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

Abstract

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. INTRODUCTIONThe differentiation and lineage commitment of stem cells are accompanied and determined by chromatin remodeling and establishment of epigenetic marks (Szutorisz and Dillon, 2005). The process of differentiation involves switching on or off genes that are expressed in specific types of cells and at specific times. This programming of gene transcription occurs in the context of their native chromatin environment and is often stably maintained throughout the life span of an organism. Many genes essential for proper development of the embryo have been discovered and most of them encode proteins that regulate transcription (Arney and Fisher, 2004;Chambers and Smith, 2004). However, the dynamic interactions between these proteins and chromatin are still areas of intensive investigation. Detailed studies of gene transcription regulatory mechanisms during cell differentiation are essential not only for understanding the fundamental mechanisms of development and differentiation, but also for harnessing the differentiation and proliferation capacity that make stem cells invaluable for regenerative medicine.Telomerase, an enzyme synthesizing TTAGGG telomere repeats, is pertinent to self-renewal potential of stem cells and is required for long-term cellular proliferation and survival (Morrison et al., 1996;Lee et al., 1998). Telomerase is tightly regulated throughout development (Kim et al., 1994;Prowse and Greider, 1995). In early embryonic tissues and stem cells, telomerase is highly expressed and telomeres are stably maintained (Wright et al., 1996;Sharpless and DePinho, 2004). Although telomerase is detec...

TERT-CLPTM1L Polymorphism rs401681 Contributes to Cancers Risk: Evidence from a Meta-Analysis Based on 29 Publications

“…For example, strikingly increased risk was found in smoking related cancers, such as lung and bladder cancers. It may be explained that CLPTM1L protein may be involved in the apoptosis response of genotoxic stress [9], [10], [11], [12]. Further more, Nan and collaborators observed a suggestive positive relationship between rs401681 C allele and shorter relative telomere length [50].…”

“…Mutations in the coding regions of TERT can affect telomerase activity and telomere length, and generate severe clinical phenotypes, including bone marrow failure syndromes and a substantive increase in cancer frequency [8]. Although the function of the CLPTM1L is largely unknown, studies have demonstrated that it may be involved in the apoptotic response to genotoxic stress induced by cisplatin, as it encodes a transcript whose over-expression has been shown to induce apoptosis in cisplatin-resistant-sensitive cells [9], [10], [11], [12]. Moreover, the CLPTM1L variants are hypothesized to enhance the metabolic activation of the reactive metabolites and/or formation and persistence of DNA adducts [13].…”

p16INK4a‐mediated suppression of telomerase in normal and malignant human breast cells

“…2006; Lee et al . 2006), as well as reduced hTERT expression, at least partly via epigenetic mechanisms (Wang et al . 2007).…”

A New Positive/Negative Selection Scheme for Precise BAC Recombineering

“…To generate reporters for studying transcriptional regulation of the TERT (telomerase reverse transcriptase) genes (16), we inserted luciferase expression cassettes into the initiation codons of the TERT genes in BAC constructs, using the two-step procedure described as follows. To make use of the recombineering method reported by Lee at al.…”