Transformation with DNA from 5-azacytidine-reactivated X chromosomes.

Abstract

5-Azacytidine (5-Aza-Cyd) can reactivate genes on the inactive human X chromosome. The 5-Aza-Cyd probably acts by causing demethylation of the DNA at specific sites, but this cannot be demonstrated directly without a cloned probe. The technique of DNA-mediated transformation was used to show that the 5-Aza-Cyd-induced reactivation occurs at the DNA level. DNA from various mouse-human or hamster-human hybrid cell lines, deficient for mouse or hamster hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8) and varying in whether they contained either an active or inactive human X chromosome, were used in transformation of HPRT- cells. DNA from the active human X chromosome-containing cell lines yielded HPRT+ transformants; DNA from the inactive X chromosome-containing cell lines did not. The inactive X chromosomal DNA was able to transform thymidine kinase-deficient mouse cells, indicating that the DNA solution was normal. Inactivation of the X chromosome involves a DNA modification. DNA from 3 cell lines with a 5-Aza-Cyd-reactivated X chromosome also transform HPRT- cells, demonstrating that the 5-Aza-Cyd has altered the DNA structure and supporting the idea that methylation plays a role in X chromosome inactivation.