Control of VDJ Recombinase Activity

Ferrier, Pierre; Krippl, B; Furley, Andrew J.; Tk, Blackwell; H, Suh; Mendelsohn, Mortimer L.; Winoto, Astar; Wd, Cook; Hood, Leroy; Costantini, Frank

doi:10.1101/sqb.1989.054.01.024

Cited by 13 publications

(4 citation statements)

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“…The implications of these results, in terms of the molecular mechanisms and nuclear factors involved, are discussed below. The V(D)J recombination-enhancing E␤ subfragments identified in this study, including the E␤98 core, promoted rearrangement and germ line transcription from the integrated transgenes in several independent transgenic mouse lines, in conformity with the correlation between the two activities generally observed at antigen receptor gene loci (20,67). In a few instances, however, we found that some E␤ subfragments (e.g., E␤200 and E␤98) were not active after transgene integration within or close to a chromosomal centromere.…”

Section: Discussionmentioning

confidence: 91%

“…However, the molecular mechanism(s) and structural basis underlying these phenomena are not yet understood. Proposed activating factors include transcription across the unrearranged (germ line) region, the down-modulation of CpG methylation, and/or other epigenetic changes in chromatin (e.g., level of histone acetylation) affecting nucleosome positioning (13,20,54).…”

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confidence: 99%

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Definition of a T-Cell Receptor β Gene Core Enhancer of V(D)J Recombination by Transgenic Mapping

Tripathi

Mathieu

Spicuglia

et al. 2000

Molecular and Cellular Biology

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V(D)J recombination in differentiating lymphocytes is a highly regulated process in terms of both cell lineage and the stage of cell development. Transgenic and knockout mouse studies have demonstrated that transcriptional enhancers from antigen receptor genes play an important role in this regulation by activating cisrecombination events. A striking example is the T-cell receptor ␤-chain (TCR␤) gene enhancer (E␤), which in the mouse consists of at least seven nuclear factor binding motifs (␤E1 to ␤E7). Here, using a well-characterized transgenic recombination substrate approach, we define the sequences within E␤ required for recombination enhancer activity. The E␤ core is comprised of a limited set of motifs (␤E3 and ␤E4) and an additional previously uncharacterized 20-bp sequence 3 of the ␤E4 motif. This core element confers cell lineage-and stage-specific recombination within the transgenic substrates, although it cannot bypass the suppressive effects resulting from transgene integration in heterochromatic centromeres. Strikingly, the core enhancer is heavily occupied by nuclear factors in immature thymocytes, as shown by in vivo footprinting analyses. A larger enhancer fragment including the ␤E1 through ␤E4 motifs but not the 3 sequences, although active in inducing germ line transcription within the transgenic array, did not retain the E␤ recombinational activity. Our results emphasize the multifunctionality of the TCR␤ enhancer and shed some light on the molecular mechanisms by which transcriptional enhancers and associated nuclear factors may impact on cis recombination, gene expression, and lymphoid cell differentiation.Immunoglobulin (Ig) and T-cell receptor (TCR) genes are assembled from separate variable (V), diversity (D), or joining (J) gene segments in a process known as V(D)J recombination (4, 44, 55). Normally, V(D)J recombination is restricted to, and required for, early B and T lymphocyte development. It depends on a unique activity, called the V(D)J recombinase, which targets recombination signal sequences (RSSs; consisting of a heptamer, a spacer of 12 or 23 bp, and a nonamer) flanking the rearranging sides of all V, D, and J segments. Rearrangement events primarily involve pairs of segments with RSSs of asymmetrical spacer length (12/23 rule).The functional core of the V(D)J recombinase consists of the lymphoid-restricted RAG-1 and RAG-2 gene products which recognize, pair off, and cleave the RSSs from two rearranging segments, a step also involving architectural proteins from the high-mobility-group family (22, 61). These cleavages consist of DNA double-strand breaks (DSBs) introduced precisely at the junction between the heptamer and adjacent coding sequences, yielding two distinct products: the hairpinsealed coding ends (CEs) and the phosphorylated, blunt-ended signal ends (SEs). Eventually, the cleaved products are assembled together, a process which requires, in addition to the RAG factors, components of the general DNA DSB repair machinery (26, 54). The two CEs are assembled to form a ...

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Section: Discussionmentioning

confidence: 91%

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confidence: 99%

Definition of a T-Cell Receptor β Gene Core Enhancer of V(D)J Recombination by Transgenic Mapping

Tripathi

Mathieu

Spicuglia

et al. 2000

Molecular and Cellular Biology

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“…Studies using transgenic mice and cell lines suggest that specific gene segment transcription correlates with differential accessibility of each locus to the recombinase during development (8,7). Production of sterile transcripts from unrearranged gene segments, in particular, has been found to precede gene rearrangement.…”

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Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression.

Kingsley¹,

Winoto²

1992

Mol. Cell. Biol.

514

384

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Analysis of a T-cell antigen receptor (TCR) a promoter from a variable gene segment (V) revealed a critical GT box element which is also found in upstream regions of several Va genes, TCR enhancer, and regulatory elements of other genes. This element is necessary for TCR gene expression and binds several proteins. These GT box-binding proteins were identified as members of a novel Spl multigene family. Two regions (for reviews, see references 5 and 27). The process of V-D-J gene rearrangements is developmentally and tissue specifically regulated. The TCR a and 8 genes are particularly interesting because the 8 locus is located within the TCR a-gene segments. In T cells committed to the a,3 lineage, the a-specific V-gene segments are rearranged to the a locus, whereas in cells committed to the -yb lineage, the b-specific V-gene segments are rearranged to their respective D/Jb gene segments. Some V gene segments, however, can rearrange to either the a or 8 locus (these gene segments are also called Va).Studies using transgenic mice and cell lines suggest that specific gene segment transcription correlates with differential accessibility of each locus to the recombinase during development (8, 7). Production of sterile transcripts from unrearranged gene segments, in particular, has been found to precede gene rearrangement. It is hypothesized that sterile transcription is either the cause or the consequence of differential opening of chromatin structure and hence accessibility of the gene segment to the recombination enzymes (references 7 and 28 and references therein). Since each of the V-gene segments contains its own promoter, it is possible that the specificity of sterile transcription from these V promoters could contribute to differential accessibility to recombinases during T-cell development, which could result in specific gene segment usage in aot and/or 9y T cells (35). * Corresponding author.Therefore, the study of TCR a promoters can serve the dual purpose of analyzing TCR gene expression as well as regulation of rearrangement.We have focused on the well-characterized Vall.1 gene segment, which is the gene segment predominantly used in T-cell response to pigeon cytochrome c (9, 38). We have analyzed the critical elements for the Vall.1 promoter and found one element which we called the GT box. This GT box is also present in most other available Va upstream sequences (presumably promoters) as well as in the TCR ax enhancer. Homologous regulatory elements (AC boxes) were previously defined in the ,-globin enhancers, promoters, and locus-controlling region (LCR) as well as in the bovine papillomavirus (BPV) promoters and simian virus (SV40) enhancer. This novel element for T-cell gene expression was shown to bind ubiquitously expressed proteins, one of which was identified as the transcription factor Spl (18). Although Spl is one of the earliest-characterized transcription factors which also binds to a consensus GC box sequence, we have made the assumption that additional GT box-binding proteins contain se...

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“…Highly homologous heptamer-nonamer recombination signal sequences (RSSs) flank both immunoglobulin and TCR genes (Tonegawa 1983). Rearrangement reporter constructs containing TCR RSSs rearrange upon transfection into pre-B cells (Yancopoulos et al 1986), and transgenic immunoglobulin gene segments can rearrange in T cells (Ferrier et al 1989;Ferrier et al 1990). Finally, the recently cloned recombination-activating genes RAG-1 and RAG-2 are expressed in both pre-B and pre-T cells (Schatz et al 1989;Oettinger et al 1990).…”

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

Schlissel¹,

Voronova²,

Baltimore³

1991

Genes Dev.

218

145

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E47 is a helix-loop-helix transcription factor that binds to sites in the immunoglobulin heavy-chain and K 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 K 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.

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Control of VDJ Recombinase Activity

Cited by 13 publications

References 0 publications

Definition of a T-Cell Receptor β Gene Core Enhancer of V(D)J Recombination by Transgenic Mapping

Definition of a T-Cell Receptor β Gene Core Enhancer of V(D)J Recombination by Transgenic Mapping

Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression.

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

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