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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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Human TCR-δ gene minilocus. (A) Diagram of the three  Tα1,2-containing minilocus constructs. Solid boxes, exons, open boxes, protein binding sites. Wild-type and mutant Tα2 sequences are shown. (B)  PCR products generated from Vδ1 rearrangements are depicted along  with the Vδ1 and Jδ1 primers (arrows) used. Similar products are generated  using Vδ2 and Jδ1 primers. Specific PCR products are not generated from  unrearranged templates because of the large distances between primers.  The primers do not amplify products from the endogenous murine TCR-δ  locus.
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Figure 1: Human TCR-δ gene minilocus. (A) Diagram of the three Tα1,2-containing minilocus constructs. Solid boxes, exons, open boxes, protein binding sites. Wild-type and mutant Tα2 sequences are shown. (B) PCR products generated from Vδ1 rearrangements are depicted along with the Vδ1 and Jδ1 primers (arrows) used. Similar products are generated using Vδ2 and Jδ1 primers. Specific PCR products are not generated from unrearranged templates because of the large distances between primers. The primers do not amplify products from the endogenous murine TCR-δ locus.

Mentions: The rearrangement substrate used in the present study has been previously described as a 22.5-kb human TCR-δ gene minilocus consisting of germline Vδ1, Vδ2, Dδ3, Jδ1, Jδ3, and Cδ gene segments (24). Frameshift mutations within the Vδ1 and Vδ2 coding segments prevent the rearranged transgene from encoding a functional TCR polypeptide that could alter normal T cell development in transgenic mice. The initial step of transgene rearrangement, V to D, is enhancer independent (24). The second step of transgene rearrangement, VD to J, is dependent on the presence of a functional enhancer in the Jδ3-Cδ intron. Thus, we infer that the enhancer is required to promote J segment accessibility to the recombinase (24). The 1.4-kb Eα has been shown to efficiently activate VD to J rearrangement in this system (25). To determine whether Tα1,2, the 116-bp core fragment of Eα, was also sufficient to activate minilocus VDJ recombination in vivo, we constructed a new TCR-δ gene minilocus containing this fragment in place of Eα (Fig. 1 A). Four independent transgenic lines denoted T2, T3, T5, and T7 were established and determined by slot blot analysis to carry 28, 1, 3, and 4 transgene copies, respectively.


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)

Human TCR-δ gene minilocus. (A) Diagram of the three  Tα1,2-containing minilocus constructs. Solid boxes, exons, open boxes, protein binding sites. Wild-type and mutant Tα2 sequences are shown. (B)  PCR products generated from Vδ1 rearrangements are depicted along  with the Vδ1 and Jδ1 primers (arrows) used. Similar products are generated  using Vδ2 and Jδ1 primers. Specific PCR products are not generated from  unrearranged templates because of the large distances between primers.  The primers do not amplify products from the endogenous murine TCR-δ  locus.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196107&req=5

Figure 1: Human TCR-δ gene minilocus. (A) Diagram of the three Tα1,2-containing minilocus constructs. Solid boxes, exons, open boxes, protein binding sites. Wild-type and mutant Tα2 sequences are shown. (B) PCR products generated from Vδ1 rearrangements are depicted along with the Vδ1 and Jδ1 primers (arrows) used. Similar products are generated using Vδ2 and Jδ1 primers. Specific PCR products are not generated from unrearranged templates because of the large distances between primers. The primers do not amplify products from the endogenous murine TCR-δ locus.
Mentions: The rearrangement substrate used in the present study has been previously described as a 22.5-kb human TCR-δ gene minilocus consisting of germline Vδ1, Vδ2, Dδ3, Jδ1, Jδ3, and Cδ gene segments (24). Frameshift mutations within the Vδ1 and Vδ2 coding segments prevent the rearranged transgene from encoding a functional TCR polypeptide that could alter normal T cell development in transgenic mice. The initial step of transgene rearrangement, V to D, is enhancer independent (24). The second step of transgene rearrangement, VD to J, is dependent on the presence of a functional enhancer in the Jδ3-Cδ intron. Thus, we infer that the enhancer is required to promote J segment accessibility to the recombinase (24). The 1.4-kb Eα has been shown to efficiently activate VD to J rearrangement in this system (25). To determine whether Tα1,2, the 116-bp core fragment of Eα, was also sufficient to activate minilocus VDJ recombination in vivo, we constructed a new TCR-δ gene minilocus containing this fragment in place of Eα (Fig. 1 A). Four independent transgenic lines denoted T2, T3, T5, and T7 were established and determined by slot blot analysis to carry 28, 1, 3, and 4 transgene copies, respectively.

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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