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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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Analysis of minilocus rearrangement by genomic Southern  blot. PstI plus EcoRI-digested tail and thymus genomic DNA samples  from Eα line L, Tα1,2 line T2, Tα1,2mTCF line JI, and Tα1,2mEts line  JO mice (all 4 wk old) were analyzed by Southern blot using a radiolabeled 1.0-kb Vδ1 genomic PstI fragment. Positions of the expected 1.0-kb  germline, 0.9-kb Vδ1-Dδ3, and 3.2-kb Vδ1-Dδ3-Jδ1 rearranged fragments  are indicated.
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Figure 4: Analysis of minilocus rearrangement by genomic Southern blot. PstI plus EcoRI-digested tail and thymus genomic DNA samples from Eα line L, Tα1,2 line T2, Tα1,2mTCF line JI, and Tα1,2mEts line JO mice (all 4 wk old) were analyzed by Southern blot using a radiolabeled 1.0-kb Vδ1 genomic PstI fragment. Positions of the expected 1.0-kb germline, 0.9-kb Vδ1-Dδ3, and 3.2-kb Vδ1-Dδ3-Jδ1 rearranged fragments are indicated.

Mentions: Tα1,2 and Eα minilocus rearrangement was also analyzed directly by genomic Southern blot (Fig. 4) as an independent means of corroborating results obtained by quantitative PCR. Analysis of Vδ1 rearrangements in PstI plus EcoRI-digested thymus DNA from Eα line L revealed low levels of 1.0-kb germline Vδ1 and 0.9-kb Vδ1-Dδ3 rearranged fragments, and higher levels of a 1.7-kb species resulting from Vδ1-Dδ3-Jδ1 rearrangement (Fig. 4). Similar results were obtained with Eα line J (data not shown). Analysis of similarly digested thymocyte DNA from Tα1,2 line T2 also revealed levels of the fully rearranged Vδ1Dδ3-Jδ1 fragment that were more prevalent than the 0.9-kb partially rearranged (Vδ1-Dδ3) species (Fig. 4). Hence, these results are consistent with those obtained by PCR and provide confirmation that Tα1,2 alone can efficiently activate minilocus VDJ recombination.


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)

Analysis of minilocus rearrangement by genomic Southern  blot. PstI plus EcoRI-digested tail and thymus genomic DNA samples  from Eα line L, Tα1,2 line T2, Tα1,2mTCF line JI, and Tα1,2mEts line  JO mice (all 4 wk old) were analyzed by Southern blot using a radiolabeled 1.0-kb Vδ1 genomic PstI fragment. Positions of the expected 1.0-kb  germline, 0.9-kb Vδ1-Dδ3, and 3.2-kb Vδ1-Dδ3-Jδ1 rearranged fragments  are indicated.
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Related In: Results  -  Collection

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Figure 4: Analysis of minilocus rearrangement by genomic Southern blot. PstI plus EcoRI-digested tail and thymus genomic DNA samples from Eα line L, Tα1,2 line T2, Tα1,2mTCF line JI, and Tα1,2mEts line JO mice (all 4 wk old) were analyzed by Southern blot using a radiolabeled 1.0-kb Vδ1 genomic PstI fragment. Positions of the expected 1.0-kb germline, 0.9-kb Vδ1-Dδ3, and 3.2-kb Vδ1-Dδ3-Jδ1 rearranged fragments are indicated.
Mentions: Tα1,2 and Eα minilocus rearrangement was also analyzed directly by genomic Southern blot (Fig. 4) as an independent means of corroborating results obtained by quantitative PCR. Analysis of Vδ1 rearrangements in PstI plus EcoRI-digested thymus DNA from Eα line L revealed low levels of 1.0-kb germline Vδ1 and 0.9-kb Vδ1-Dδ3 rearranged fragments, and higher levels of a 1.7-kb species resulting from Vδ1-Dδ3-Jδ1 rearrangement (Fig. 4). Similar results were obtained with Eα line J (data not shown). Analysis of similarly digested thymocyte DNA from Tα1,2 line T2 also revealed levels of the fully rearranged Vδ1Dδ3-Jδ1 fragment that were more prevalent than the 0.9-kb partially rearranged (Vδ1-Dδ3) species (Fig. 4). Hence, these results are consistent with those obtained by PCR and provide confirmation that Tα1,2 alone can efficiently activate minilocus VDJ recombination.

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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