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

Abstract

It has been shown that 5-azacytidine (5-Aza-Cyd) can reactivate genes on the inactive human X chromosome. It is assumed that the 5-Aza-Cyd acts by causing demethylation of the DNA at specific sites, but this cannot be demonstrated directly without a cloned probe. Instead, we have utilized the technique of DNA-mediated transformation to show that the 5-Aza-Cyd-induced reactivation occurs at the DNA level. DNAs 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, whereas DNA from the inactive X chromosome-containing cell lines did not. The inactive X chromosomal DNA was able to transform thymidine Idnase-deficient mouse cells, indicating that the DNA solution was normal. These results confirm that inactivation of the X chromosome involves a DNA modification. Furthermore, DNAs from three 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. Dosage compensation for most ofthe X chromosome is achieved in mammalian females early in development when one of the X chromosomes undergoes a condensation and ceases most transcriptional activity (1, 2). In eutherian mammals, either the maternal or paternal X chromosome may be inactivated in somatic cells, but once established, the pattern of inactivation remains the same for each cell and its descendants. It was originally proposed that the entire X chromosome is inactivated, but there is now evidence that the steroid sulfatase (STS; sterolsulfate sulfohydrolase, EC 3.1.6.2) locus and the Xg locus to which it is linked escape inactivation (3-6).Liskay and Evans (7) have shown that DNA from the inactive X chromosome will not function in DNA-mediated transformation of the hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) gene, suggesting that the inactive X chromosome has been modified at the DNA level. One possible DNA modification that has been proposed as a mechanism for X chromosome inactivation is methylation ofcytosine residues (8-10). Recently, a number of reports have appeared correlating hypomethylation ofspecific sites with gene expression (for reviews see refs. 11 and 12). 5-Azacytidine (5-Aza-Cyd) is an analog of cytidine known to inhibit methylation of DNA (13). It has been used in a number of studies designed to investigate the relationship between DNA methylation and gene activity. Mohandas et al. (14) treated mouse-human hybrid cells, retaining an inactive human X chromosome, with 5-Aza-Cyd and were able to demonstrate reactivation of the human HPRT locus. Some ofthe HPRT' clones obtained also showed reactivation ofother X chromosome-linked enzymes, glucose-6-phosp...

Cell cycle-specific reactivation of an inactive X-chromosome locus by 5-azadeoxycytidine.

“…(24). In addition, it has been shown that DNA from 37-26R-D cells will not mediate transformation ofHPRT-recipient mouse cells, but DNA from clones with reactivated HPRT genes after 5-Aza-Cyd treatment are competent in the transformation assay (25).…”

DNA methylation and chromatin structure

“…For example, DNA from the Xi is not able to transform HPRT -cells to HPRT' [17]. However, DNA from the Xa, or from an Xi chromosome which had previously been treated with S-azacytidine can transform the recipient cells to HPRT + .…”

5-Azadeoxycytidine-induced Chromatin Remodeling of the Inactive X-linked HPRT Gene Promoter Occurs prior to Transcription Factor Binding and Gene Reactivation

“…A variety of molecular mechanisms have been implicated in regulating the initiation, spreading, and maintenance of X inactivation (1-7). The involvement of DNA methylation in this process has been established by studies using methyl-sensitive restriction enzymes (8 -10), DNA-mediated transformation (11)(12)(13), genomic sequencing (14 -16), and the DNA demethylating agent 5-azacytidine (6,13,(17)(18)(19). All of these studies support the notion that hypermethylation of the 5Ј CpG island associated with many X-linked housekeeping genes is involved in the transcriptional silencing of these genes on the inactive X chromosome.…”