Abstract
Human telomerase gene hTERT is important for cancer and aging. hTERT promoter is regulated by multiple transcription factors (TFs) and its activity is dependent on the chromatin environment. However, it remains unsolved how the interplay between TFs and chromatin environment controls hTERT transcription. In this study, we employed the recombinase-mediated BAC targeting and BAC recombineering techniques to dissect the functions of two proximal E-box sites at −165 and +44 nt in regulating the hTERT promoter in the native genomic contexts. Our data showed that mutations of these sites abolished promoter binding by c-Myc/Max, USF1 and USF2, decreased hTERT promoter activity, and prevented its activation by overexpressed c-Myc. Upon inhibition of histone deacetylases, mutant and wildtype promoters were induced to the same level, indicating that the E-boxes functioned to de-repress the hTERT promoter and allowed its transcription in a repressive chromatin environment. Unexpectedly, knockdown of endogenous c-Myc/Max proteins activated hTERT promoter. This activation did not require the proximal E-boxes but was accompanied by increased promoter accessibility, as indicated by augmented active histone marks and binding of multiple TFs at the promoter. Our studies demonstrated that c-Myc/Max functioned in maintaining chromatin-dependent repression of the hTERT gene in addition to activating its promoter.
Highlights
The proliferative lifespan of most human somatic cells is restricted by telomeres, which serve as protective caps of chromosomal ends
As we reported previously [29], when a single-copy H(wt) was integrated into an acceptor site, the chromatinized hTERT promoter was 50–100-folds more active in Tel+ cells than in Tel− cells, as measured by Renilla luciferase (Rluc)/Firefly luciferase (Fluc) (Figure 1B) or by Rluc activities normalized to cell numbers determined by MTT assays
The difference paralleled endogenous hTERT expression in these two cell lines (Figure 3A), but was in sharp contrast to the transiently transfected hTERT reporter plasmid, pTERTLuc800WT, which was active in Tel+ and Tel− cells (Figure 1C) [23,24]. pTERTLuc800WT contained a 800-bp hTERT promoter sequence upstream of the initiation codon and included most of regulatory elements based on previously reported transient transfection studies [18]
Summary
The proliferative lifespan of most human somatic cells is restricted by telomeres, which serve as protective caps of chromosomal ends. Telomeric TTAGGG repeats are replenished by telomerase [1], a ribonucleoprotein complex consisting of a catalytic reverse transcriptase (TERT), an RNA template (TERC) and accessory proteins [2,3]. With the exception of certain stem cells, telomerase activity is either absent or very low [4,5]. Somatic cells suffer telomere attrition upon successive divisions and are destined to senescence. Whereas TERC is abundant in most human tissues [6], the hTERT gene is tightly regulated and its expression correlated with telomerase activity [7]. Ectopic hTERT expression in many cell types results in telomere stabilization and cellular immortalization [8,9]
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