Reactivation of an Inactive Human X Chromosome: Evidence for X Inactivation by DNA Methylation

TLDR

A mouse‑human somatic cell hybrid clone lacking HPRT and carrying a structurally normal inactive human X chromosome was isolated. The study aims to determine whether DNA methylation mediates human X chromosome inactivation by treating the hybrid cells with 5‑azacytidine. The authors treated the hybrid cells with 5‑azacytidine, isolated 14 HPRT‑positive clones, and examined expression of human X‑linked markers to assess reactivation. 5‑Azacytidine increased HPRT‑positive clones by 1000‑fold, and isoelectric focusing confirmed human HPRT expression, with some clones also expressing human G6PD and PGK.

Abstract

A mouse-human somatic cell hybrid clone, deficient in hypoxanthine-guanine phosphoribosyltransferase (HPRT) and containing a structurally normal inactive human X chromosome, was isolated. The hybrid cells were treated with 5-azacytidine and tested for the reactivation and expression of human X-linked genes. The frequency of HPRT-positive clones after 5-azacytidine treatment was 1000-fold greater than that observed in untreated hybrid cells. Fourteen independent HPRT-positive clones were isolated and analyzed for the expression of human X markers. Isoelectric focusing showed that the HPRT expressed in these clones is human. One of the 14 clones expressed human glucose-6-phosphate dehydrogenase and another expressed human phosphoglycerate kinase. Since 5-azacytidine treatment results in hypomethylation of DNA, DNA methylation may be a mechanism of human X chromosome inactivation.