HYPOMETHYLATION OF THE T-CELL RECEPTOR β CHAIN GENE PRECEDES REARRANGEMENT

Abstract

In order to define a functional role for methylation and hypomethylation during T-cell receptor Beta chain (Tcrβ) rearrangement, the status of the TcR gene was examined during different stages of rearrangement using Southern analysis. T-cells rearranged at the TcRβ genetic locus (such as most thymocytes) are over 95% hypomethylated in the J-cluster. Most T-cell precursors (triple negative (TN) cells) that have rearranged TcR loci are completely methylated in CCGG sequences. In contrast, other hematopoietic cells such as a mastocytoma cell line or mature B-cell lines, have over 90% of the CCGG sequences methylated. Hematopoietic precursor cells (lineage negative (Lin-) cells) along with αβ transgenic thymocytes are also highly methylated in the Tcr0 locus. Embryonal tissue was analyzed in order to capture precursor T-cells at the transition from methylation to hypomethylation. T-cells rearrange VD-J sequences and become hypomethylated at the J cluster region between day 14 and 16. Although 99% of day 14 thymocytes are not VD-J rearranged, 50% of the DNA shows hypomethylation in the TcrOβ gene. Hypomethylation was also observed in vitro as T-cell DNA rearranged. Moreover, treatment of day 14 fetal thymus organ cultures with the hypomethylating drug 5-deoxy-azacytidine and the cytokine interleukin 7 (IL 7), strongly enhanced Tcrg rearrangement.