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B lineage-specific regulation of V(D)J recombinase activity is established in common lymphoid progenitors.

Borghesi L, Hsu LY, Miller JP, Anderson M, Herzenberg L, Herzenberg L, Schlissel MS, Allman D, Gerstein RM - J. Exp. Med. (2004)

Bottom Line: Evidence of this recombinase activity is detectable in all four progeny lineages (B, T, and NK, and DC), and rag2 levels are the highest in progenitor subsets immediately downstream of the CLP.By single cell PCR, we demonstrate that V(D)J rearrangements are detectable at IgH loci in approximately 5% of splenic natural killer cells.As activity of the Erag enhancer is restricted to the B cell lineage, this provides the first molecular evidence for establishment of a lineage-specific transcription program in multipotent progenitors.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester 01655, USA.

ABSTRACT
Expression of V(D)J recombinase activity in developing lymphocytes is absolutely required for initiation of V(D)J recombination at antigen receptor loci. However, little is known about when during hematopoietic development the V(D)J recombinase is first active, nor is it known what elements activate the recombinase in multipotent hematopoietic progenitors. Using mice that express a fluorescent transgenic V(D)J recombination reporter, we show that the V(D)J recombinase is active as early as common lymphoid progenitors (CLPs) but not in the upstream progenitors that retain myeloid lineage potential. Evidence of this recombinase activity is detectable in all four progeny lineages (B, T, and NK, and DC), and rag2 levels are the highest in progenitor subsets immediately downstream of the CLP. By single cell PCR, we demonstrate that V(D)J rearrangements are detectable at IgH loci in approximately 5% of splenic natural killer cells. Finally, we show that recombinase activity in CLPs is largely controlled by the Erag enhancer. As activity of the Erag enhancer is restricted to the B cell lineage, this provides the first molecular evidence for establishment of a lineage-specific transcription program in multipotent progenitors.

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(A) The transgenic H2-SVEX substrate contains VEX (white rectangle) driven by the murine H2K promoter (black rectangle). VEX within the substrate is initially in the antisense orientation and is flanked by V(D)J recombination signal sequences (triangles) which direct inversional recombination. Primers used to discriminate H2-SVEX before and after rearrangement are indicated (2011, 200, 586, and 2165; as described in Materials and Methods). (B) Splenocytes from SB110 and SB88 H2-SVEX animals were stained with antibodies to detect CD19+ IgM+ B cells, CD3+ T cells, CD11b+CD3−CD19−NK1.1− myeloid cells, or Gr-1+ CD19−CD3−NK1.1− granulocytes and subsequently examined for VEX expression. The percentage of VEX+ cells in the gate is given, and outliers are shown. (C) H2-SVEX recombination depends on RAG1. B220+ B cells in the bone marrow were examined for VEX expression in H2-SVEX SB110, H2-SVEX RAG1−/−, RAG−/−, and nontransgenic C57BL/6 control mice. The percentage of VEX+ cells in the gate is given. H2-SVEX RAG1−/− mice were identified by PCR analysis of the SVEX cassette as depicted in A and Fig. 2 A. Identical results were obtained with SB88 H2-SVEX RAG1−/− mice (not depicted). The data presented are representative of six independent experiments.
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fig1: (A) The transgenic H2-SVEX substrate contains VEX (white rectangle) driven by the murine H2K promoter (black rectangle). VEX within the substrate is initially in the antisense orientation and is flanked by V(D)J recombination signal sequences (triangles) which direct inversional recombination. Primers used to discriminate H2-SVEX before and after rearrangement are indicated (2011, 200, 586, and 2165; as described in Materials and Methods). (B) Splenocytes from SB110 and SB88 H2-SVEX animals were stained with antibodies to detect CD19+ IgM+ B cells, CD3+ T cells, CD11b+CD3−CD19−NK1.1− myeloid cells, or Gr-1+ CD19−CD3−NK1.1− granulocytes and subsequently examined for VEX expression. The percentage of VEX+ cells in the gate is given, and outliers are shown. (C) H2-SVEX recombination depends on RAG1. B220+ B cells in the bone marrow were examined for VEX expression in H2-SVEX SB110, H2-SVEX RAG1−/−, RAG−/−, and nontransgenic C57BL/6 control mice. The percentage of VEX+ cells in the gate is given. H2-SVEX RAG1−/− mice were identified by PCR analysis of the SVEX cassette as depicted in A and Fig. 2 A. Identical results were obtained with SB88 H2-SVEX RAG1−/− mice (not depicted). The data presented are representative of six independent experiments.

Mentions: The H2-SVEX transgene was constructed by placing the RSS-VEX-RSS fragment (Fig. 1 A) into the H2K (HIL) transgenic vector using a unique NotI restriction site located between the H2 promoter and the H2 exon fragment. The H2K cassette vector expresses genes under the control of the H2K promoter/enhancer and Moloney MuLV enhancer/poly(A), typically at high levels in HSC and all hematolymphoid cells (32–35). Heterologous promoter activation has been shown to be sufficient for directing rearrangement of chromosomal recombination substrates (36). Transgenic mice were made at the UMMS transgenic facility using standard procedures. From the injected C57BL/6 embryos, 13 of 136 mice were positive for the transgene as analyzed by PCR. Of these potential founders, six expressed VEX in peripheral white blood cells and four such mice were used to establish permanent transgenic lines: SB68, SB88, SB110, and SB114. VEX is from MFG-hu-VEX-2 (37–39). The recombination signal sequence (RSS) fragments contain consensus RSS and 16 bp from the murine DFL16.1 coding region (12-RSS) or 17 bp from the murine JH1 coding region (23-RSS) (40).


B lineage-specific regulation of V(D)J recombinase activity is established in common lymphoid progenitors.

Borghesi L, Hsu LY, Miller JP, Anderson M, Herzenberg L, Herzenberg L, Schlissel MS, Allman D, Gerstein RM - J. Exp. Med. (2004)

(A) The transgenic H2-SVEX substrate contains VEX (white rectangle) driven by the murine H2K promoter (black rectangle). VEX within the substrate is initially in the antisense orientation and is flanked by V(D)J recombination signal sequences (triangles) which direct inversional recombination. Primers used to discriminate H2-SVEX before and after rearrangement are indicated (2011, 200, 586, and 2165; as described in Materials and Methods). (B) Splenocytes from SB110 and SB88 H2-SVEX animals were stained with antibodies to detect CD19+ IgM+ B cells, CD3+ T cells, CD11b+CD3−CD19−NK1.1− myeloid cells, or Gr-1+ CD19−CD3−NK1.1− granulocytes and subsequently examined for VEX expression. The percentage of VEX+ cells in the gate is given, and outliers are shown. (C) H2-SVEX recombination depends on RAG1. B220+ B cells in the bone marrow were examined for VEX expression in H2-SVEX SB110, H2-SVEX RAG1−/−, RAG−/−, and nontransgenic C57BL/6 control mice. The percentage of VEX+ cells in the gate is given. H2-SVEX RAG1−/− mice were identified by PCR analysis of the SVEX cassette as depicted in A and Fig. 2 A. Identical results were obtained with SB88 H2-SVEX RAG1−/− mice (not depicted). The data presented are representative of six independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211824&req=5

fig1: (A) The transgenic H2-SVEX substrate contains VEX (white rectangle) driven by the murine H2K promoter (black rectangle). VEX within the substrate is initially in the antisense orientation and is flanked by V(D)J recombination signal sequences (triangles) which direct inversional recombination. Primers used to discriminate H2-SVEX before and after rearrangement are indicated (2011, 200, 586, and 2165; as described in Materials and Methods). (B) Splenocytes from SB110 and SB88 H2-SVEX animals were stained with antibodies to detect CD19+ IgM+ B cells, CD3+ T cells, CD11b+CD3−CD19−NK1.1− myeloid cells, or Gr-1+ CD19−CD3−NK1.1− granulocytes and subsequently examined for VEX expression. The percentage of VEX+ cells in the gate is given, and outliers are shown. (C) H2-SVEX recombination depends on RAG1. B220+ B cells in the bone marrow were examined for VEX expression in H2-SVEX SB110, H2-SVEX RAG1−/−, RAG−/−, and nontransgenic C57BL/6 control mice. The percentage of VEX+ cells in the gate is given. H2-SVEX RAG1−/− mice were identified by PCR analysis of the SVEX cassette as depicted in A and Fig. 2 A. Identical results were obtained with SB88 H2-SVEX RAG1−/− mice (not depicted). The data presented are representative of six independent experiments.
Mentions: The H2-SVEX transgene was constructed by placing the RSS-VEX-RSS fragment (Fig. 1 A) into the H2K (HIL) transgenic vector using a unique NotI restriction site located between the H2 promoter and the H2 exon fragment. The H2K cassette vector expresses genes under the control of the H2K promoter/enhancer and Moloney MuLV enhancer/poly(A), typically at high levels in HSC and all hematolymphoid cells (32–35). Heterologous promoter activation has been shown to be sufficient for directing rearrangement of chromosomal recombination substrates (36). Transgenic mice were made at the UMMS transgenic facility using standard procedures. From the injected C57BL/6 embryos, 13 of 136 mice were positive for the transgene as analyzed by PCR. Of these potential founders, six expressed VEX in peripheral white blood cells and four such mice were used to establish permanent transgenic lines: SB68, SB88, SB110, and SB114. VEX is from MFG-hu-VEX-2 (37–39). The recombination signal sequence (RSS) fragments contain consensus RSS and 16 bp from the murine DFL16.1 coding region (12-RSS) or 17 bp from the murine JH1 coding region (23-RSS) (40).

Bottom Line: Evidence of this recombinase activity is detectable in all four progeny lineages (B, T, and NK, and DC), and rag2 levels are the highest in progenitor subsets immediately downstream of the CLP.By single cell PCR, we demonstrate that V(D)J rearrangements are detectable at IgH loci in approximately 5% of splenic natural killer cells.As activity of the Erag enhancer is restricted to the B cell lineage, this provides the first molecular evidence for establishment of a lineage-specific transcription program in multipotent progenitors.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester 01655, USA.

ABSTRACT
Expression of V(D)J recombinase activity in developing lymphocytes is absolutely required for initiation of V(D)J recombination at antigen receptor loci. However, little is known about when during hematopoietic development the V(D)J recombinase is first active, nor is it known what elements activate the recombinase in multipotent hematopoietic progenitors. Using mice that express a fluorescent transgenic V(D)J recombination reporter, we show that the V(D)J recombinase is active as early as common lymphoid progenitors (CLPs) but not in the upstream progenitors that retain myeloid lineage potential. Evidence of this recombinase activity is detectable in all four progeny lineages (B, T, and NK, and DC), and rag2 levels are the highest in progenitor subsets immediately downstream of the CLP. By single cell PCR, we demonstrate that V(D)J rearrangements are detectable at IgH loci in approximately 5% of splenic natural killer cells. Finally, we show that recombinase activity in CLPs is largely controlled by the Erag enhancer. As activity of the Erag enhancer is restricted to the B cell lineage, this provides the first molecular evidence for establishment of a lineage-specific transcription program in multipotent progenitors.

Show MeSH