Variant repeats are interspersed throughout the telomeres and recruit nuclear receptors in ALT cells

Dimitri Conomos, et al., Variant repeats are interspersed throughout the telomeres and recruit nuclear receptors in ALT cells, J. Cell Biol. 199, 2012

Telomeres in cells that use recombination-mediated alternative lengthening of telomeres (ALT) pathway elicit a DNA damage response that is partly independent of telomere length.

Here we used next generation sequencing to analyze the DNA content of ALT telomeres. We discovered that variant repeats were interspersed throughout the telomeres of ALT cells. We found that the C-type (TCAGGG) variant repeat predominated and created a high-affinity binding site for the nuclear receptors COUP-TF2 and TR4.

We used next generation sequencing to quantitatively determine the identity and extent of variant sequence within telomere arrays of WI38-VA13/2RA as well as the telomerase-positive HeLa cell line. Samples were paired-end sequenced, and reads containing greater than six nonconsecutive telomeric repeats in the format TBAGGG were considered to be reads derived from telomeres.

Structure and Variability of Human Chromosome Ends

Titia de Lange, et al., Structure and Variability of Human Chromosome Ends, Molecular and Cellular Biology 10, 1990,

The minimal size of the subtelomeric repeat is 4 kilo bases (kb); it shows a high frequency of restriction fragment length polymorphisms and undergoes extensive de novo methylation in somatic cells. Distal to the subtelomeric repeat, the chromosomes terminate in a long region (up to 14kb) that may be entirely composed of TTAGGG repeats. This terminal segment is unusually variable. Although sperm telomeres are 10 to 14kb long, telomeres in somatic cells are several kilo base pairs shorter and very heterogeneous in length.

Human telomeres contain at least three types of G-rich repeat distributed non-randomly

Robin C. Allshire, et al., Human telomeres contain at least three types of G-rich repeat distributed non-randomly,  Nucleic Acids Research 17, 1989

Human telomeres do not contain a pure uniform 6 base pair repeat unit but that there are at least three types of repeat.

The distribution of each type of repeat appears to be non-random. Each human telomere has a similar arrangement of these repeats relative to the ends of the chromosome.

Analysing the change in length of the telomeric repeat region between an individuals blood and gremlin DNA reveals that this is due to variable amounts of the TTAGGG repeat and not the other repeat types.

There can only be, on average, a maximum of 8.3kb of TTAGGG like repeats per sperm telomere. In addition approximately 1.2kb of TTGGGG like repeats must be distal to all MnlI and HphI site. The most proximal 1.9kb of each telomeric repeat region is made up of TTGGGG and TGAGGG like repeats, this leaves approximately 3.6kb of the human telomeric repeat region unaccounted for.

 

Assessing telomeric DNA content in pediatric cancers using whole-genome sequencing data

Matthew Parker, et al., Assessing telomeric DNA content in pediatric cancers using whole-genome sequencing data, Genome Biology, 2012, 13:R113.

To measure telomeric DNA content, we counted telomeric reads containing TTAGGGx4 or CCCTAAx4 and normalized to the average genomic coverage.

We found that the pattern of telomeric DNA alteration varies dramatically across the landscape of pediatric malignancies: telomere gain was found in 32% of solid tumors, 4% of brain tumors and 0% of hematopoietic malignancies.

A high proportion of osteosarcoma had long heterogeneous telomeres with ALT, … and some of the high grade gliomas had ALT and long telomeres.

They used qPCR, FISH and TRF for validation of telomere length measuring from WGS data.

Telomere extension by telomerase and ALT generates variant repeats by mechanistically distinct processes

Michael Lee, et al., Telomere extension by telomerase and ALT generates variant repeats by mechanistically distinct processes, Nucleic Acids Research, 2014, vol. 42, No. 3, 1733-1746.

We have evaluated telomere sequence content in human cells using whole-genome sequencing followed by telomere read extraction in a panel of mortal cell strains and immortal cell lines. We identified a wide range of telomere variant repeats in human cells, and found evidence that variant repeats are generated by mechanistically distinct processes during telomerase- and ALT-mediated telomere lengthening. Telomerase-mediated telomere extension resulted in biased repeat synthesis of variant repeats that differed from the canonical sequence at positions 1 and 3, but not at positions 2, 4, 5 or 6.

In contrast, cell lines that use the ALT pathway contained a large range of variant repeats that varied greatly between lines.

The presence of unexpectedly large numbers of variant repeats in cells utilizing either telomere maintenance mechanism suggests a conserved role for variant sequences at human telomeres.

We identified a significant increase in GCAGGG variants, specific to the HT1080 hTR TCA cell line.