Limits...
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.

Show MeSH
Time course of Tα1,2 minilocus rearrangement during fetal  ontogeny. Genomic DNA samples from Tα1,2 line T2 thymi isolated on  days 14.5–17.5 of gestation, as well as from a postnatal (PN) T2 mouse  (4 wk old), were amplified by PCR and probed as in Fig. 2.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196107&req=5

Figure 5: Time course of Tα1,2 minilocus rearrangement during fetal ontogeny. Genomic DNA samples from Tα1,2 line T2 thymi isolated on days 14.5–17.5 of gestation, as well as from a postnatal (PN) T2 mouse (4 wk old), were amplified by PCR and probed as in Fig. 2.

Mentions: The 1.4-kb Eα has been previously shown to confer physiologically appropriate developmental control to the enhancer-dependent VD to J step of minilocus rearrangement. Because the 116-bp Tα1,2 core enhancer fragment proved sufficient to activate minilocus rearrangement, we asked whether the core enhancer fragment is sufficient to impart precise developmental control as well. Accordingly, we examined the timing of minilocus VDJ rearrangement during ontogeny in Tα1,2 transgenic animals. PCR analysis of fetal thymus genomic DNA templates from line T2 timed pregnancies revealed that the VD to J step of minilocus rearrangement began on fetal day 15.5 (Fig. 5), one day earlier than that previously noted in Eα line J (25). Identical results were obtained from analysis of Tα1,2 line T5 (data not shown). Thus, Tα1,2 appeared to be activated slightly earlier during fetal thymic ontogeny than the intact Eα.


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)

Time course of Tα1,2 minilocus rearrangement during fetal  ontogeny. Genomic DNA samples from Tα1,2 line T2 thymi isolated on  days 14.5–17.5 of gestation, as well as from a postnatal (PN) T2 mouse  (4 wk old), were amplified by PCR and probed as in Fig. 2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196107&req=5

Figure 5: Time course of Tα1,2 minilocus rearrangement during fetal ontogeny. Genomic DNA samples from Tα1,2 line T2 thymi isolated on days 14.5–17.5 of gestation, as well as from a postnatal (PN) T2 mouse (4 wk old), were amplified by PCR and probed as in Fig. 2.
Mentions: The 1.4-kb Eα has been previously shown to confer physiologically appropriate developmental control to the enhancer-dependent VD to J step of minilocus rearrangement. Because the 116-bp Tα1,2 core enhancer fragment proved sufficient to activate minilocus rearrangement, we asked whether the core enhancer fragment is sufficient to impart precise developmental control as well. Accordingly, we examined the timing of minilocus VDJ rearrangement during ontogeny in Tα1,2 transgenic animals. PCR analysis of fetal thymus genomic DNA templates from line T2 timed pregnancies revealed that the VD to J step of minilocus rearrangement began on fetal day 15.5 (Fig. 5), one day earlier than that previously noted in Eα line J (25). Identical results were obtained from analysis of Tα1,2 line T5 (data not shown). Thus, Tα1,2 appeared to be activated slightly earlier during fetal thymic ontogeny than the intact Eα.

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