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Developmental regulation of VDJ recombination by the core fragment of the T cell receptor alpha enhancer.

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

Bottom Line: We demonstrate that the 116-bp T alpha 1,2 core enhancer fragment of the 1.4-kb E alpha is sufficient to activate the enhancer-dependent step of minilocus rearrangement, and that within T alpha 1,2, intact binding sites for TCF/LEF and Ets family transcription factors are essential.We conclude that the core fragment of E alpha can establish accessibility to the recombinase in developing thymocytes in vivo in a fashion that is dependent on the binding of TCF/LEF and Ets family transcription factors, but that these and other factors that bind to the E alpha core cannot account for the precise developmental onset of accessibility that is provided by the intact E alpha.Rather, our data suggests a critical role for factors that bind E alpha outside of the core T alpha 1,2 region in establishing the precise developmental onset of TCR alpha rearrangement in vivo.

View Article: PubMed Central - PubMed

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

ABSTRACT
The role of T cell receptor alpha enhancer (E alpha) cis-acting elements in the developmental regulation of VDJ recombination at the TCR alpha/delta locus was examined in transgenic mice containing variants of a minilocus VDJ recombination substrate. We demonstrate that the 116-bp T alpha 1,2 core enhancer fragment of the 1.4-kb E alpha is sufficient to activate the enhancer-dependent step of minilocus rearrangement, and that within T alpha 1,2, intact binding sites for TCF/LEF and Ets family transcription factors are essential. Although minilocus rearrangement under the control of the 1.4-kb E alpha initiates at fetal day 16.5 and is strictly limited to alpha beta T cells, we find that rearrangement under the control of T alpha 1,2 initiates slightly earlier during ontogeny and occurs in both gamma delta and alpha beta T cells. We conclude that the core fragment of E alpha can establish accessibility to the recombinase in developing thymocytes in vivo in a fashion that is dependent on the binding of TCF/LEF and Ets family transcription factors, but that these and other factors that bind to the E alpha core cannot account for the precise developmental onset of accessibility that is provided by the intact E alpha. Rather, our data suggests a critical role for factors that bind E alpha outside of the core T alpha 1,2 region in establishing the precise developmental onset of TCR alpha rearrangement in vivo.

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PCR analysis of Eα, Tα1,2, and Tα1,2mEts minilocus rearrangement. Genomic DNA templates from unfractionated thymocytes of  an Eα mouse from line J, of Tα1,2 mice from lines T2 and T5, and of  Tα1,2mEts mice from lines JN, JR, and JO (all 4 wk old) were amplified  by PCR and probed as in Fig. 2.
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Figure 3: PCR analysis of Eα, Tα1,2, and Tα1,2mEts minilocus rearrangement. Genomic DNA templates from unfractionated thymocytes of an Eα mouse from line J, of Tα1,2 mice from lines T2 and T5, and of Tα1,2mEts mice from lines JN, JR, and JO (all 4 wk old) were amplified by PCR and probed as in Fig. 2.

Mentions: VDJ recombination in the four Tα1,2 transgenic lines was assessed by quantitative PCR of thymic genomic DNA templates that were amplified using primers specific for minilocus Vδ1, Vδ2, and Jδ1 gene segments (24). PCR using primer combinations Vδ1-Jδ1 or Vδ2-Jδ1 yields a 0.3-kb product resulting from transgene VDJ rearrangement and a 1.2-kb fragment resulting from VD rearrangement (Fig. 1 B), both of which can be detected on Southern blots probed with radiolabeled Vδ1- or Vδ2-specific DNA fragments. Amplification of a 0.3-kb rearrangement-independent product with a pair of Cδ primers serves as an internal control for PCR efficiency and allows quantitative comparison of rearrangement patterns between different templates. As seen in Fig. 2 and Table 1, low levels of Vδ1-Dδ3 and high levels of Vδ1-Dδ3-Jδ1 rearrangement were observed in three of four Tα1,2 lines (T2, T5, and T7). Levels of Vδ1Dδ3-Jδ1 rearrangement in thymocytes from lines T2 and T5 were 60 and 38%, respectively, of that found in thymocytes from Eα line J, which includes the intact 1.4-kb Eα (Fig. 3, Table 2). However, VD and VDJ rearranged products were barely detectable in Tα1,2 line T3 (Fig. 2, Table 1). Similar variability in rearrangement phenotype among different lines of animals bearing an identical construct has been noted in our previous studies (24, 39) and is likely due to inherent differences in transgene integration sites. We suggest that the minilocus is integrated into a relatively inactive region of chromatin in line T3; in support of this notion, preliminary in vivo footprinting studies have demonstrated markedly diminished protection of protein binding sites within Tα1,2 in thymus DNA of T3 mice as compared with thymus DNA of T2, T5, or T7 mice (HernandezMunain, C., personal communication). Taken as a whole, our results demonstrate that the 116-bp Tα1,2 fragment of Eα is, in most contexts, sufficient to mediate activation of the enhancer-dependent VD to J step of minilocus rearrangement. The apparently less efficient conversion of Vδ2Dδ3 to Vδ2-Dδ3-Jδ1 in these lines is probably related to our previous observation that Vδ2 rearrangement is only ∼10% as efficient as Vδ1 rearrangement in this system (24).


Developmental regulation of VDJ recombination by the core fragment of the T cell receptor alpha enhancer.

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

PCR analysis of Eα, Tα1,2, and Tα1,2mEts minilocus rearrangement. Genomic DNA templates from unfractionated thymocytes of  an Eα mouse from line J, of Tα1,2 mice from lines T2 and T5, and of  Tα1,2mEts mice from lines JN, JR, and JO (all 4 wk old) were amplified  by PCR and probed as in Fig. 2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196107&req=5

Figure 3: PCR analysis of Eα, Tα1,2, and Tα1,2mEts minilocus rearrangement. Genomic DNA templates from unfractionated thymocytes of an Eα mouse from line J, of Tα1,2 mice from lines T2 and T5, and of Tα1,2mEts mice from lines JN, JR, and JO (all 4 wk old) were amplified by PCR and probed as in Fig. 2.
Mentions: VDJ recombination in the four Tα1,2 transgenic lines was assessed by quantitative PCR of thymic genomic DNA templates that were amplified using primers specific for minilocus Vδ1, Vδ2, and Jδ1 gene segments (24). PCR using primer combinations Vδ1-Jδ1 or Vδ2-Jδ1 yields a 0.3-kb product resulting from transgene VDJ rearrangement and a 1.2-kb fragment resulting from VD rearrangement (Fig. 1 B), both of which can be detected on Southern blots probed with radiolabeled Vδ1- or Vδ2-specific DNA fragments. Amplification of a 0.3-kb rearrangement-independent product with a pair of Cδ primers serves as an internal control for PCR efficiency and allows quantitative comparison of rearrangement patterns between different templates. As seen in Fig. 2 and Table 1, low levels of Vδ1-Dδ3 and high levels of Vδ1-Dδ3-Jδ1 rearrangement were observed in three of four Tα1,2 lines (T2, T5, and T7). Levels of Vδ1Dδ3-Jδ1 rearrangement in thymocytes from lines T2 and T5 were 60 and 38%, respectively, of that found in thymocytes from Eα line J, which includes the intact 1.4-kb Eα (Fig. 3, Table 2). However, VD and VDJ rearranged products were barely detectable in Tα1,2 line T3 (Fig. 2, Table 1). Similar variability in rearrangement phenotype among different lines of animals bearing an identical construct has been noted in our previous studies (24, 39) and is likely due to inherent differences in transgene integration sites. We suggest that the minilocus is integrated into a relatively inactive region of chromatin in line T3; in support of this notion, preliminary in vivo footprinting studies have demonstrated markedly diminished protection of protein binding sites within Tα1,2 in thymus DNA of T3 mice as compared with thymus DNA of T2, T5, or T7 mice (HernandezMunain, C., personal communication). Taken as a whole, our results demonstrate that the 116-bp Tα1,2 fragment of Eα is, in most contexts, sufficient to mediate activation of the enhancer-dependent VD to J step of minilocus rearrangement. The apparently less efficient conversion of Vδ2Dδ3 to Vδ2-Dδ3-Jδ1 in these lines is probably related to our previous observation that Vδ2 rearrangement is only ∼10% as efficient as Vδ1 rearrangement in this system (24).

Bottom Line: We demonstrate that the 116-bp T alpha 1,2 core enhancer fragment of the 1.4-kb E alpha is sufficient to activate the enhancer-dependent step of minilocus rearrangement, and that within T alpha 1,2, intact binding sites for TCF/LEF and Ets family transcription factors are essential.We conclude that the core fragment of E alpha can establish accessibility to the recombinase in developing thymocytes in vivo in a fashion that is dependent on the binding of TCF/LEF and Ets family transcription factors, but that these and other factors that bind to the E alpha core cannot account for the precise developmental onset of accessibility that is provided by the intact E alpha.Rather, our data suggests a critical role for factors that bind E alpha outside of the core T alpha 1,2 region in establishing the precise developmental onset of TCR alpha rearrangement in vivo.

View Article: PubMed Central - PubMed

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

ABSTRACT
The role of T cell receptor alpha enhancer (E alpha) cis-acting elements in the developmental regulation of VDJ recombination at the TCR alpha/delta locus was examined in transgenic mice containing variants of a minilocus VDJ recombination substrate. We demonstrate that the 116-bp T alpha 1,2 core enhancer fragment of the 1.4-kb E alpha is sufficient to activate the enhancer-dependent step of minilocus rearrangement, and that within T alpha 1,2, intact binding sites for TCF/LEF and Ets family transcription factors are essential. Although minilocus rearrangement under the control of the 1.4-kb E alpha initiates at fetal day 16.5 and is strictly limited to alpha beta T cells, we find that rearrangement under the control of T alpha 1,2 initiates slightly earlier during ontogeny and occurs in both gamma delta and alpha beta T cells. We conclude that the core fragment of E alpha can establish accessibility to the recombinase in developing thymocytes in vivo in a fashion that is dependent on the binding of TCF/LEF and Ets family transcription factors, but that these and other factors that bind to the E alpha core cannot account for the precise developmental onset of accessibility that is provided by the intact E alpha. Rather, our data suggests a critical role for factors that bind E alpha outside of the core T alpha 1,2 region in establishing the precise developmental onset of TCR alpha rearrangement in vivo.

Show MeSH