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The human telomerase reverse transcriptase hTERT is highly expressed in undifferentiated embryonic cells and silenced in the majority of somatic cells. To investigate the mechanisms of hTERT silencing, we have developed a novel reporter using a bacterial artificial chromosome (BAC) that contained the entire hTERT gene and its neighboring loci, hCRR9 and hXtrp2. Firefly and Renilla luciferases were used to monitor transcription from the hTERT and hCRR9 promoters, respectively. In mouse embryonic stem cells stably integrated with the BAC reporter, both hTERT and hCRR9 promoters were highly expressed. Upon differentiation into embryoid bodies and further into mineral-producing osteogenic cells, the hTERT promoter activity decreased progressively, whereas the hCRR9 promoter remained highly active, both resembling their endogenous counterparts. In fully differentiated cells, the hTERT promoter was completely silenced and adopted a chromatin structure that was similar to its native counterpart in human cells. Inhibition of histone deacetylases led to the opening of the hTERT promoter and partially relieved repression, suggesting that histone deacetylation was necessary but not sufficient for hTERT silencing. Thus, our result demonstrated that developmental silencing of the human TERT locus could be recapitulated in a chromosomal position-independent manner during the differentiation of mouse embryonic stem cells.

Transcriptional Silencing of a Novel hTERT Reporter Locus during In Vitro Differentiation of Mouse Embryonic Stem Cells