The chromatin remodelling factor ATRX suppresses R‐loops in transcribed telomeric repeats

Abstract

ATRX is a chromatin remodelling factor found at a wide range of tandemly repeated sequences including telomeres (TTAGGG) ATRX mutations are found in nearly all tumours that maintain their telomeres via the alternative lengthening of telomere (ALT) pathway, and ATRX is known to suppress this pathway. Here, we show that recruitment of ATRX to telomeric repeats depends on repeat number, orientation and, critically, on repeat transcription. Importantly, the transcribed telomeric repeats form RNA-DNA hybrids (R-loops) whose abundance correlates with the recruitment of ATRX Here, we show loss of ATRX is also associated with increased R-loop formation. Our data suggest that the presence of ATRX at telomeres may have a central role in suppressing deleterious DNA secondary structures that form at transcribed telomeric repeats, and this may account for the increased DNA damage, stalling of replication and homology-directed repair previously observed upon loss of ATRX function.

ATRX loss induces telomere dysfunction and necessitates induction of alternative lengthening of telomeres during human cell immortalization

“…Telomeres are composed of repetitive DNA with high G/C contents and thus prone to form secondary structures including telomeric RNA‐DNA hybrids (R‐loop) and G‐quadruplex structures (Sundquist & Klug, ; Balk et al , ). These high‐order structures are known to pose threats to the processivity of the replication machinery and can recruit ATRX (Law et al , ; Clynes et al , ; Nguyen et al , ). Those realizations, together with the finding that re‐expression of ATRX in ATRX ‐null cells could strongly repress ALT‐associated activities (Clynes et al , ; Napier et al , ), fueled a speculation that ATRX might function directly to suppress ALT by resolving those secondary structures during telomere replication (Leung et al , ; Clynes et al , ; Nguyen et al , ).…”

“…Functionally, ATRX and its binding partner DAXX are known to form a histone variant H3.3‐specific chaperone complex that orchestrates replication‐independent nucleosome assembly at repetitive heterochromatic DNA regions including telomeres (Drane et al , ; Goldberg et al , ; Lewis et al , ; Elsasser et al , ; Voon et al , ). Besides its histone H3.3 depositing function, ATRX has also been implicated in the process of telomere DNA replication by facilitating resolution of telomere cohesion, R‐loops, and G‐quadruplex structures (Law et al , ; Clynes et al , ; Ramamoorthy & Smith, ; Nguyen et al , ). Given the fact that telomere length maintenance is essential for cell immortalization and tumorigenesis, the high concordance of ATRX deficiency with ALT activation has hence prompted the speculation that ATRX may exert its tumor suppressor function by directly repressing ALT (Pickett & Reddel, ; Maciejowski & de Lange, ).…”

“…Indeed, restoration of ATRX expression in ATRX‐negative ALT lines suppresses many ALT‐associated phenotypes (Clynes et al , ; Napier et al , ). Along this line, a variety of ATRX nuclear functions have been invoked to explain its participation in ALT suppression, including its involvement in telomeric H3.3 loading (Wong et al , ), telomere cohesion resolution (Ramamoorthy & Smith, ), guarding the DNA replication against replication stress or G‐quadruplex structure formation (Law et al , ; Leung et al , ; Clynes et al , ), and telomeric R‐loop suppression (Nguyen et al , ). But despite the extensive efforts, the crucial demonstration of a causal role between the ATRX or DAXX loss and ALT activation has not been reported.…”

DNA:RNA hybrids at telomeres – when it is better to be out of the (R) loop

“…In line with this prediction, in vitro studies demonstrated that plasmids containing human telomeric repeats form RLs with telomere‐containing RNA transcribed off the C‐rich strand . Utilizing the S9.6‐DNA:RNA hybrid‐specific antibody, human TRLs were observed in cancer cells, in particular those that maintain their telomeres by the ALT pathway , as well as in cells of patients with immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome . In both ALT and ICF cells, abnormally high levels of TERRA are transcribed , explaining the high levels of TRLs detected in these cells.…”

“…D). Recently, it was demonstrated that the damaging secondary structures that ATRX acts to resolve are actually products of TRLs . This study revealed that factors that influence TRL generation such as telomere length, repeat orientation, and TERRA transcription levels impact the degree by which ATRX is recruited to telomeres.…”