Regulation of human and mouse telomerase genes by genomic contexts and transcription factors during embryonic stem cell differentiation

De Cheng,Jiuhong Kang,Fan Zhang,Wenwen Jia,Yuanjun Zhao,Jiyue Zhu,Shuwen Wang

doi:10.1038/s41598-017-16764-w

Abstract

Differential regulation of telomerase reverse transcriptase (TERT) genes contribute to distinct aging and tumorigenic processes in humans and mice. To study TERT regulation, we generated mouse embryonic stem cell (ESC) lines containing single-copy bacterial artificial chromosome (BAC) reporters, covering hTERT and mTERT genes and their neighboring loci, via recombinase-mediated BAC targeting. ESC lines with chimeric BACs, in which two TERT promoters were swapped, were also generated. Using these chromatinized BACs, we showed that hTERT silencing during differentiation to embryoid bodies (EBs) and to fibroblast-like cells was driven by the human-specific genomic context and accompanied by increases of repressive epigenetic marks, H3K9me3 and H3K27me3, near its promoter. Conversely, the mouse genomic context did not repress TERT transcription until late during differentiation. The hTERT promoter was more active than its mouse counterpart when compared in the same genomic contexts. Mutations of E-box and E2F consensus sites at the promoter had little effect on hTERT transcription in ESCs. However, the mutant promoters were rapidly silenced upon EB differentiation, indicating that transcription factors (TFs) bound to these sites were critical in maintaining hTERT transcription during differentiation. Together, our study revealed a dynamic hTERT regulation by chromatin environment and promoter-bound TFs during ESC differentiation.

Highlights

Telomeres are protective caps of chromosomal ends that are critical for genomic stability[1]
We report the development of recombinase-mediated BAC targeting (RMBT) technique in embryonic stem cell (ESC) to investigate of hTERT and mTERT gene regulation in ESCs and during differentiation
The de novo chromatin around bacterial artificial chromosome (BAC) reporters was assembled from transfected naked BAC DNAs without going through the same stepwise epigenetic modifications as those of endogenous loci[19,27]

Summary

Introduction

Telomeres are protective caps of chromosomal ends that are critical for genomic stability[1]. We showed that the hTERT promoter in randomly integrated H(wt) was highly active in mouse embryonic stem cells, and was efficiently repressed upon in vitro differentiation[13,19] This was in contrast with the endogenous mTERT mRNA, which was only moderately down regulated during the same differentiation process and widely expressed in mouse tissues, indicating the significant differences in the regulation of TERT genes in human and mice[16]. To study the species-specific TERT regulation during differentiation, we developed a new recombinase-mediated BAC targeting (RMBT) protocol to integrate H(wt) and M(wt), a BAC reporter containing a mouse genomic region equivalent to that in H(wt), into a chromosomal acceptor site in mouse ESCs. We showed that the hTERT promoter in H(wt), but not the mTERT promoter in M(wt), was progressively down-regulated during EB differentiation and further silenced upon differentiation into osteogenic and fibroblast-like cells. We demonstrated that the RMBT system in ESCs should facilitate the study of species-specific developmental regulation of human and mouse TERT genes

Methods

Results

Conclusion