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Gene expression profile of Huh7 cells with long and short telomeres

ABSTRACT: We sorted H2O2–treated HCC Huh7-cells harboring telomeres in the top 20th percentile (long telomeres) and bottom 20th percentile of telomere length (short telomeres) according to fluorescence-activated cell sorting based on the fluorescence intensity associated with the telomere-binding protein, followed by whole-genome mRNA-expression microarray analysis. In detail, a stable Huh7 cell line expressing GFP-tagged telomere-binding protein adrenocortical dysplasia protein homolog (TPP1) was established using 1 mg/mL G418 as a selection antibiotic. The telomere lengths in living cells were measured by assessing the expression of TPP1 by fluorescence-activated cell sorting (FACS) under the assumption that the expression level of the telomere binding protein is proportional to telomere length. The cells were dissociated at 37°C in 0.2 % w/v trypsin-EDTA for 10 min. After gentle pipetting to induce dissociation, the cells were washed twice with cold PBS solution containing 0.6 % w/v ultra-pure bovine serum albumins (A0100-010) and filtered using a cell strainer (BD Bioscience). HCC cells displaying weak or strong GFP expression were isolated from the mock- or H2O2-treated cells using a FACS Aria II (BD Bioscience), respectively. A whole genome mRNA expression microarray (Macrogen, Seoul, Korea) was used to compare the gene expression patterns between HCC cells possessing short or long telomeres.

ORGANISM(S): Homo sapiens

PROVIDER: GSE114004 | GEO | 2019/06/10

REPOSITORIES: GEO

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Project description:Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1/Timeless, a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Î mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of the FPC in regulation of telomere stability in cancer cells. Genome-wide distributions of Rad52 in wild type and in swi1 deletion in fission yeast The'SP1173_WT ChIP-seq' is an input sample (non-tagged data).

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Gene expression profile of Huh7 cells with long and short telomeres

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