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Mol Cell Biol. 1993 February; 13(2): 891-901

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.

Hubert H. Humphrey Center for Experimental Medicine and Cancer Research, Hadassah Medical School, Hebrew University, Jerusalem, Israel.

ABSTRACT

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.

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