depletion of the DNA-PK cofactor InsP6 inhibits Ku mobility.

Repair of DNA double-strand breaks (DSBs) in mammalian cells by nonhomologous end-joining (NHEJ) is initiated by the DNA-PK protein complex. Recent studies have shown inositol hexakisphosphate (InsP6) is a potent cofactor for DNA-PK activity in NHEJ. Specifically, InsP6 binds to the Ku component of DNA-PK, where it induces a conformational change and a corresponding increase in DNA end-joining activity. However, the effect of InsP6 on the dynamics of Ku, such as its mobility in the nucleus, is unknown. Importantly, these dynamics reflect the character of Ku's interactions with other molecules. To address this question, the diffusion of Ku was measured by fluorescence photobleaching experiments using cells expressing green fluorescent protein (GFP)-labeled Ku. InsP6 was depleted by treating cells with calmodulin inhibitors, which included the compounds W7 and chlorpromazine. These treatments caused a 50% reduction in the mobile fraction of Ku-GFP, and this could be reversed by replenishing cells with InsP6. By expressing deletion mutants of Ku-GFP, it was determined that its W7-sensitive region occurred at the N-terminus of the dimerization domain of Ku70. These results therefore show that InsP6 enhances Ku mobility through a discrete region of Ku70, and modulation of InsP6 levels in cells represents a potential avenue for regulating NHEJ by affecting the dynamics of Ku and hence its interaction with other nuclear proteins.