Importance of TRF1 for Functional Telomere Structure

Michael Reth,Tomohiko Iwano,Yoichi Shinkai,Makoto Tachibana

doi:10.1074/jbc.m309138200

Michael Reth, Tomohiko Iwano + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m309138200

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Abstract

Telomeres are comprised of telomeric DNA sequences and associated binding molecules. Their structure functions to protect the ends of linear chromosomes and ensure chromosomal stability. One of the mammalian telomere-binding factors, TRF1, localizes telomeres by binding to double-stranded telomeric DNA arrays. Because the overexpression of wild-type and dominant-negative TRF1 induces progressive telomere shortening and elongation in human cells, respectively, a proposed major role of TRF1 is that of a negative regulator of telomere length. Here we report another crucial function of TRF1 in telomeres. In conditional mouse TRF1 null mutant embryonic stem cells, TRF1 deletion induced growth defect and chromosomal instability. Although no clear telomere shortening or elongation was observed in short term cultured TRF1-deficient cells, abnormal telomere signals were observed, and TRF1-interacting telomere-binding factor, TIN2, lost telomeric association. Furthermore, another double-stranded telomeric DNA-binding factor, TRF2, also showed decreased telomeric association. Importantly, end-to-end fusions with detectable telomere signals at fusion points accumulated in TRF1-deficient cells. These results strongly suggest that TRF1 interacts with other telomere-binding molecules and integrates into the functional telomere structure.

Highlights

Telomeres are specialized DNA structures positioned at the termini of eukaryotic chromosomes [1]
We demonstrate that murine TRF1 is absolutely required for normal cell growth, telomere structure, and chromosomal stability
Generation of Conditional murine TRF1 (mTRF1)-deficient ES Cells—To generate mTRF1-deficient ES cells, we introduced a mTRF1 targeting plasmid, pLNTK-mTRF1TV (Fig. 1A), into E14 mouse ES cells. pLNTK-mTRF1TV potentially introduces a deletion of the initiation codon containing exon 1

Summary

MATERIALS AND METHODS

TRF1 Targeting and Expression Constructs—13.9 kb of genomic DNA containing the mTRF1 gene was isolated from a 129SV genomic phage clone. After rehydration in phosphate-buffered saline, fixed cells were treated with 2 N HCl, 0.5% Triton X-100 for 20 min, washed extensively in blocking buffer (1% bovine serum albumin, 0.5% Tween 20 in phosphate-buffered saline), and stained with anti-BrdUrd Ab (Becton Dickinson) diluted 1:100 in the blocking buffer for 20 min. The cells were incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG Ab (e-bioscience) diluted 1:200 in the blocking buffer for 20 min. For examining TRFs containing telomeric sequences, the gel was dried at 60 °C for 3 h, denatured in a 1.5 M NaCl, 0.5 M NaOH solution for 30 min, neutralized in a 1.5 M NaCl, 0.5 M Tris-HCl pH 8.0 buffer for 30 min, and probed with ␥-32P-labeled (C3TA2) telomeric DNA oligonucleotides in 5ϫ SSC, 5ϫ Denhardt’s solution at 37 °C overnight. For examination of the status of the 3Ј overhangs of the telomeric sequences, the denaturing and neutralization steps were omitted

RESULTS AND DISCUSSION

No of metaphases analyzed

Containing TTAGGG