TERRA, CpG methylation and telomere heterochromatin: lessons from ICF syndrome cells

Abstract

Self-reinforcing negative feedback loops are commonly observed in biological systems. RNA-mediated negative feedback loops have been described in the formation of heterochromatin at centromeres in fission yeast and the inactive X chromosome in mammalian cells. The telomere repeat-containing RNA (TERRA) has also been implicated in the formation of telomeric heterochromatin through a self-reinforcing negative feedback loop. In cells derived from human ICF syndrome, TERRA levels are abnormally elevated and telomeres are abnormally shortened. We now show that telomere heterochromatin is also abnormal in ICF cells. We propose that ICF cells fail to reinforce the TERRA-dependent negative feedback loop as a result of the inability to establish heterochromatin at subtelomeres. This failure is likely due to the lack of DNMT3b and DNA methylation, which is a genetic lesion associated with ICF syndrome. Failure of this feedback mechanism leads to catastrophic telomere dysfunction and chromosome instability.

Figure 1

Loss of telomeric heterochromatin formation in ICF cells. (A) RNA dot-blot analysis of total RNA isolated from ICF LCLs (GM08714) or WT control LCLs from the father (GM08729) in two independent experiments. RNA was detected by hybridization with probes containing (CCCTAA)₆, GAPDH, or (TTAGGG)₆, as indicated. RNase A treatment (+) was used as an RNA specificity control. (B) Representative telomere DNA FISH analysis on metaphase spreads for telomere defects in WT and ICF LCLs. Arrows indicate ends with telomeric signals loss. (C) Methylated DNA Immuoprecipitation (MeDIP) analysis of Cytosine methylation in WT (red) or ICF (black) LCLs. Real-time PCR was used to quantify methylated DNA signals by primers specific for indicated subtelomeric regions (2p, 18p, region 6, 10q or 16p) or controls β-actin and GAPDH loci. The bar graph represents the value of MeDIP relative to IgG IP.

Figure 2

Decreased association of TR F2 with subtelomeres in ICF cells. (A) ChIP analysis by TR F2 antibody at indicated subtelomeric regions or at the control Actin loci in WT or ICF LCLs. ChIP DNA was assayed by real-time PCR with specific primers as indicated. TRF2 binding was first normalized to corresponding input DNA and then shown as fold change relative to IgG. (B) Same as in (A), except that TRF1 antibody was used for ChIP analysis.

Figure 3

DNA hypomethylation correlates with increased H3K4 methylation and reduced H3K9 methylation at subtelomeres of ICF cells. (A–C) ChIP analysis of subtelomeric histone modifications with antibodies specific for histone H3 K4 di-methylation (H3 K4me2), H3 K9 tri-methylation (H3 K9me3), or H3 K27 tri-methylation (H3 K27me3) in WT (red) or ICF (black) LCLs, respectively.

Figure 4

Schematic model for dynamic changes of subtelomeric and telomeric heterochromatin regulated by TERRA RNA transcription in ICF cells. In normal cells, TERRA facilitates heterochromatin formation through a negative feedback looping mechanism, which is likely mediated by the recruitment of DNMT3b and the generation of CpG methylation at subtelomeres. TERRA RNA further stabilizes the telomeric heterochromatin through physical interactions with ORC, MeCP2, and heterochromatin factors including HP1. In ICF cells, loss of CpG methylation at subtelomeres due to DNMT3b mutations results in an irreversible chromatin state, as well as a loss of negative feedback looping by TERRA. This leads to an “open” chromatin state enriched with H3 K4 dimethylation and allows transcription factor assembly and RNA polymerase II unimpeded access to subtelomeres. Eventually, highly induced TERRA levels may contribute to telomere dysfunction in ICF cells either by inhibiting telomerase activity, forming RNA-DNA hybrids with telomeres, or by interactions with telomere binding proteins.