Human Telomerase Expression is under Direct Transcriptional Control of the Telomere-binding-factor TRF2

Abstract

Tight regulatory mechanisms to maintain repression of human Telomerase (hTERT), the sole protein that synthesizes telomeres, is crucial for normal adult somatic cells. In contrast, enhanced telomerase activity and resulting pathological maintenance of telomeres, is widely understood as causal in >90% of human cancers. These implicate underlying mechanisms connecting hTERT regulation and telomeres, possibly through telomeric proteins, that remain unclear. In light of of recent work by us and others showing non-telomeric function of the telomere-binding protein TRF2, here we examined whether and how TRF2 affected hTERT regulation. Direct binding of TRF2spanning ~450 bp of the hTERT promoter from the Transcriptional Start Site (TSS)led to TRF2-dependent recruitment of the polycomb repressor complex PRC2 in both normal and cancer cells. This induced repressor histone modifications resulting in TRF2-dependent hTERT repression.Mutations in the hTERT promoter, found frequently in aggressive glioblastoma and reported to destabilize the G-quadruplex structure, resulted in loss of TRF2 binding and consequent hTERT over-expression. Conversely, using G-quadruplex-stabilizing ligands we regained TRF2 binding, hTERT re-suppression, in highly proliferating glioblastoma cells with telomerase hyperactivation due to hTERT promoter mutations. Together, results herein demonstrate direct control of hTERT through TRF2 in a G-quadruplex-dependent mannerimplicating mechanisms of how telomerase regulation might be linked to telomeres in normal and cancer cells. IntroductionTelomeres, comprising of (GGGTTA) n repeats in complex with telomere-binding proteins, at the end of human chromosomes are essential for genome stability 1-6 . The only protein that can replicate telomeric DNA is telomerase -a ribonucleoprotein complex of the reverse transcriptase (hTERT) and RNA the template (hTERC). In adult somatic cells, hTERT is kept trasncriptionally repressed.Resulting loss of telomeres with each replication cycle leads to replicative senescence, much like a 'molecular clock' that helps maintain cellular homeostasis. Deregulation or loss of hTERT repression, which results in aberrant maintenance of telomeres, has been causally associated to initiation/progression of more than 90% human cancers 7,8 . Although, these suggest tight control of telomerase might be linked to telomeres -the role of telomeres or telomere-binding factors in regulation of hTERT remains poorly explored.Relatively recent work by others and us showing non-telomeric binding of telomere-binding proteins, TRF1, TRF2 and RAP1, is of interest in this context. Genome wide RAP1 association has been reported in mouse and human cells while TRF1/TRF2 binding has been demonstrated in human cells 9-11 . Notably, we found about 20000 TRF2 binding sites spread throughout the genome where TRF2-mediated promoter epigenetics and gene regulation was evident. A large fraction of the TRF2 binding sites coincided with potential DNA secondary structure G-quadruplex-forming sequences 12 .Further, ...

Extra-telomeric impact of telomeres: Emerging molecular connections in pluripotency or stemness

“…More recent work, on the other hand, suggested that hTERT regulation is under direct transcriptional control of TRF2 (118). Here, TRF2 presence on the hTERT promoter was independent of telomeres (i.e.…”

“…there was involvement of extra-telomeric TRF2). This was also clear from TRF2 occupancy at the exogenously inserted hTERT promoter ;46 Mb away from telomeres (118)-where looping due to physical proximity like the 5p telomere end was unlikely. Together, these leads suggest the involvement of the TSP model discussed above in hTERT regulation, where the presence of extra-telomeric TRF2 on the hTERT promoter is of interest and depends on how much TRF2 is free or sequestered at the telomere ends.…”

“…Telomerase-the only protein that synthesizes telomeres-is overexpressed in most cancers. Recent findings suggest that telomeres exert control over telomerase through telomeric or extra-telomeric mechanisms (33,118). Teasing out molecular details of these controls, including whether and how the TSP model might be involved in telomere-dependent control of telomerase, remains to be studied in further detail, considering its broad and significant implications.…”

TERRA G-quadruplex RNA interaction with TRF2 GAR domain is required for telomere integrity

“…The TRF2 GAR domain has been shown to bind the 4-way junctions formed at T-loops and restrict promiscuous telomere recombination 96 . TRF2 was also shown to inhibit telomerase expression in normal cells via its interaction with G4 structures in the TERT promoter, although it is not clear if the GAR domain was required for this activity 97 . TRF2 may interact with G4 DNA through domains other than GAR, and the GAR domain is known to have functions in addition to TERRA binding.…”

Emerging Molecular Connections between NM23 Proteins, Telomeres and Telomere-Associated Factors: Implications in Cancer Metastasis and Ageing

“…Therefore, NM23-H2-TRF2 interaction suggests potential co-regulation through association with G-quadruplexes. An example of this is the regulation of hTERT, where independently both NM23-H2 [ 12 ] and TRF2 [ 96 ] have been observed to control hTERT by binding to promoter G-quadruplexes.…”