Extinction of Oct-3/4 gene expression in embryonal carcinoma x fibroblast somatic cell hybrids is accompanied by changes in the methylation status, chromatin structure, and transcriptional activity of the Oct-3/4 upstream region.
In this study we evaluate, for the first time, the molecular mechanism that underlies the extinction of a tissue-specific transcription factor, Oct-3/4, in somatic cell hybrids and compared it with its down-regulation in retinoic acid (RA)-treated embryonal carcinoma (EC) cells. The Oct-3/4 gene, which belongs to the POU family of transcription factors and is abundantly expressed in EC (OTF9-63) cells, provides an excellent model system with which to study the extinction phenomenon. Unlike other genes whose expression has been repressed in hybrid cells but not during in vivo differentiation, Oct-3/4 expression is dramatically repressed in OTF9-63 x fibroblast hybrids and also during embryogenesis. The ectopic expression of Oct-3/4 in hybrid cells under a constitutive promoter is sufficient for transcriptional activation of an octamer-dependent promoter. These results argue against the possibility that fibroblasts contain a direct repressor which binds directly to the octamer sequence and prevents Oct-3/4 protein from binding. The extinction of Oct-3/4 binding activity in the hybrid cells occurs at the level of mRNA transcription, similarly to the repression of Oct-3/4 transcription during in vivo differentiation. This shutdown of Oct-3/4 transcription in hybrid cells and in RA-treated EC cells is accompanied by de novo methylation of its 1.3-kb upstream region. In contrast to EC cells, in which this region is sensitive to MspI digestion, in hybrid cells and in RA-treated EC cells, the Oct-3/4 upstream region is resistant to MspI digestion, which suggests a change in its chromatin structure. Furthermore, extinction is not restricted to the endogenous Oct-3/4 gene but is also exerted upon a transiently transfected reporter gene driven by the Oct-3/4 upstream region. Thus, changes in the cellular activity of trans-acting factors acting on the upstream region also contribute to the inability of the hybrid and RA-treated EC cells to generate Oct-3/4 transcripts. In conclusion, this study draws a connection between the shutdown of Oct-3/4 expression in RA-differentiated EC cells and its extinction in hybrid cells. In both systems, repression of Oct-3/4 expression is achieved through changes in the methylation status, chromatin structure, and transcriptional activity of the Oct-3/4 upstream regulatory region.
Ben-Shushan
E
E
Hubert H. Humphrey Center for Experimental Medicine and Cancer Research, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
Pikarsky
E
E
Klar
A
A
Bergman
Y
Y
D000818:Animals; D015139:Blotting, Southern; D002843:Chromatin / Q000378:metabolism / Q000648:ultrastructure; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D054278:Embryonal Carcinoma Stem Cells; D005347:Fibroblasts / Q000166:cytology; D005786:Gene Expression Regulation / Q000187:drug effects; D006822:Hybrid Cells; D007739:L Cells; D008745:Methylation; D051379:Mice; D014411:Neoplastic Stem Cells; D050814:Octamer Transcription Factor-3; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology
D002843:Chromatin; D004268:DNA-Binding Proteins; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D014157:Transcription Factors; D014212:Tretinoin; D004247:DNA; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific