Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells

Abstract

cMutations in ATRX (alpha thalassemia/mental retardation syndrome X-linked), a chromatin-remodeling protein, are associated with the telomerase-independent ALT (alternative lengthening of telomeres) pathway of telomere maintenance in several types of cancer, including human gliomas. In telomerase-positive glioma cells, we found by immunofluorescence that ATRX localized not far from the chromosome ends but not exactly at the telomere termini. Chromatin immunoprecipitation (ChIP) experiments confirmed a subtelomeric localization for ATRX, yet short hairpin RNA (shRNA)-mediated genetic inactivation of ATRX failed to trigger the ALT pathway. Cohesin has been recently shown to be part of telomeric chromatin. Here, using ChIP, we showed that genetic inactivation of ATRX provoked diminution in the amount of cohesin in subtelomeric regions of telomerasepositive glioma cells. Inactivation of ATRX also led to diminution in the amount of TERRAs, noncoding RNAs resulting from transcription of telomeric DNA, as well as to a decrease in RNA polymerase II (RNAP II) levels at the telomeres. Our data suggest that ATRX might establish functional interactions with cohesin on telomeric chromatin in order to control TERRA levels and that one or the other or both of these events might be relevant to the triggering of the ALT pathway in cancer cells that exhibit genetic inactivation of ATRX.T he linear chromosomes of eukaryotic organisms require particular protection at their extremities. Telomeres, which represent the ends of these chromosomes and contain repeated TGrich sequences that do not code for proteins, as well as proteins of the shelterin complex, are essential for this protection (1). Telomeres protect chromosome ends from DNA repair activities that reseal chromosome internal DNA breaks occurring during DNA damage (2). Telomere sequences naturally erode with ongoing cell divisions, due to intrinsic mechanisms associated with the fixed 5=-to-3= polarity of replication of the DNA of the genome. Below a certain threshold, shortened telomeres result in DNA damageinduced cell cycle arrest, which is the equivalent of replicative senescence in cultured cells.By limiting the replicative potential of cells, telomere length acts as a biological clock, and telomere erosion serves as a barrier against tumorigenesis in healthy tissue. Paradoxically, telomere erosion or telomere dysfunction also induces chromosomal instability and favors the emergence of tumors (3). However, following cancer initiation, tumor cells must overcome the telomere-controlled replicative-senescence barrier, and all have an absolute necessity to maintain functional telomeres in order to sustain continuous and unlimited cell proliferation. In around 85 to 90% of cancer types, this occurs through upregulation of telomerase, a reverse transcriptase with a built-in RNA template specialized in telomeric DNA replication that is naturally repressed in most somatic tissues (4). In the remaining 10 to 15% of cancer types, an alternative pathway called the ALT (alternativ...

Enhancer-associated long non-coding RNA LEENE regulates endothelial nitric oxide synthase and endothelial function

“…Antibodies used for RIP assays include anti-RNA Pol II (mouse monoclonal to mouse and human RNA Pol II CTD repeat YSPTSPS, ab817, Abcam), anti-KLF4 (rabbit monoclonal to residues near the carboxy terminus of human KLF4 protein, 12173, Cell Signaling Technology), and anti-MED1 (rabbit polyclonal to residues between 1525 and carboxy terminus of MED1, A300-793A, Bethyl Laboratories). All of the antibodies have been previously authenticated for ChIP use 59 – 61 and we used 5 μl antibody for chromatins isolated from 1 mg input total protein. Following three times of wash to remove non-specific binding, RNA was extracted by TRizol and reverse transcribed for qPCR analysis.…”

Roles of telomeres and telomerase in cancer, and advances in telomerase-targeted therapies

“…The mammalian cell lines harboring active ALT have higher TERRA levels compared with telomerase-positive cells [ 20 ]. However, the exact role of TERRA in activation of the ALT mechanism is not clear [ 21 ].…”

ALTernative Telomere Maintenance and Cancer

“…Alterations in such chromatin factors may create an environment permissive for HR at telomeres. For example, ATRX depletion alters cell cycle regulation of the long-noncoding RNA telomeric repeat-containing RNA (TERRA) and RPA at ALT telomeres, but does not enhance all ALT characteristics [56,60–62]. Regardless, the enrichment of certain mutations in ALT-positive tumors may provide a useful diagnostic or prognostic tool.…”