Abstract
Telomere repeat DNA forms a nucleo-protein structure that can obstruct chromosomal DNA replication, especially under conditions of replication stress. Transcription of telomere repeats can initiate at subtelomeric CTCF-binding sites to generate telomere repeat-encoding RNA (TERRA), but the role of transcription, CTCF, and TERRA in telomere replication is not known. Here, we have used CRISPR/Cas9 gene editing to mutate CTCF-binding sites at the putative start site of TERRA transcripts for a class of subtelomeres. Under replication stress, telomeres lacking CTCF-driven TERRA exhibit sister-telomere loss and upon entry into mitosis, exhibit the formation of ultra-fine anaphase bridges and micronuclei. Importantly, these phenotypes could be rescued by the forced transcription of TERRA independent of CTCF binding. Our findings indicate that subtelomeric CTCF facilitates telomeric DNA replication by promoting TERRA transcription. Our findings also demonstrate that CTCF-driven TERRA transcription acts in cis to facilitate telomere repeat replication and chromosome stability.
Highlights
Telomere repeat DNA forms a nucleo-protein structure that can obstruct chromosomal DNA replication, especially under conditions of replication stress
In this study we address the regulation and function for telomere repeat-encoding RNA (TERRA) by CRISPR/CAS9 gene editing of a subtelomeric CTCF-binding motif implicated in TERRA transcription initiation
A series of highly conserved CTCF-binding motifs found on most subtelomeres within a few kilobases from the telomere repeats has been implicated in TERRA transcriptional regulation[9, 25]
Summary
Telomere repeat DNA forms a nucleo-protein structure that can obstruct chromosomal DNA replication, especially under conditions of replication stress. Telomeres lacking CTCF-driven TERRA exhibit sister-telomere loss and upon entry into mitosis, exhibit the formation of ultra-fine anaphase bridges and micronuclei These phenotypes could be rescued by the forced transcription of TERRA independent of CTCF binding. TERRA transcripts have been identified in multiple organisms, including yeast and human, and are known to initiate within subtelomeric repeat regions and proceed into the terminal (TTAGGG)n repeats[13,14,15] The function of these transcripts has been addressed in multiple studies, but it remains unclear whether these transcripts function directly on the telomeres from which they are transcribed (cis-acting) or whether they work remotely (trans-acting) on other telomeres or locations. We show that CTCF-dependent transcription of the 17p subtelomere is important for completion of replication of that telomere under stress, and in the absence of CTCF and TERRA transcription, these sites form ultra-fine anaphase bridges and micronuclei during and after mitosis, respectively
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