Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription

Virus-transformed pre-B cells undergo ordered immunoglobulin (Ig) gene rearrangements during culture. We devised a series of highly sensitive polymerase chain reaction assays for Ig gene rearrangement and unrearranged Ig gene segment transcription to study both the possible relationship between these processes in cultured pre-B cells and the role played by heavy (H) chain (mu) protein in regulating gene rearrangement. Our analysis of pre-B cell cultures representing various stages of maturity revealed that transcription of each germline Ig locus precedes or is coincident with its rearrangement. Cell lines containing one functional rearranged H chain allele, however, continue to transcribe and to rearrange the allelic, unrearranged H chain locus. These cell lines appear to initiate but not terminate rearrangement events and therefore provide information about the requirements for activating rearrangement but not about allelic exclusion mechanisms

Helix-loop-helix transcription factor E47 activates germ-line immunoglobulin heavy-chain gene transcription and rearrangement in a pre-T-cell line

E47 is a helix-loop-helix transcription factor that binds to sites in the immunoglobulin heavy-chain and κ light-chain gene enhancers. Other proteins of this type are involved in cell-type determination. A possible role for E47 in B-cell development was tested by overexpressing a cDNA encoding E47 in the pre-T-cell line 2017. We found a dramatic activation of a germ-line heavy-chain gene transcript in these stable transfectants and an equally large induction of immunoglobulin D-to-J rearrangement, the first recognized step in B-cell development. Germ-line κ light-chain gene transcription and rearrangement were unaffected, but transcription of the recombination-activating genes RAG-1 and RAG-2 and the lymphoid-specific transcription factor Oct-2 was increased. These T cells did not transcribe their rearranged DJ alleles, however, and failed to progress to the next stage of heavy-chain gene assembly, V-to-DJ rearrangement. Because transcription factor E47 can induce pre-T cells to carry out events of B-cell differentiation, it may be a crucial determinant of the earliest stages of B-cell development

Deletion of Germline Promoter PDβ1 from the TCRβ Locus Causes Hypermethylation that Impairs Dβ1 Recombination by Multiple Mechanisms

AbstractThe role of the germline transcriptional promoter, PDβ1, in V(D)J recombination at the T cell receptor β locus was investigated. Deletion of PDβ1 caused reduced germline transcription and DNA hypermethylation in the Dβ1-Jβ1 region and decreased Dβ1 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′ Dβ1 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

Stimulation of κ Light-Chain Gene Rearrangement by the Immunoglobulin, µ Heavy Chain in a Pre-B-Cell Line

B-lymphocyte development exhibits a characteristic order of immunoglobulin gene rearrangements. Previous work has led to the hypothesis that expression of the immunoglobulin µ heavy chain induces rearrangement activity at the K light-chain locus. To examine this issue in more detail, we isolated five matched pairs of µ^- and endogenously rearranged µ^+ cell lines from the Abelson murine leukemia virus-transformed pro-B-cell line K.40. In four of the five µ^+ cell lines, substantial expression of µ protein on the cell surface was observed, and this correlated with an enhanced frequency of K immunoglobulin gene rearrangement compared with that in the matched µ^- cell lines. This increased K gene rearrangement frequency was not due to a general increase in the amount of V(D)J recombinase activity in the µ^+ cells. Consistently, introduction of a functionally rearranged µ gene into one of the µ^- pre-B-cell lines resulted in a fivefold increase in K gene rearrangements. In three of the four clonally matched pairs with increased K gene rearrangements, the increase in rearrangement frequency was not accompanied by a significant increase in germ line transcripts from the C_K locus. However, in the fourth pair, K.40D, we observed an increase in germ line transcription of the kappa locus after expression of µ protein encoded by either an endogenously rearranged or a transfected functional heavy-chain allele. In these cells, the amount of the germ line C_K transcript correlated with the measured frequency of rearranged K genes. These results support a regulated model of B-cell development in which µ protein expression in some way targets the V(D)J recombinase to the K gene locus

Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription

Virus-transformed pre-B cells undergo ordered immunoglobulin (Ig) gene rearrangements during culture. We devised a series of highly sensitive polymerase chain reaction assays for Ig gene rearrangement and unrearranged Ig gene segment transcription to study both the possible relationship between these processes in cultured pre-B cells and the role played by heavy (H) chain (mu) protein in regulating gene rearrangement. Our analysis of pre-B cell cultures representing various stages of maturity revealed that transcription of each germline Ig locus precedes or is coincident with its rearrangement. Cell lines containing one functional rearranged H chain allele, however, continue to transcribe and to rearrange the allelic, unrearranged H chain locus. These cell lines appear to initiate but not terminate rearrangement events and therefore provide information about the requirements for activating rearrangement but not about allelic exclusion mechanisms

Functional immunoglobulin transgenes guide ordered B-cell differentiation in Rag-1-deficient mice

We have examined the regulatory role of the individual components of the immunoglobulin antigen receptor in B-cell development by transgenic complementation of Rag-1 deficient (Rag-1⁻) mice. Complementation with a membrane µ heavy chain (µHC) gene allows progression of developmentally arrested Rag-1⁻ pro-B-cells to the small pre-B cell stage, whereas the introduction of independently integrated µHC and κ light chain (κLC) transgenes promotes the appearance of peripheral lymphocytes which, however, remain unresponsive to external stimuli. Complete reconstitution of the B-cell lineage and the emergence of functionally nature Rag-1⁻ peripheral B cells is achieved by the introduction of cointegrated heavy and light chain transgenes encoding an anti-H-2^k antibody. This experimental system demonstrates the competence of the µHC and κLC to direct and regulate the sequential stages of B-cell differentiation, defines the time at which negative selection of self-reactive B cells occurs, and shows that elimination of these cells occurs equally well in the absence of Rag-1 as in its presence. These data also support the hypothesis that Rag-1 directly participates in the V(D)J recombination process

RAG2's non-core domain contributes to the ordered regulation of V(D)J recombination

Variable (diversity) joining [V(D)J] recombination of immune gene loci proceeds in an ordered manner with D to J portions recombining first and then an upstream V joins that recombinant. We present evidence that the non-core domain of recombination activating gene (RAG) protein 2 is involved in the regulation of recombinatorial order. In mice lacking the non-core domain of RAG2 the ordered rearrangement is disturbed and direct V to D rearrangements are 10- to 1000-times increased in tri-partite immune gene loci. Some forms of inter-chromosomal translocations between TCRβ and TCRδ D gene segments are also increased in the core RAG2 animals as compared with their wild-type (WT) counterparts. In addition, the concise use of proper recombination signal sequences (RSSs) appears to be disturbed in the core RAG2 mice as compared with WT RAG2 animals