A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival.

The role of DNA methylation and of the maintenance DNA methyltransferase Dnmt1 in the epigenetic regulation of developmental stage- and cell lineage-specific gene expression in vivo is uncertain. This is addressed here through the generation of mice in which Dnmt1 was inactivated by Cre/loxP-mediated deletion at sequential stages of T cell development. Deletion of Dnmt1 in early double-negative thymocytes led to impaired survival of TCRalphabeta(+) cells and the generation of atypical CD8(+)TCRgammadelta(+) cells. Deletion of Dnmt1 in double-positive thymocytes impaired activation-induced proliferation but differentially enhanced cytokine mRNA expression by naive peripheral T cells. We conclude that Dnmt1 and DNA methylation are required for the proper expression of certain genes that define fate and determine function in T cells.

Liver-derived DEC205+B220+CD19- dendritic cells regulate T cell responses.

Leukocytes resident in the liver may play a role in immune responses. We describe a cell population propagated from mouse liver nonparenchymal cells in IL-3 and anti-CD40 mAb that exhibits a distinct surface immunophenotype and function in directing differentiation of naive allogeneic T cells. After culture, such cells are DEC-205(bright)B220+CD11c-CD19-, and negative for T (CD3, CD4, CD8alpha), NK (NK 1.1) cell markers, and myeloid Ags (CD11b, CD13, CD14). These liver-derived DEC205+B220+ CD19- cells have a morphology and migratory capacity similar to dendritic cells. Interestingly, they possess Ig gene rearrangements, but lack Ig molecule expression on the cell surface. They induce low thymidine uptake of allogeneic T cells in MLR due to extensive apoptosis of activated T cells. T cell proliferation is restored by addition of the common caspase inhibitor peptide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). T cells stimulated by liver-derived DEC205+B220+D19- cells release both IL-10 and IFN-gamma, small amounts of TGF-beta, and no IL-2 or IL-4, a cytokine profile resembling T regulatory type 1 cells. Expression of IL-10 and IFN-gamma, but not bioactive IL-12 in liver DEC205+B220+CD19- cells was demonstrated by RNase protection assay. In vivo administration of liver DEC205+B220+CD19- cells significantly prolonged the survival of vascularized cardiac allografts in an alloantigen-specific manner.

Target DNA chromatinization modulates nicking by L1 endonuclease.

L1 elements are human transposons which replicate via an RNA intermediate. At least 15% of the human genome is composed of L1 sequence. An important initial step in the transposition reaction is nicking of the genomic DNA by L1 endonuclease (L1 EN). In vivo much of the genome exists in the form of chromatin or is undergoing biochemical transactions such as transcription, replication or repair, which may alter the accessibility of the L1 transposition machinery to DNA. To investigate this possibility we have examined the effect of substrate chromatinization on the ability of L1 EN to nick DNA. We find that DNA incorporated into nucleosomes is generally refractory to nicking by L1 EN. Interestingly, nicking of a minority of DNA sequences is enhanced when included in chromatin. Thus, dynamic epigenetic factors such as chromatinization are likely to influence the relatively permanent placement of L1 and other retroelements in the human genome.

Annexin V binds to positively selected B cells.

Recombinant annexin V (rAnV) has been used in flow cytometry to identify cells undergoing apoptosis, based on its ability to bind to phosphatidylserine, a negatively charged lipid normally restricted to the cytoplasmic face of the plasma membrane but externalized early during apoptosis. When we stained murine bone marrow (BM) cells with fluorescently labeled rAnV, we found that a surprisingly large fraction of BM B cells bearing selectable transgenic Ag receptors bind significant amounts of rAnV, but that these cells are not apoptotic. Here, we show that binding of rAnV to developing B cells in normal mice correlates with B cell receptor-dependent selection events at several stages of development within both B-1 and B-2 cell subsets. In fact, nearly all B-1 B cells and splenic marginal zone B cells bind rAnV, suggesting that the externalization of phosphatidylserine occurs once mature B cells are selected through BCR-mediated signaling. However, this plasma membrane alteration is apparently not shared by all lymphocytes, because we did not find a parallel population of rAnV-binding viable T cells in vivo in normal or TCR transgenic mice. We also show that BM stromal cell lines can influence the extent of rAnV binding by viable BM B cells during coculture in vitro. We suggest that rAnV detects a potentially important membrane alteration that occurs as B cells develop in the BM and are readied for export to the peripheral lymphoid organs and again among mature B cells recruited to the marginal zone or the B-1 compartment.

Deletion of germline promoter PD beta 1 from the TCR beta locus causes hypermethylation that impairs D beta 1 recombination by multiple mechanisms.

The role of the germline transcriptional promoter, PD beta 1, in V(D)J recombination at the T cell receptor beta locus was investigated. Deletion of PD beta 1 caused reduced germline transcription and DNA hypermethylation in the Dbeta1-J beta 1 region and decreased D beta 1 rearrangement. Analyses of methylation levels surrounding recombination signal sequences (RSS) before, during, and after recombination revealed that under physiological conditions cleavage of hypomethylated alleles was preferred over hypermethylated alleles. Methylation of a specific CpG site within the heptamer of the 3' D beta 1 RSS was incompatible with cleavage by the V(D)J recombinase. These findings suggest that methylation can regulate V(D)J recombination both at a general level by influencing regional chromatin accessibility and specifically by blocking RSS recognition or cleavage by the V(D)J recombinase.

c-Myb binds to a sequence in the proximal region of the RAG-2 promoter and is essential for promoter activity in T-lineage cells.

The RAG-2 gene encodes a component of the V(D)J recombinase which is essential for the assembly of antigen receptor genes in B and T lymphocytes. Previously, we reported that the transcription factor BSAP (PAX-5) regulates the murine RAG-2 promoter in B-cell lines. A partially overlapping but distinct region of the proximal RAG-2 promoter was also identified as an important element for promoter activity in T cells; however, the responsible factor was unknown. In this report, we present data demonstrating that c-Myb binds to a Myb consensus site within the proximal promoter and is critical for its activity in T-lineage cells. We show that c-Myb can transactivate a RAG-2 promoter-reporter construct in cotransfection assays and that this transactivation depends on the proximal promoter Myb consensus site. By using a chromatin immunoprecipitation (ChIP) strategy, fractionation of chromatin with anti-c-Myb antibody specifically enriched endogenous RAG-2 promoter DNA sequences. DNase I genomic footprinting revealed that the c-Myb site is occupied in a tissue-specific fashion in vivo. Furthermore, an integrated RAG-2 promoter construct with mutations at the c-Myb site was not enriched in the ChIP assay, while a wild-type integrated promoter construct was enriched. Finally, this lack of binding of c-Myb to a chromosomally integrated mutant RAG-2 promoter construct in vivo was associated with a striking decrease in promoter activity. We conclude that c-Myb regulates the RAG-2 promoter in T cells by binding to this consensus c-Myb binding site.

Pre-B and pre-T-cell receptors: conservation of strategies in regulating early lymphocyte development.

Early lymphocyte development is characterized by the regulated activity of the V(D)J recombinase and the positive and negative selection of cells based on the structure of their assembled antigen receptor genes. Developing B and T cells use remarkably similar signaling complexes, the pre-B-cell receptor (pre-BCR) and the pre-T-cell receptor (pre-TCR) respectively, to monitor the progress of antigen receptor gene assembly This review will compare and contrast the regulation and activities of the pre-BCR and pre-TCR signaling complexes. In addition, we will consider a number of critical but as yet unanswered questions prompted by such an analysis.

The interleukin 7 receptor is required for T cell receptor gamma locus accessibility to the V(D)J recombinase.

Defects in the interleukin (IL)-7 signal transduction pathway lead to severe immunodeficiency in humans and in mice. In IL-7 receptor-deficient (IL-7R-/-) mice, lymphoid precursors show a reduced survival rate and variable/diversity/joining region V(D)J recombination is variously affected in different loci, being arrested in the T cell receptor (TCR)-gamma locus, aberrant in the immunoglobulin heavy chain (IgH) locus, and delayed in the TCR-beta locus. Here, we analyze the recombination defect of the TCR-gamma locus. Using ligation-mediated polymerase chain reaction, we sought intermediates of the recombination process. In the absence of the IL-7 signal, no initiation of recombination of the TCR-gamma locus was observed, whereas recombination intermediates at the TCR-beta locus could be detected. Thus, the failure to rearrange the TCR-gamma locus is due to a failure to initiate cleavage rather than a failure to religate broken DNA ends. V(D)J recombination was previously thought to begin at the pro-T2 stage of T cell development after the arrest of IL-7R-/- thymocytes at the pro-T1 stage. However, here we show that both TCR-gamma and -beta recombination intermediates are readily detectable in normal T1 cells, but only TCR-beta intermediates were detected in IL-7R-/- T1 cells, supporting a mechanistic role for IL-7 in TCR-gamma locus rearrangement. Since reduced recombination activating gene (rag) expression has been reported in the absence of the IL-7 signal, we directly tested whether the TCR-gamma locus is accessible to cleavage by recombinant Rag proteins in vitro. We found a reduction in chromatin accessibility for Rag-mediated cleavage in IL-7R-/- thymocytes compared with wild-type. Thus, IL-7 controls recombination at the TCR-gamma locus by regulating locus accessibility.

Annexin V binds to viable B cells and colocalizes with a marker of lipid rafts upon B cell receptor activation.

Recombinant annexin V (rAnV) has been used to identify apoptotic cells based on its ability to bind phosphatidylserine (PS), a lipid normally restricted to the cytoplasmic face of the plasma membrane, but externalized early during apoptosis. However, this association of rAnV binding and apoptosis is not an obligatory one. We demonstrate that rAnV binds to a large fraction of murine B cells bearing selectable Ag receptors despite the fact that these cells are not apoptotic. Phosphatidylserine, which is uniformly distributed on resting B cells, is mobilized to co-cap with IgM on anti-IgM-treated B cells and to colocalize with GM1, a marker of lipid rafts. Cross-linking PS before anti-IgM treatment sequesters this lipid and alters signaling through IgM. Thus, PS exposed on the majority of B cells in vivo does not reflect early apoptosis, but, instead, plays a role in receptor-mediated signaling events.

Pro-B-cell-specific transcription and proapoptotic function of protein kinase Ceta.

Using a subtractive cloning scheme on cDNA prepared from primary pro-B and pre-B cells, we identified several genes whose products regulate apoptosis. We further characterized one of these genes, encoding protein kinase Ceta (PKCeta). PKCeta transcripts were readily detected in pro-B cells but were absent in pre-B cells. Although both a full-length and a truncated form of PKCeta were detectable in bone marrow pro-B cells, transition to the pre-B-cell stage was associated with increased relative levels of truncated PKCeta. We found that PKCeta is proteolyzed in apoptotic lymphocytes, generating a kinase-active fragment identical to the truncated form which is capable of inducing apoptosis when expressed in a pro-B cell line. Caspase-3 can generate an identical PKCeta cleavage product in vitro, and caspase inhibitors prevent the generation of this product during apoptosis in transfected cell lines. Inducible overexpression of either the full-length or truncated form of PKCeta results in cell cycle arrest at the G(1)/S transition. These results suggest that the expression and proteolytic activation of PKCeta play an important role in the regulation of cell division and cell death during early B-cell development.

Nucleosome structure completely inhibits in vitro cleavage by the V(D)J recombinase.

Lineage specificity and temporal ordering of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement are reflected in the accessibility of recombination signal sequences (RSSs) within chromatin to in vitro cleavage by the V(D)J recombinase. In this report, we investigated the basis of this regulation by testing the ability of purified RAG1 and RAG2 proteins to initiate cleavage on positioned nucleosomes containing RSS substrates. We found that nicking and double-strand DNA cleavage of RSSs positioned on the face of an unmodified nucleosome are entirely inhibited. This inhibition was independent of translational position or rotational phase and could not be overcome either by addition of the DNA-bending protein HMG-1 or by the use of hyperacetylated histones. We suggest that the nucleosome could act as the stable unit of chromatin which limits recombinase accessibility to potential RSS targets, and that actively rearranging gene segments might be packaged in a modified or disrupted nucleosome structure.

Overexpression of RelA causes G1 arrest and apoptosis in a pro-B cell line.

NF-kappaB/Rel family proteins form a network of post-translationally regulated transcription factors that respond to a variety of extracellular stimuli and mediate distinct cellular responses. These responses include cytokine gene expression, regulated cell cycle activation, and both the protection from and induction of the cell death program. To examine the function of individual Rel family proteins in B cell development and resolve their role in the signaling of apoptosis, we used a tetracycline-regulated gene expression system to overexpress either c-Rel or RelA in the transformed pro-B cell line 220-8. Elevated levels of RelA, but not c-Rel, induced a G1 cell cycle arrest followed by apoptosis. Both the DNA binding and transactivation domains of RelA were required for this effect. When RelA was overexpressed in the immature B cell line WEHI 231 or the mature B cell line M12, neither cell cycle arrest nor apoptosis was evident. The differential effects of elevated RelA levels in these cell lines suggests that susceptibility to NF-kappaB-induced apoptosis may reflect a relevant selection event during B cell development.

Distinct factors regulate the murine RAG-2 promoter in B- and T-cell lines.

The recombination activating genes RAG-1 and RAG-2 are expressed in a lymphoid-cell-specific and developmentally regulated fashion. To understand the transcriptional basis for this regulation, we have cloned and characterized the murine RAG-2 promoter. The promoter was lymphoid cell specific, showing activity in various B- and T-cell lines but little activity in nonlymphoid cells. To our surprise, however, the promoter was regulated differently in B and T cells. Using nuclear extracts from B-cell lines, we found that the B-cell-specific transcription factor BSAP (Pax-5) could bind to a conserved sequence critical for promoter activity. BSAP activated the promoter in transfected cells, and the BSAP site was occupied in a tissue-specific manner in vivo. An overlapping DNA sequence binding to a distinct factor was necessary for promoter activity in T cells. Full promoter activity in T cells was also dependent on a more distal DNA sequence whose disruption had no effect on B-cell activity. The unexpected finding that a B-cell-specific factor regulates the RAG-2 promoter may explain some of the recently observed differences in the regulation of RAG transcription between B and T cells.

Enhancer control of V(D)J recombination at the TCRbeta locus: differential effects on DNA cleavage and joining.

Deletion of the TCRbeta transcriptional enhancer (Ebeta) results in nearly complete inhibition of V(D)J recombination at the TCRbeta locus and a block in alpha beta T cell development. This result, along with previous work from many laboratories, has led to the hypothesis that transcriptional enhancers affect V(D)J recombination by regulating the accessibility of the locus to the recombinase. Here we test this hypothesis by performing a detailed analysis of the recombination defect in Ebeta-deleted (Ebeta-/-) mice using assays that detect various reaction intermediates and products. We found double-strand DNA breaks at recombination signal sequences flanking Dbeta and Jbeta gene segments in Ebeta-/- thymuses at about one-third to one-thirtieth the level found in thymuses with an unaltered TCRbeta locus. These sites are also subject to in vitro cleavage by the V(D)J recombinase in both Ebeta-/- and Ebeta+/+ thymocyte nuclei. However, the corresponding Dbeta-to-Jbeta coding joints are further reduced (by 100- to 300-fold) in Ebeta-/- thymuses. Formation of extrachromosomal Dbeta-to-Jbeta signal joints appears to be intermediately affected and nonstandard Dbeta-to-Dbeta joining occurs at the Ebeta-deleted alleles. These data indicate that, unexpectedly, loss of accessibility alone cannot explain the loss of TCRbeta recombination in the absence of the Ebeta element and suggest an additional function for Ebeta in the process of DNA repair at specific TCRbeta sites during the late phase of the recombination reaction.

Structure of nonhairpin coding-end DNA breaks in cells undergoing V(D)J recombination.

The V(D)J recombinase recognizes a pair of immunoglobulin or T-cell receptor gene segments flanked by recombination signal sequences and introduces double-strand breaks, generating two signal ends and two coding ends. Broken coding ends were initially identified as covalently closed hairpin DNA molecules. Before recombination, however, the hairpins must be opened and the ends must be modified by nuclease digestion and N-region addition. We have now analyzed nonhairpin coding ends associated with various immunoglobulin gene segments in cells undergoing V(D)J recombination. We found that these broken DNA ends have different nonrandom 5'-strand deletions which were characteristic for each locus examined. These deletions correlate well with the sequence characteristics of coding joints involving these gene segments. In addition, unlike broken signal ends, these nonhairpin coding-end V(D)J recombination reaction intermediates have 3' overhanging ends. We discuss the implications of these results for models of how sequence modifications occur during coding-joint formation.

Growth retardation and leaky SCID phenotype of Ku70-deficient mice.

Ku70, Ku80, and DNA-PKcs are subunits of the DNA-dependent protein kinase (DNA-PK), an enzyme implicated in DNA double-stranded break repair and V(D)J recombination. Our Ku70-deficient mice were about 50% the size of control littermates, and their fibroblasts were ionizing radiation sensitive and displayed premature senescence associated with the accumulation of nondividing cells. Ku70-deficient mice lacked mature B cells or serum immunoglobulin but, unexpectedly, reproducibly developed small populations of thymic and peripheral alpha/beta T lineage cells and had a significant incidence of thymic lymphomas. In association with B and T cell developmental defects, Ku70-deficient cells were severely impaired for joining of V(D)J coding and recombination signal sequences. These unanticipated features of the Ku70-deficient phenotype with respect to lymphocyte development and V(D)J recombination may reflect differential functions of the three DNA-PK components.

V(D)J recombinase activity in a subset of germinal center B lymphocytes.

Reexpression of the V(D)J recombinase-activating genes RAG1 and RAG2 in germinal center B cells creates the potential for immunoglobulin gene rearrangement and the generation of new antigen receptor specificities. Intermediate products of V(D)J recombination are abundant in a subset of germinal center B cells, demonstrating that the kappa immunoglobulin light-chain locus becomes a substrate for renewed V(D)J recombinase activity. This recombinationally active cell compartment contains many heavy-chain VDJ rearrangements that encode low-affinity or nonfunctional antibody. In germinal centers, secondary V(D)J recombination may be induced by diminished binding to antigen ligands, thereby limiting abrupt changes in receptor specificity to B cells that are usually eliminated from the germinal center reaction. This restriction preserves efficient antigen-driven selection in germinal centers while allowing for saltations in the somatic evolution of B cells.

A truncated heavy chain protein relieves the requirement for surrogate light chains in early B cell development.

Early B cell development depends upon the surface expression of Ig heavy chain protein (mu) in a signaling complex known as the pre-B cell receptor (pre-BCR). In addition to mu, the pre-BCR consists of the surrogate light chains VpreB and lambda5 and the transmembrane signal transduction proteins Ig-alpha and Ig-beta. Expression of this complex is associated with changes in surface marker expression, gene transcription, and Ig gene rearrangement. Mutations preventing the expression of either mu or lambda5 result in developmental arrest, but the precise roles of the various components of the pre-BCR remain unclear. Using mice transgenic for a surface-expressed, but truncated, form of mu that cannot associate with surrogate light chains, we have studied the role of surrogate light chains in B cell development. We found that expression of the truncated mu transgene resulted in changes in surface marker expression, germline kappa locus transcription, and V(D)J recombinase targeting indistinguishable from those induced by intact mu protein. These experiments lead us to conclude that surrogate light chains, while necessary for the assembly of the wild-type pre-BCR, are not directly involved in pre-BCR signaling or otherwise required for early B cell development.

Accessibility and the developmental regulation of V(D)J recombination.

Antigen receptor genes are assembled from their component gene segments by a highly regulated series of site-specific DNA recombination reactions known as V(D)J recombination. Proteins encoded by the RAG1 and RAG2 genes are responsible for the recognition and double-stranded cleavage of a highly conserved DNA sequence flanking all rearranging gene segments. It remains uncertain how this common lymphoid recombinase is targeted to distinct loci in developing B and T cells and to specific loci at successive stages of lymphocyte development. This review considers evidence that DNA sequences which regulate the transcription of antigen receptor genes also regulate the recombination reaction by determining the accessibility of individual loci to the V(D)J recombinase.