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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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Detection of V(D)J recombinase activity in natural killer cells. (A) The CD4−CD8−CD90− subset of bone marrow was examined for CD122 and DX5 or NK1.1 expression in control B6 (top) or SB110 H2-SVEX transgenic mice (bottom). Immature CD122+DX5− NK1.1− pNK cells (gated population in left and middle) were subsequently analyzed for VEX expression (right). (B) CD122+NK1.1+ cells lacking CD3, CD4, CD8, and CD19 were examined for VEX expression in bone marrow (left) or spleen (right) obtained from SB88 H2-SVEX transgenic (top) or control B6 mice (bottom). Identical results were obtained using SB110 H2-SVEX transgenic mice (not depicted). (C) Bone marrow CD3−CD4−CD8−CD122+NK1.1−DX5− pNK (top) and CD3− CD4−CD8−CD122+NK1.1+CD3−CD19− NK (bottom) were examined for GFP expression in RAG2 GFP NG transgenic (left) or control B6 mice (right). The percentage of cells in each gate is given. (D) NK1.1+DX5+CD19−CD3−CD4−CD8− spleen cells were sorted from C57BL/6 or βδ-TCR−/−mice were analyzed by PCR for D-JH joins. PCR analysis of bulk sorted cells is depicted in the gel on the top. SPL refers to total splenic cells. Two cells lines with known rearrangement patterns were used as controls. The AMLV-transformed cell line A12 is derived from a RAG1−/− mouse and therefore retains germline Ig configuration (61). The AMLV-transformed “DJH fixed” 300–35 line bears a D-JH1 rearrangement (62). D-JH4 was not examined. By PCR analysis of single, sorted NK cells from C57BL/6 mice, we determined that the frequency of splenic NK that harbor D-JH rearrangements was 4.8% (3 D-JH+ cells out of 62 NK cells analyzed) as indicated on the bottom. The data in A–C are representative of two to five independent experiments. The data in D are representative of at least two experiments using independently sorted cells.
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fig4: Detection of V(D)J recombinase activity in natural killer cells. (A) The CD4−CD8−CD90− subset of bone marrow was examined for CD122 and DX5 or NK1.1 expression in control B6 (top) or SB110 H2-SVEX transgenic mice (bottom). Immature CD122+DX5− NK1.1− pNK cells (gated population in left and middle) were subsequently analyzed for VEX expression (right). (B) CD122+NK1.1+ cells lacking CD3, CD4, CD8, and CD19 were examined for VEX expression in bone marrow (left) or spleen (right) obtained from SB88 H2-SVEX transgenic (top) or control B6 mice (bottom). Identical results were obtained using SB110 H2-SVEX transgenic mice (not depicted). (C) Bone marrow CD3−CD4−CD8−CD122+NK1.1−DX5− pNK (top) and CD3− CD4−CD8−CD122+NK1.1+CD3−CD19− NK (bottom) were examined for GFP expression in RAG2 GFP NG transgenic (left) or control B6 mice (right). The percentage of cells in each gate is given. (D) NK1.1+DX5+CD19−CD3−CD4−CD8− spleen cells were sorted from C57BL/6 or βδ-TCR−/−mice were analyzed by PCR for D-JH joins. PCR analysis of bulk sorted cells is depicted in the gel on the top. SPL refers to total splenic cells. Two cells lines with known rearrangement patterns were used as controls. The AMLV-transformed cell line A12 is derived from a RAG1−/− mouse and therefore retains germline Ig configuration (61). The AMLV-transformed “DJH fixed” 300–35 line bears a D-JH1 rearrangement (62). D-JH4 was not examined. By PCR analysis of single, sorted NK cells from C57BL/6 mice, we determined that the frequency of splenic NK that harbor D-JH rearrangements was 4.8% (3 D-JH+ cells out of 62 NK cells analyzed) as indicated on the bottom. The data in A–C are representative of two to five independent experiments. The data in D are representative of at least two experiments using independently sorted cells.

Mentions: In addition to B and T lymphocytes, the CLP gives rise to NK and DC, and it is possible that some VEX+ CLPs may give rise to these two lineages. Because VEX permanently marks cells that have expressed the V(D)J recombinase, we had the opportunity to examine whether some NK and DC have a history of recombinase activity. We first examined VEX in NK precursors (pNK; CD122+ CD4−CD8−CD90−DX5−) (42, 43). Approximately 60% of pNK from H2-SVEX mice express VEX versus 0% from control B6 (Fig. 4 A, right). We also examined expression of rag2 transcription in this subset using the NG RAG2 transgenic reporter line in which GFP replaces the major exon of RAG2 (17). The pNK population from these reporter mice had high levels of GFP expression (Fig. 4 C, top, third row, left panel), indicative of RAG2 transcription, supporting the idea that the pNK may have (or may have had) an active V(D)J recombinase.


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)

Detection of V(D)J recombinase activity in natural killer cells. (A) The CD4−CD8−CD90− subset of bone marrow was examined for CD122 and DX5 or NK1.1 expression in control B6 (top) or SB110 H2-SVEX transgenic mice (bottom). Immature CD122+DX5− NK1.1− pNK cells (gated population in left and middle) were subsequently analyzed for VEX expression (right). (B) CD122+NK1.1+ cells lacking CD3, CD4, CD8, and CD19 were examined for VEX expression in bone marrow (left) or spleen (right) obtained from SB88 H2-SVEX transgenic (top) or control B6 mice (bottom). Identical results were obtained using SB110 H2-SVEX transgenic mice (not depicted). (C) Bone marrow CD3−CD4−CD8−CD122+NK1.1−DX5− pNK (top) and CD3− CD4−CD8−CD122+NK1.1+CD3−CD19− NK (bottom) were examined for GFP expression in RAG2 GFP NG transgenic (left) or control B6 mice (right). The percentage of cells in each gate is given. (D) NK1.1+DX5+CD19−CD3−CD4−CD8− spleen cells were sorted from C57BL/6 or βδ-TCR−/−mice were analyzed by PCR for D-JH joins. PCR analysis of bulk sorted cells is depicted in the gel on the top. SPL refers to total splenic cells. Two cells lines with known rearrangement patterns were used as controls. The AMLV-transformed cell line A12 is derived from a RAG1−/− mouse and therefore retains germline Ig configuration (61). The AMLV-transformed “DJH fixed” 300–35 line bears a D-JH1 rearrangement (62). D-JH4 was not examined. By PCR analysis of single, sorted NK cells from C57BL/6 mice, we determined that the frequency of splenic NK that harbor D-JH rearrangements was 4.8% (3 D-JH+ cells out of 62 NK cells analyzed) as indicated on the bottom. The data in A–C are representative of two to five independent experiments. The data in D are representative of at least two experiments using independently sorted cells.
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Related In: Results  -  Collection

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fig4: Detection of V(D)J recombinase activity in natural killer cells. (A) The CD4−CD8−CD90− subset of bone marrow was examined for CD122 and DX5 or NK1.1 expression in control B6 (top) or SB110 H2-SVEX transgenic mice (bottom). Immature CD122+DX5− NK1.1− pNK cells (gated population in left and middle) were subsequently analyzed for VEX expression (right). (B) CD122+NK1.1+ cells lacking CD3, CD4, CD8, and CD19 were examined for VEX expression in bone marrow (left) or spleen (right) obtained from SB88 H2-SVEX transgenic (top) or control B6 mice (bottom). Identical results were obtained using SB110 H2-SVEX transgenic mice (not depicted). (C) Bone marrow CD3−CD4−CD8−CD122+NK1.1−DX5− pNK (top) and CD3− CD4−CD8−CD122+NK1.1+CD3−CD19− NK (bottom) were examined for GFP expression in RAG2 GFP NG transgenic (left) or control B6 mice (right). The percentage of cells in each gate is given. (D) NK1.1+DX5+CD19−CD3−CD4−CD8− spleen cells were sorted from C57BL/6 or βδ-TCR−/−mice were analyzed by PCR for D-JH joins. PCR analysis of bulk sorted cells is depicted in the gel on the top. SPL refers to total splenic cells. Two cells lines with known rearrangement patterns were used as controls. The AMLV-transformed cell line A12 is derived from a RAG1−/− mouse and therefore retains germline Ig configuration (61). The AMLV-transformed “DJH fixed” 300–35 line bears a D-JH1 rearrangement (62). D-JH4 was not examined. By PCR analysis of single, sorted NK cells from C57BL/6 mice, we determined that the frequency of splenic NK that harbor D-JH rearrangements was 4.8% (3 D-JH+ cells out of 62 NK cells analyzed) as indicated on the bottom. The data in A–C are representative of two to five independent experiments. The data in D are representative of at least two experiments using independently sorted cells.
Mentions: In addition to B and T lymphocytes, the CLP gives rise to NK and DC, and it is possible that some VEX+ CLPs may give rise to these two lineages. Because VEX permanently marks cells that have expressed the V(D)J recombinase, we had the opportunity to examine whether some NK and DC have a history of recombinase activity. We first examined VEX in NK precursors (pNK; CD122+ CD4−CD8−CD90−DX5−) (42, 43). Approximately 60% of pNK from H2-SVEX mice express VEX versus 0% from control B6 (Fig. 4 A, right). We also examined expression of rag2 transcription in this subset using the NG RAG2 transgenic reporter line in which GFP replaces the major exon of RAG2 (17). The pNK population from these reporter mice had high levels of GFP expression (Fig. 4 C, top, third row, left panel), indicative of RAG2 transcription, supporting the idea that the pNK may have (or may have had) an active V(D)J recombinase.

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