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A TRF1-controlled common fragile site containing interstitial telomeric sequences

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Abstract

Mouse telomeres have been suggested to resemble common fragile sites (CFS), showing disrupted TTAGGG fluorescent in situ hybridization signals after aphidicolin treatment. This “fragile” telomere phenotype is induced by deletion of TRF1, a shelterin protein that binds telomeric DNA and promotes efficient replication of the telomeric ds[TTAGGG]n tracts. Here we show that the chromosome-internal TTAGGG repeats present at human chromosome 2q14 form an aphidicolin-induced CFS. TRF1 binds to and stabilizes CFS 2q14 but does not affect other CFS, establishing 2q14 as the first CFS controlled by a sequence-specific DNA binding protein. The data show that telomeric DNA is inherently fragile regardless of its genomic position and imply that CFS can be caused by a specific DNA sequence.

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Acknowledgments

We thank Agnel Sfeir for the help in the early stages of this work and members of the de Lange lab for comments on this manuscript. We are grateful to Eugene Rudensky for the help with qPCR. This work was supported by a grant from the NIH (AG16642) to TdL. TdL is an American Cancer Society Research Professor. NB is supported by “The Alessandro & Catherine di Montezemolo” American-Italian Cancer Foundation Post-Doctoral Research Fellowship.

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None declared.

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  1. Laboratory for Cell Biology and Genetics, The Rockefeller University, Box 159, 1230 York Avenue, New York, NY, 10065, USA

    Nazario Bosco & Titia de Lange

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  1. Nazario Bosco
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  2. Titia de Lange
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Correspondence to Titia de Lange.

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Communicated by Jan Karlseder

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Supplementary Table 1

Frequency of CFS 2q14 (Fig. 3b), N-FRA, FRA3B, FRA7H and FRA16D expression (Fig. 3i) after shTRF1 treatment. (DOCX 64 kb)

Supplementary Table 2

Frequency of CFS 2q14 (Fig. 3d), N-FRA, FRA3B, FRA7H and FRA16D expression (Fig. 3j) after shTRF2 treatment. (DOCX 54 kb)

Supplementary Table 3

Frequency of CFS 2q14 after treatment with shBLM and shTRF1 (Fig. 4e). (DOCX 32 kb)

Supplementary Table 4

Frequency of NFRA, FRA3B, FRA7H and FRA16D after treatment with shBLM and shTRF1 (Fig. 4f). (DOCX 64 kb)

Supplementary Table 5

Frequency of CFS 2q14 (Fig. 5d), FRA3B and FRA16D (Fig. 5c) after treatment with shATR. (DOCX 42 kb)

Supplementary Table 6

Frequency of CFS 2q14 (Fig. 5g), FRA3B and FRA16D expression (Fig. 5f) in HCT116 ATRflox/- cells after deletion of ATR. (DOCX 47 kb)

Fig. S1

The human chromosome 2q14 region. a. DNA sequence from 2q14.1 containing the telomere fusion of two ancestral ape chromosomes. Highlighted are the recognition sites for the TRF1 Myb/SANT DNA binding domain (TAGGGTT or AACCCTA in the other strand). b. Sequence from the genomic locus encoding the Rap1 (TERF1, TRF2 interacting factor 1) gene on chromosome 16, which does not contain TRF1 binding sites. The underlined sequences in both panels indicate the positions of the qPCR primers given in the methods section. (JPEG 951 kb)

High resolution image (TIFF 989 kb)

Fig. S2

Mapping the breakages on chromosome arm 2q. a, b. Example of a metaphase obtained from BJ/hTERT/SV40 after aphidicolin treatment. The two chromosomes 2 are highlighted, one of which is detailed in Fig. 1 (bottom box). The other chromosome 2, enlarged in panel b, shows two breakages centromeric to the CFS 2q14, which could be FRA2A and FRA2B. (JPEG 159 kb)

High resolution image (TIFF 3610 kb)

Fig. S3

Monitoring the effect of TRF1 and TRF2 shRNAs. a. Quantification of the effect of shRNA treatment on TRF1 levels at telomeres determined by IF-FISH. b. Quantification of TIF formation upon inhibition of TRF1 with shRNAs. (c,d) Quantification of the frequency of fragile telomeres induced by repression of TRF1 (c) or TRF2 (d) with shRNAs. For all panels the bars represent mean values of three independent experiments with SD. At least 50 nuclei or 30 metaphases were analyzed per IF or FISH experiment. Brackets with asterisks indicate p values below 0.05 (student’s t-test). (JPEG 141 kb)

High resolution image (TIFF 358 kb)

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Bosco, N., de Lange, T. A TRF1-controlled common fragile site containing interstitial telomeric sequences. Chromosoma 121, 465–474 (2012). https://doi.org/10.1007/s00412-012-0377-6

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