Reactivation of an inactive human X chromosome introduced into mouse embryonal carcinoma cells by microcell fusion with persistent expression of XIST.
An inactive human X chromosome was introduced by microcell fusion into two mouse embryonal carcinoma cell lines, PSA1-TG8 and OTF9-63, each of which has a single X chromosome. The donor cell line was a mouse-human somatic cell hybrid, CF150, retaining one or more inactive human X chromosome(s) per cell as its only human element. Twenty hybrid clones isolated retained EC morphology and contained the intact human X chromosome(s) or its truncated derivative(s). Replication banding analysis showed that the introduced human X chromosome(s) or its derivative(s) replicated synchronously with other mouse chromosomes, suggesting reactivation of the human X chromosomal elements after transfer. Reversal of inactivation was further confirmed by the expression of five human X-linked genes repressed in CF150, although the XIST (X inactive specific transcript) gene continued to be active. The level of XIST expression in our hybrid cells was almost identical to that of parental CF150 cells. Methylation status of 5' end of the active XIST gene varied considerably from almost full methylation to unmethylation in these hybrids. Thus, mouse EC cells used in this study were capable of altering methylation status of the human XIST gene in a manner lacking consistency and unable to repress its transcription. Furthermore, we failed to obtain any positive evidence for the occurrence of X chromosome inactivation in differentiating monochromosome EC hybrids. Taken together, these findings suggest that the human X chromosome inactivation center including the XIST gene is unable to function effectively in mouse cells.
Yoshida
I
I
Research Center for Molecular Genetics, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan. ikuya@noah.eesbio.hokudai.ac.jp
Nishita
Y
Y
Mohandas
T K
TK
Takagi
N
N
D000818:Animals; D002459:Cell Fusion; D002460:Cell Line; D004303:Dosage Compensation, Genetic; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D006822:Hybrid Cells; D008745:Methylation; D051379:Mice; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D014157:Transcription Factors / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014960:X Chromosome
D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D014157:Transcription Factors; C077885:XIST non-coding RNA