RAG-2 Promoter Research Articles

Identification of a third evolutionarily conserved gene within theRAG locus and itsRAG1-dependent and -independent regulation

Recombination-activating gene (RAG)1 and RAG2 encode T and B lymphocyte-specific endonucleases indispensable for rearrangements of antigen-receptor gene segments but also capable of causing deleterious chromosome rearrangements. The mechanisms regulating RAG expression and repression are not clear. Here we identify NWC, a third evolutionarily conserved gene within the RAG locus, and show that it is ubiquitously expressed, with the notable exception of RAG-nonexpressing immature and mature T and B lymphocytes because in lymphocytes it is regulated by the RAG1 promoter and transcribed as RAG1-NWC hybrid mRNA molecules. We also show that in all other cells NWC is controlled by the RAG2 intragenic promoter, which in immature and mature T and B lymphocytes is silent. The possible implications of these findings for understanding the activation and inactivation of RAG genes in lymphocytes and their repression in other cells are discussed.

Activation of mouse RAG-2 promoter by Myc-associated zinc finger protein

Recombination activating gene-1 ( RAG-1) and RAG-2 are expressed specifically in lymphocytes undergoing the antigen receptor gene rearrangement during the lymphocyte development. Our previous study showed that the −41 to −17 nucleotides (nt) 5 ′-upstream region of mouse RAG-2 were pre-requisite for the core promoter activity and that Pax-5/c-Myb/LEF-1 protein–protein complex was responsible for its activity in immature B cells. In this study, we show that the −65/−42 sequence, the non-conserved sequence between human and mouse RAG-2 promoter, is necessary for the full promoter activity for mouse RAG-2. Electrophoresis mobility shift assay revealed that Myc-associated zinc finger protein (MAZ) as well as SP1/3 binds a GA box in this region. Using chromatin immunoprecipitation, we show that MAZ binds the RAG-2 promoter region in pre-B cells. Furthermore, we show that MAZ synergistically activates the murine RAG-2 promoter with Pax-5/c-Myb/LEF-1 complex. These results first demonstrate that MAZ participates in activation of mouse RAG-2 promoter.

T cell receptor revision does not solely target recent thymic emigrants.

CD4(+)Vbeta5(+) T cells enter one of two tolerance pathways after recognizing a peripherally expressed superantigen encoded by an endogenous retrovirus. One pathway leads to deletion, while the other, termed TCR revision, results in cellular rescue upon expression of an alternate TCR that no longer recognizes the tolerogen. TCR revision requires the rearrangement of novel TCR beta-chain genes and depends on recombinase-activating gene (RAG) expression in peripheral T cells. In line with recent findings that RAG(+) splenic B cells are immature cells that have maintained RAG expression, it has been hypothesized that TCR revision is limited to recent thymic emigrants that have maintained RAG expression and TCR loci in a recombination-permissive configuration. Using mice in which the expression of green fluorescent protein is driven by the RAG2 promoter, we now show that in vitro stimulation can drive reporter expression in noncycling, mature, peripheral CD4(+) T cells. In addition, thymectomized Vbeta5 transgenic RAG reporter mice are used to demonstrate that TCR revision can target peripheral T cells up to 2 mo after thymectomy. Both sets of experiments strongly suggest that reinduction of RAG genes triggers TCR revision. Approximately 3% of CD4(+)Vbeta5(+) T cells in thymectomized Vbeta5 transgenic reporter mice have undergone TCR revision within the previous 4-5 days. TCR revision can also occur in Vbeta5(+) T cells from nontransgenic mice, illustrating the relevance of this novel tolerance mechanism in unmanipulated animals.

Lymphoid enhancer-binding factor-1 binds and activates the recombination-activating gene-2 promoter together with c-Myb and Pax-5 in immature B cells.

The recombination-activating gene (RAG)-1 and RAG-2 are expressed specifically in immature lymphoid cells undergoing the recombination of Ag receptor genes. We studied the regulation of murine RAG-2 promoter and revealed that -41/-17 RAG-2 promoter region, which was indispensable for the RAG-2 promoter activity in B cell lines, contained binding sites for lymphoid enhancer-binding factor-1 (LEF-1), c-Myb, and Pax-5. We showed that these three transcription factors bound the promoter region in vitro and in vivo. Cotransfection assays using a human embryonic kidney cell line (293T) showed that LEF-1, c-Myb, and Pax-5 cooperatively activated the RAG-2 promoter, via their synergistic DNA binding. We also showed that LEF-1, c-Myb, and Pax-5 physically interact in the cells. Finally, we demonstrated that a dominant-negative LEF-1 protein, which lacks the binding site for beta-catenin, suppressed the RAG-2 promoter activity as well as the endogenous RAG-2 expression in a pre-B cell line (18.81). These results suggest that LEF-1/beta-catenin complex regulates the RAG-2 promoter activation in concert with c-Myb and Pax-5 in immature B cells. The link between LEF-1/beta-catenin and Wnt signaling in B lineage cells will be discussed.

Characterization of chromatin structure and enhancer elements for murine recombination activating gene-2.

Recombination-activating genes (RAGs) play a critical role in V(D)J recombination machinery and their expression is specifically regulated during lymphocyte ontogeny. To elucidate the molecular mechanisms regulating murine RAG-2 expression, we examined a chromatin structure of 25-kb DNA segment adjacent to murine RAG-2 by analyzing DNase I hypersensitive (HS) sites. In a RAG-2-expressing murine pre-B cell line, three lymphoid cell-specific HS sites (HS1, HS2, and HS3) were identified. Among these HS sites, one HS site (HS3) that locates in the RAG-2 promoter was associated only with RAG-2-expressing cell lines. Using the transient enhanced green fluorescence protein reporter gene assays, we identified two enhancer elements in the 5'-upstream region of RAG-2 that corresponded to HS1 and HS2. One of the enhancer elements (D3) exhibited enhancer activity only in the lymphoid cell lines. Analysis of the transgenic mice carrying the enhanced green fluorescence protein-reporter gene linked with D3 revealed that D3 activated the reporter gene-expression in the primary lymphoid tissues, but not in the secondary lymphoid tissues or nonlymphoid tissues. D3 was active in CD4(-)CD8(-), but not in CD4(+)CD8(+) or CD4(+)CD8(-) thymocytes in the thymus, and also active in B220(+)IgM(-), but not in B220(+)IgM(+), cells in the bone marrow. Finally, our data suggested that C/EBP may bind to the D3 enhancer and function as one of the transcription factor(s) responsible for the enhancer activity. These results show that the tissue- and stage-specific expression of murine RAG-2 is regulated by alteration of the chromatin structure as well as cis-regulatory enhancer elements.

Combinatorial regulation of the murine RAG-2 promoter by Sp1 and distinct lymphocyte-specific transcription factors

The recombination activation genes, RAG-1 and RAG-2, encode the critical components of the recombinase complex responsible for the generation of functional antigen receptor genes. In order to gain an insight into the transcription factors and cis-acting elements that regulate the lymphocyte-specific expression of RAG-2, the promoter-region of this gene was isolated and characterized. This analysis demonstrated that a relatively small promoter fragment could confer lymphocyte-restricted expression to a reporter construct. Our work and that of others subsequently revealed that RAG-2 promoter expression is positively regulated by BSAP (PAX-5) and c- Myb transcription factors in B- and T-lineage cells, respectively. Although BSAP and c- Myb were deemed necessary for lymphocyte-specific expression, our analysis also uncovered a G-rich region at the 5′-end of the core promoter that was essential for full activity in lymphocyte cell lines. Site-directed mutagenesis revealed that a GA-box within the G-rich region was required for full promoter activity and subsequent DNA binding assays demonstrated that this element was specifically recognized by Sp1. Apart from showing that Sp1 interacts within the RAG-2 promoter, we also demonstrate that the Sp1-binding site is necessary for the high-level activation of this promoter.

Cooperative binding of c-Myb and Pax-5 activates theRAG-2 promoter in immature B cells

AbstractThe recombination activating gene-1 (RAG-1) andRAG-2 are expressed specifically in immature lymphoid cells undergoing the recombination of antigen receptor genes. The regulation of murine RAG-2 promoter was studied and it was revealed that the −41/−17 RAG-2 promoter region, which is conserved between humans and mice, was indispensable for theRAG-2 promoter activity in B-cell lines. The region contained 2 cis elements that bound c-Myb and Pax-5. Mutation in the c-Myb–binding site in the promoter reduced the promoter activity in B-cell lines. Cooperative activation of theRAG-2 promoter was seen by a combination of c-Myb and Pax-5 in a human embryonic kidney cell line (293T), via their synergistic DNA-binding. Deletion experiments showed that the C-terminus of c-Myb was responsible for their interaction. Furthermore, the dominant-negative c-Myb mutant suppressed the activation of theRAG-2 promoter in a pre–B-cell line as well as in 293T cells. These results suggest that cooperative binding of c-Myb and Pax-5 to the RAG-2 promoter is one of the mechanisms to direct the restricted expression of the RAG-2 in immature B cells.

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.

Lineage-specific regulation of the murine RAG-2 promoter: GATA-3 in T cells and Pax-5 in B cells

Recombination activating gene-1 (RAG-1) and RAG-2 are expressed in lymphoid cells undergoing the antigen receptor gene rearrangement. A study of the regulation of the mouse RAG-2 promoter showed that the lymphocyte-specific promoter activity is conferred 80 nucleotide (nt) upstream of RAG-2. Using an electrophoretic mobility shift assay, it was shown that a B-cell-specific transcription protein, Pax-5, and a T-cell-specific transcription protein, GATA-3, bind to the -80 to -17 nt region in B cells and T cells, respectively. Mutation of the RAG-2 promoter for Pax-5- and GATA-3-binding sites results in the reduction of promoter activity in B cells and T cells. These results indicate that distinct DNA binding proteins, Pax-5 and GATA-3, may regulate the murine RAG-2 promoter in B and T lineage cells, respectively. (Blood. 2000;95:3845-3852)

Lineage-specific regulation of the murine RAG-2 promoter: GATA-3 in T cells and Pax-5 in B cells

AbstractRecombination activating gene-1 (RAG-1) andRAG-2 are expressed in lymphoid cells undergoing the antigen receptor gene rearrangement. A study of the regulation of the mouse RAG-2 promoter showed that the lymphocyte-specific promoter activity is conferred 80 nucleotide (nt) upstream of RAG-2. Using an electrophoretic mobility shift assay, it was shown that a B-cell–specific transcription protein, Pax-5, and a T-cell–specific transcription protein, GATA-3, bind to the −80 to −17 nt region in B cells and T cells, respectively. Mutation of the RAG-2 promoter for Pax-5– and GATA-3–binding sites results in the reduction of promoter activity in B cells and T cells. These results indicate that distinct DNA binding proteins, Pax-5 and GATA-3, may regulate the murine RAG-2 promoter in B and T lineage cells, respectively.

Functional analysis of the human RAG 2 promoter

Recombination activating genes RAG1 and RAG2 are essential components of V(D)J recombination, a process that generates the specific antigen receptors in lymphocytes. To understand the mechanisms underlying the lineage and developmental regulation of transcription of RAG2, we have characterized the human RAG2 exon 1A promoter. In this study, a series of deletion constructs were used to isolate the promoter while a linker scanning approach was taken to assess functionally relevant cis elements within the promoter. Two regulatory domains were identified. The −140 to −123 region is critical for promoter activity in all cell lines tested. Mutations to the putative Ets (−122 to −118) or to the C/EBP (−137 to −129) consensus core sequences did abrogate promoter activity, although specific DNA/protein interactions remained, as determined by EMSA. The −69 to −48 region demonstrates lineage specific promoter activity. Mutations to an overlapping, BSAP-myb-Ikaros-myb site (−65 to −39) resulted in differential promoter activity in human B and T cells. EMSA analysis of this region showed a B cell specific protein complex. Transfection of BSAP into cell lines trans-activates the human RAG2 promoter. We conclude that transcriptional regulation of the human RAG2 gene is complex, involving both tissue specific and ubiquitous factors, and both proximal and distal regulatory elements.

RAG2 is regulated differentially in B and T cells by elements 5' of the promoter.

To study RAG2 gene regulation in vivo, we developed a blastocyst complementation method in which RAG2-deficient embryonic stem cells were transfected with genomic clones containing RAG2 and then assessed for their ability to generate lymphocytes. A RAG2 genomic clone that contained only the RAG2 promoter sequences rescued V(D)J recombination in RAG2-deficient pro-B cell lines, but did not rescue development of RAG2-deficient lymphocytes in vivo. However, inclusion of varying lengths of sequences 5' of the RAG2 promoter generated constructs capable of rescuing only in vivo B cell development, as well as other constructs that rescued both B and T cell development. In particular, the 2-kb 5' region starting just upstream of the RAG2 promoter, as well as the region from 2-7 kb 5', could independently drive B cell development, but not efficient T cell development. Deletion of the 2-kb 5' region from the murine germ line demonstrated that this region was not required for RAG expression sufficient to generate normal B or T cell numbers, implying redundancy among 5' elements. We conclude that RAG2 expression in vivo requires elements beyond the core promoter, that such elements contribute to differential regulation in the B vs. T lineages, and that sequences sufficient to direct B cell expression are located in the promoter-proximal 5' region.

Cloning and characterization of the human recombination activating gene 1 (RAG1) and RAG2 promoter regions.

Recombination activating gene 1 (RAG1) and RAG2 are the essential and tissue-specific components of V(D)J recombination. We have characterized the genomic organization of the human RAG locus, mapped the transcriptional initiation sites, and partially sequenced and performed functional reporter assays on the 5' flanking regions of human RAG1 and RAG2. Transcription initiation sites were mapped by rapid amplification of 5' cDNA ends, primer extension, and/or RNase protection in normal thymocytes, three pre-B cell lines, and a mature B cell line. A single promoter region was used for RAG1 transcription. In contrast, transcription of RAG2 initiates at two distinct regions of the genome. The 5'-flanking region of the human RAG2 gene is TATA-less; however, there is a GATAA consensus at position -34 with respect to the major transcriptional initiation site of RAG1. Promoter regions of human RAG1 and RAG2 are active in both lymphoid and nonlymphoid cell lines, suggesting that an outside regulatory element is probably involved in the tissue-specific transcriptional regulation of the RAG genes.

Identification and characterization of the murine Rag1 promoter

Rag1 and Rag2 are required for the somatic rearrangement of immunoglobulin genes and T-cell receptor genes and the subsequent development of B and T cells. We describe the pattern of DNase I hypersensitive sites surrounding the Rag1 gene that accompanies mouse B-cell development and show that one of these sites corresponds to the murine Rag1 promoter. Transcription initiates over a 30 bp region, with approximately 70% of the transcripts initiating within a 5 bp region. The promoter contains neither a consensus TATA box nor an initiator, but does contain an AT rich sequence that could serve as a non-consensus TATA box. The Rag1 promoter directs only negligible levels of expression in transient transfection assays, but when combined with a heterologous enhancer, it is capable of driving significant levels of expression in pre-B cells, pre-T cells, and mature B cells. Methylation interference and mutation analysis reveal that the Rag1 promoter contains binding sites for E-box binding proteins, NF-Y proteins, and Ikaros proteins. These findings are discussed with respect to B-cell development and regulation of differential Rag1 expression by the promoter in pre-B, pre-T, and CNS cells.

Regulation of the RAG-1 promoter by the NF-Y transcription factor.

Abstract We have cloned the RAG-1 promoter region and have determined that almost all detectable promoter activity resides within a 208-bp fragment. Sequence analysis of this promoter region has identified potential recognition motifs for a number of lymphocyte-restricted and ubiquitous transcription factors. Subsequent assays have revealed that the NF-Y transcription factor interacts with a CCAAT site within the RAG-1 promoter and appears to play an important role in the positive transcriptional regulation of this gene.

Chromatin Structure and Transcriptional Regulation of Human RAG-1 Gene

The recombination activating genes (RAGs) play a critical role in V(D)J recombination machinery and lymphocyte development. Their expression is strictly regulated during lymphocyte ontogeny, with expression being rapidly lost as the lymphoid precursors differentiate into their progeny. To elucidate molecular mechanisms of regulation of human RAG-1 gene expression, we examined a chromatin structure of a approximately 24-kb DNA segment adjacent to a human RAG-1 promoter region in various cell lines by analyzing DNase I hypersensitive (DNase I HS) sites. In a RAG-1-expressing human pre-B-cell line, at least four DNase I HS sites (HS1, HS2, HS3, and HS4) were identified. Among these HS sites, one HS site (HS1) was ubiquitously detected in all cell lines examined, but the other three HS sites (HS2, HS3, and HS4) were associated only with RAG-1-expressing lymphoid cell lines. Using transient expression assays, we showed that the 5' upstream region of the major transcription start site showed low but significant promoter activity and that a DNA segment within HS3 located in the promoter region was indispensable to its basal promoter activity. Importantly, this promoter region was shown to be active in both RAG-1-expressing and RAG-1-nonexpressing cell lines. These results suggest that alteration of chromatin structure in the promoter region, in addition to other control elements outside of the promoter region, is one of the mechanisms regulating tissue- and stage-specific expression of human RAG-1 gene.