The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats

Abstract

The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences.

Figure 1

(A) Slot blot showing the telomeric enrichment of DNA immunoprecipitated with anti-TRF1 or TRF2 antibodies. DNA immunoprecipitated by a total H3 antibody and pulled down by protein G alone was used as a control. Half of the precipitated DNA was loaded, along with an input scale (2 500 ng to 10 ng, corresponding to 10% to 0.04% of the total input), and hybridized sequentially to a telomeric probe and a genomic probe. For each probe, we quantified the fraction of the immunoprecipitated DNA. The ratio of the value obtained for the telomeric probe to the genomic probe is the telomeric enrichment factor. (B) Fold enrichment of the fraction of raw reads containing only (TTAGGG)_n sequences from TRF ChIP-Seq as normalized to the reads obtained through immunoprecipitation of protein G.

Figure 2

(A) TRF1, TRF2m, and TRF2p ChIP-Seq peaks. The peaks largely coincide, as shown on the Venn diagram. Assessment of overlaps was performed by visual inspection in the Integrated Genome Browser. (B) Visualization of TRF peaks and TRF binding sites. Regions of significant read enrichment (P < 0.001) for each ChIP analysis (over the protein G background) are shown for human chromosome 1, along with the (TTAGGG)_n repeats extracted from RepeatMasker UCSC files . The upper line (TRF binding sites) displays the positions of the common peaks obtained with the three TRF antibodies. The criterion is one peak with a P value < 0.001 and two peaks with P < 0.05. For the individual antibodies (TRF1, TRF2p, and TRF2m), only the peaks with a P value < 0.001 are shown.

Figure 3

Validation of the TRF binding sites by ChIP-qPCR with TRF1 and TRF2m antibodies. Enrichment (quantified as the IP/input ratio minus the background ratio (obtained from the protein G ChIP analysis)) of the different loci was normalized to the value for a GADPH gene sequence. Three ChIP-qPCR analyses were performed using BJ-HELTRas^mc cells and SNG28 cells.

Figure 4

(A) Motif prediction analysis of the 68 TRF binding sites, performed using MEME software. The telomeric (TTAGGG)_n motif and the (ATTCC)n motif present in satellite DNA families 2 and 3 were identified. (B) An example of a TRF peak associated with a (TTAGGG)_n repeat. (C) An example of a TRF peak associated with a Satellite 2/3 sequence. (D) An example of a TRF peak associated with an alphoid satellite sequence. (E) An example of a (TTAGGG)_n repeat not enriched by Chip-Seq analysis performed using the three anti-TRF antibodies.

Figure 5

(A) Classification of the peaks according to their location relative to genic sequences. Note the significant bias in the location of the TRF peaks, and more generally that of the ITSs, such that they tended to occur in genic regions of the genome (defined as sequences located less than 100 kb from any gene) as opposed to gene desert regions (sequences located more than 100 kb from any gene). (B) Schematic representations of the SNAP25 and PLXNB2 gene regions showing the TRF and REST peaks.