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.

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.

Stem cell pluripotency and transcription factor Oct4

“…Expression of Oct4 is regulated at the transcription level by cis-acting elements located upstream of the Oct4 gene and methylation of chromatin structure (Fig 2B) [24]. By analyzing the expression of the LacZ reporter gene under the control of a 18Kb fragment from Oct4 genomic locus, Yeom et al identified two elements, which they named proximal enhancer (PE) and distal enhancer (DE) that may regulate the cell -type-specific expression of Oct4 ( Fig 2B) [25].…”

“…One hypothesis is that the steady state level of Oct4 in totipotent cells may be a consequence of the establishment of active chromatin rather than the function of transcription activators. Ben-Shushan et al reported that the extinction of Oct4 activity in stem cells-fibroblast hybrid cells was accompanied by rapid methylation of regulatory sequences such as PE and DE in the Oct4 promoter/enhancer region [24]. Jaenisch suggested that there must be a wave of de novo methylation occurring in the somatic cells of the embryo [29].…”

A Unique Developmental Pattern of Oct-3/4DNA Methylation Is Controlled by a cis-demodification Element

“…Oct-3/4 Methylation and Transcription-The Oct-3/4 gene undergoes de novo methylation both during embryogenesis (described above) and also in EC cells as a consequence of RA treatment (22). To find out if methylation plays a significant role in the regulation of Oct-3/4 gene expression, two kinds of experiments were performed.…”

“…We have shown previously (22) that treatment of EC cells with RA reduces Oct-3/4 expression that is associated with increased methylation and changes in chromatin structure in the immediate upstream regulatory region that includes the promoter (P) and proximal enhancer (PE) elements. In this study we show that in vivo the Oct-3/4 gene is unmethylated from the blastula stage and starts to undergo de novo methylation at 6.5 dpc.…”

Rex-1, a Gene Encoding a Transcription Factor Expressed in the Early Embryo, Is Regulated via Oct-3/4 and Oct-6 Binding to an Octamer Site and a Novel Protein, Rox-1, Binding to an Adjacent Site

“…Therefore, it is very likely that expression of Oct-3/4 plays an important role in determining early steps in embryogenesis and differentiation. Support for this notion was gained by showing that in EC ϫ fibroblast somatic cell hybrids, Oct-3/4 expression is suppressed, and reexpression of Oct-3/4 in these cells correlated with differentiation potential (2,61).…”