Figure 2. Effect of Runx site mutations on DJ recombination and germline transcription.

(A) Histograms showing the expression of intracellular TCRβ (i.c.TCRβ) in a CD25⁺ CD44⁻ DN3 subset from mice with the indicated genotype. (B) Semi-quantitative DNA-PCR analyses for analyzing Dβ to Jβ rearrangement in CD25⁺ CD44⁻ DN3 subsets from Eβ^+/Δ(lane 1), Eβ^Δ/Δ (lane 2), Eβ^M1/Δ (lane 3), Eβ^M2/Δ (lane 4) and Eβ^M3/Δ (lane 5) mice. The bar indicates the position corresponding to the germline (GL) configuration. (C) The germline transcript of the Dβ1 region in CD4⁻CD8⁻ DN cells isolated from Rag2^−/−, Eβ^M1/Δ, Eβ^M3/M3 and Eβ^Δ/Δ mice are shown. The germline transcript of Cδ region was used as control. RNA without reverse-transcriptase reaction is shown in lanes indicated as (−). (D) Chromatin immunoprecipitation assay (ChIP) to detect Runx1 binding to Eβ and Cd4 silencer (S4). CD4⁻CD8⁻ DN cells isolated from wild-type and Eβ^M3/M3 mice were used to prepare chromatin DNA. Chromatin DNA was immunoprecipitated with the IgG control or anti-Runx1 antibody and was used as the template for qPCR amplification. The Cd4 silencer region as well as peripheral CD4⁺ and CD8⁺ T cells (WT) were used as controls for Runx1 binding. Combined data from three independent ChIP experiments is shown.