Expression and differential regulation of human TERRA at several chromosome ends

Abstract

The telomeric long noncoding RNA TERRA has been implicated in regulating telomere maintenance by telomerase and homologous recombination, and in influencing telomeric protein composition during the cell cycle and the telomeric DNA damage response. TERRA transcription starts at subtelomeric regions resembling the CpG islands of eukaryotic genes extending toward chromosome ends. TERRA contains chromosome-specific subtelomeric sequences at its 5' end and long tracts of UUAGGG-repeats toward the 3' end. Conflicting studies have been published as to whether TERRA is expressed from one or several chromosome ends. Here, we quantify TERRA species by RT-qPCR in normal and several cancerous human cell lines. By using chromosome-specific subtelomeric DNA primers, we demonstrate that TERRA is expressed from a large number of telomeres. Deficiency in DNA methyltransferases leads to TERRA up-regulation only at the subset of chromosome ends that contain CpG-island sequences, revealing differential regulation of TERRA promoters by DNA methylation. However, independently of the differences in TERRA expression, short telomeres were uniformly present in a DNA methyltransferase deficient cell line, indicating that telomere length was not dictated by TERRA expression in cis Bioinformatic analyses indicated the presence of a large number of putative transcription factors binding sites at TERRA promoters, and we identified a subset of them that repress TERRA expression. Altogether, our study confirms that TERRA corresponds to a large gene family transcribed from multiple chromosome ends where we identified two types of TERRA promoters, only one of which is regulated by DNA methylation.

Keywords: TERRA; long noncoding RNA; telomere length; telomeres.

FIGURE 1.

Absolute quantification of TERRA in human cell lines. (A) HLF, HeLa and U2OS. (B) HCT116 and HCT116 DKO. Quantification was performed by RT-qPCR using standard curves prepared with plasmid standards containing subtelomeric sequences of the indicated telomeres. RT efficiency was assumed to be 100% efficient.

FIGURE 2.

Relative TERRA expression levels in HCT116 WT and DKO cells. (A) Relative quantification of TERRA expression in HCT116 WT and DKO cells. WT TERRA levels were adjusted to 1. (**) P < 0.01, (***) P < 0.001, n = 3. (B) Representation of position and CG content of subtelomeric CpG islands at the indicated chromosome ends.

FIGURE 3.

TERRA expression and telomere length at individual chromosome ends do not correlate in HCT116 DKO cells. 9p (A), 20q (B), 15q (C), and 17p (D) TERRA expression was measured by RT-qPCR in HCT116 WT and DKO cells. TERRA levels were normalized to GAPDH. N = 3. STELA was performed to measure telomere length of 9p, 20q (E), 15q (F), and 17p (G) telomeres. Each lane corresponds to an independent small pool PCR done with genomic DNA of the indicated cell line. Negative controls were done without telorette ligation (lanes marked with an asterisk). (H) Scattered plot (geometric mean ± 95% confidence interval) representing the same results as in E, F, and G. Each band is represented by one dot. (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001.

FIGURE 4.

ZNF148 (C), ZFX (D), EGR1 (E), and PLAG1 (F) repress TERRA transcription. HeLa cells were transfected with siRNAs against transcription factors predicted to bind to subtelomeric DNA sequences. CTCF (A) and ATRX (B) depletion served as positive controls for a positive and negative regulator of TERRA transcription. (*) P < 0.05, n = 3.