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Regulation of T cell receptor delta gene rearrangement by CBF/PEBP2.

Lauzurica P, Zhong XP, Krangel MS, Roberts JL - J. Exp. Med. (1997)

Bottom Line: Because mutation of the deltaE3 binding site for the transcription factor c-Myb had previously established a similar role for c-Myb, and because a 60-bp fragment of E(delta) carrying deltaE3 and deltaE4 binding sites for CBF/PEBP2, c-Myb, and GATA-3 displays significant enhancer activity in transient transfection experiments, we tested whether this fragment of E(delta) is sufficient to activate VDJ recombination in vivo.This fragment failed to efficiently activate the enhancer-dependent VD to J step of minilocus rearrangement in all three transgenic lines examined, indicating that the binding of CBF/PEBP2 and c-Myb to their cognate sites within E(delta), although necessary, is not sufficient for the activation of VDJ recombination by E(delta).These results imply that CBF/PEBP2 and c-Myb collaborate with additional factors that bind elsewhere within E(delta) to modulate local accessibility to the VDJ recombinase in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
We have analyzed transgenic mice carrying versions of a human T cell receptor (TCR)-delta gene minilocus to study the developmental control of VDJ (variable/diversity/joining) recombination. Previous data indicated that a 1.4-kb DNA fragment carrying the TCR-delta enhancer (E(delta)) efficiently activates minilocus VDJ recombination in vivo. We tested whether the transcription factor CBF/PEBP2 plays an important role in the ability of E(delta) to activate VDJ recombination by analyzing VDJ recombination in mice carrying a minilocus in which the deltaE3 element of E(delta) includes a mutated CBF/PEBP2 binding site. The enhancer-dependent VD to J step of minilocus rearrangement was dramatically inhibited in three of four transgenic lines, arguing that the binding of CBF/PEBP2 plays a role in modulating local accessibility to the VDJ recombinase in vivo. Because mutation of the deltaE3 binding site for the transcription factor c-Myb had previously established a similar role for c-Myb, and because a 60-bp fragment of E(delta) carrying deltaE3 and deltaE4 binding sites for CBF/PEBP2, c-Myb, and GATA-3 displays significant enhancer activity in transient transfection experiments, we tested whether this fragment of E(delta) is sufficient to activate VDJ recombination in vivo. This fragment failed to efficiently activate the enhancer-dependent VD to J step of minilocus rearrangement in all three transgenic lines examined, indicating that the binding of CBF/PEBP2 and c-Myb to their cognate sites within E(delta), although necessary, is not sufficient for the activation of VDJ recombination by E(delta). These results imply that CBF/PEBP2 and c-Myb collaborate with additional factors that bind elsewhere within E(delta) to modulate local accessibility to the VDJ recombinase in vivo.

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Quantification of  minilocus VDJ rearrangement.  Serially diluted samples of thymus genomic DNA from Eδ line  A were amplified by PCR using  primers 1 and 3 or primers 5 and  6, and Southern blots were developed using radiolabeled Vδ1  and Cδ cDNA probes. The Vδ1Dδ3-Jδ1 (solid line) and Cδ (dotted  line) hybridization signals were  quantified using a PhosphorImager. The results are expressed  log hybridization signal in arbitrary units versus log DNA concentration  in μg/ml. The second most concentrated DNA sample in this experiment corresponds to the amount of DNA used for analysis of single copy  integrants in all subsequent experiments. Lower amounts of DNA were  used for multicopy integrants.
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Figure 2: Quantification of minilocus VDJ rearrangement. Serially diluted samples of thymus genomic DNA from Eδ line A were amplified by PCR using primers 1 and 3 or primers 5 and 6, and Southern blots were developed using radiolabeled Vδ1 and Cδ cDNA probes. The Vδ1Dδ3-Jδ1 (solid line) and Cδ (dotted line) hybridization signals were quantified using a PhosphorImager. The results are expressed log hybridization signal in arbitrary units versus log DNA concentration in μg/ml. The second most concentrated DNA sample in this experiment corresponds to the amount of DNA used for analysis of single copy integrants in all subsequent experiments. Lower amounts of DNA were used for multicopy integrants.

Mentions: Analysis of wild-type and EδmCore minilocus VDJ recombination was performed by PCR from thymus genomic DNA templates, using specific Vδ1, Vδ2, Jδ1, and Jδ3 primers (primers 1, 2, 3 and 4; Fig. 1 C) as described previously (13). Previous studies have shown the wild-type minilocus to rearrange stepwise, first V to D, and then VD to J (13). Amplification with Vδ and Jδ1 primers yields 0.3kb fragments that represent complete VDJ rearrangements, and in addition, 1.2-kb fragments that represent the VD rearrangement intermediates (Fig. 1 D). Amplification with Vδ and Jδ3 primers yields 0.3-kb VDJ products only. PCR reactions were also performed with a pair of Cδ primers (primers 5 and 6; Fig. 1 C), to control for differences in transgene copy number and PCR efficiency between samples; the amount of genomic DNA template used in PCR reactions was typically adjusted to obtain similar Cδ amplification signals in each sample. PCR products were detected and quantified by agarose gel electrophoresis followed by blotting and hybridization with appropriate 32P-labeled probes. In agreement with our previous studies (13), quantification of PCR signals revealed amplification to be linear over a broad range of template concentrations (Fig. 2).


Regulation of T cell receptor delta gene rearrangement by CBF/PEBP2.

Lauzurica P, Zhong XP, Krangel MS, Roberts JL - J. Exp. Med. (1997)

Quantification of  minilocus VDJ rearrangement.  Serially diluted samples of thymus genomic DNA from Eδ line  A were amplified by PCR using  primers 1 and 3 or primers 5 and  6, and Southern blots were developed using radiolabeled Vδ1  and Cδ cDNA probes. The Vδ1Dδ3-Jδ1 (solid line) and Cδ (dotted  line) hybridization signals were  quantified using a PhosphorImager. The results are expressed  log hybridization signal in arbitrary units versus log DNA concentration  in μg/ml. The second most concentrated DNA sample in this experiment corresponds to the amount of DNA used for analysis of single copy  integrants in all subsequent experiments. Lower amounts of DNA were  used for multicopy integrants.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196263&req=5

Figure 2: Quantification of minilocus VDJ rearrangement. Serially diluted samples of thymus genomic DNA from Eδ line A were amplified by PCR using primers 1 and 3 or primers 5 and 6, and Southern blots were developed using radiolabeled Vδ1 and Cδ cDNA probes. The Vδ1Dδ3-Jδ1 (solid line) and Cδ (dotted line) hybridization signals were quantified using a PhosphorImager. The results are expressed log hybridization signal in arbitrary units versus log DNA concentration in μg/ml. The second most concentrated DNA sample in this experiment corresponds to the amount of DNA used for analysis of single copy integrants in all subsequent experiments. Lower amounts of DNA were used for multicopy integrants.
Mentions: Analysis of wild-type and EδmCore minilocus VDJ recombination was performed by PCR from thymus genomic DNA templates, using specific Vδ1, Vδ2, Jδ1, and Jδ3 primers (primers 1, 2, 3 and 4; Fig. 1 C) as described previously (13). Previous studies have shown the wild-type minilocus to rearrange stepwise, first V to D, and then VD to J (13). Amplification with Vδ and Jδ1 primers yields 0.3kb fragments that represent complete VDJ rearrangements, and in addition, 1.2-kb fragments that represent the VD rearrangement intermediates (Fig. 1 D). Amplification with Vδ and Jδ3 primers yields 0.3-kb VDJ products only. PCR reactions were also performed with a pair of Cδ primers (primers 5 and 6; Fig. 1 C), to control for differences in transgene copy number and PCR efficiency between samples; the amount of genomic DNA template used in PCR reactions was typically adjusted to obtain similar Cδ amplification signals in each sample. PCR products were detected and quantified by agarose gel electrophoresis followed by blotting and hybridization with appropriate 32P-labeled probes. In agreement with our previous studies (13), quantification of PCR signals revealed amplification to be linear over a broad range of template concentrations (Fig. 2).

Bottom Line: Because mutation of the deltaE3 binding site for the transcription factor c-Myb had previously established a similar role for c-Myb, and because a 60-bp fragment of E(delta) carrying deltaE3 and deltaE4 binding sites for CBF/PEBP2, c-Myb, and GATA-3 displays significant enhancer activity in transient transfection experiments, we tested whether this fragment of E(delta) is sufficient to activate VDJ recombination in vivo.This fragment failed to efficiently activate the enhancer-dependent VD to J step of minilocus rearrangement in all three transgenic lines examined, indicating that the binding of CBF/PEBP2 and c-Myb to their cognate sites within E(delta), although necessary, is not sufficient for the activation of VDJ recombination by E(delta).These results imply that CBF/PEBP2 and c-Myb collaborate with additional factors that bind elsewhere within E(delta) to modulate local accessibility to the VDJ recombinase in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
We have analyzed transgenic mice carrying versions of a human T cell receptor (TCR)-delta gene minilocus to study the developmental control of VDJ (variable/diversity/joining) recombination. Previous data indicated that a 1.4-kb DNA fragment carrying the TCR-delta enhancer (E(delta)) efficiently activates minilocus VDJ recombination in vivo. We tested whether the transcription factor CBF/PEBP2 plays an important role in the ability of E(delta) to activate VDJ recombination by analyzing VDJ recombination in mice carrying a minilocus in which the deltaE3 element of E(delta) includes a mutated CBF/PEBP2 binding site. The enhancer-dependent VD to J step of minilocus rearrangement was dramatically inhibited in three of four transgenic lines, arguing that the binding of CBF/PEBP2 plays a role in modulating local accessibility to the VDJ recombinase in vivo. Because mutation of the deltaE3 binding site for the transcription factor c-Myb had previously established a similar role for c-Myb, and because a 60-bp fragment of E(delta) carrying deltaE3 and deltaE4 binding sites for CBF/PEBP2, c-Myb, and GATA-3 displays significant enhancer activity in transient transfection experiments, we tested whether this fragment of E(delta) is sufficient to activate VDJ recombination in vivo. This fragment failed to efficiently activate the enhancer-dependent VD to J step of minilocus rearrangement in all three transgenic lines examined, indicating that the binding of CBF/PEBP2 and c-Myb to their cognate sites within E(delta), although necessary, is not sufficient for the activation of VDJ recombination by E(delta). These results imply that CBF/PEBP2 and c-Myb collaborate with additional factors that bind elsewhere within E(delta) to modulate local accessibility to the VDJ recombinase in vivo.

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