Human telomerase reverse transcriptase (hTERT) is expressed in most cancer cells. Paradoxically, its promoter is embedded in a hypermethylated CpG island. A short region escapes to this alteration, allowing a basal level of transcription. However, the methylation of adjacent regions may play a role in the maintenance of low hTERT expression. It is now well established that methyl-CpG binding domain proteins mediate the transcriptional silencing of hypermethylated genes. The potential involvement of these proteins in the control of hTERT expression was firstly investigated in HeLa cells. Chromatin immunoprecipitation assays showed that only methyl-CpG-binding domain protein 2 (MBD2) associated the hypermethylated hTERT promoter. In MBD2 knockdown HeLa cells, constitutively depleted in MBD2, neither methyl CpG binding protein 2 (MeCP2) nor MBD1 acted as substitutes for MBD2. MBD2 depletion by transient or constitutive RNA interference led to an upregulation of hTERT transcription that can be downregulated by expressing mouse Mbd2 protein. Our results indicate that MBD2 is specifically and directly involved in the transcriptional repression of hTERT in HeLa cells. This specific transcriptional repression was also observed in breast, liver and neuroblastoma cancer cell lines. Thus, MBD2 seems to be a general repressor of hTERT in hTERT-methylated telomerase-positive cells.

Germline TP53 mutations predispose to multiple cancers defining Li-Fraumeni/Li-Fraumeni-like syndrome (LFS/LFL), a disease with large individual disparities in cancer profiles and age of onset. G-quadruplexes (G4s) are secondary structural motifs occurring in guanine tracks, with regulatory effects on DNA and RNA. We analyzed 85 polymorphisms within or near five predicted G4s in TP53 in search of modifiers of penetrance of LFS/LFL in Brazilian cancer families with (n = 35) or without (n = 110) TP53 mutations. Statistical analyses stratified on family structure showed that cancer tended to occur ~15 years later in mutation carriers who also carried the variant alleles of two polymorphisms within predicted G4-forming regions, rs17878362 (TP53 PIN3, 16 bp duplication in intron 3; P = 0.082) and rs17880560 (6 bp duplication in 3' flanking region; P = 0.067). Haplotype analysis showed that this inverse association was driven by the polymorphic status of the remaining wild-type (WT) haplotype in mutation carriers: in carriers with a WT haplotype containing at least one variant allele of rs17878362 or rs17880560, cancer occurred ~15 years later than in carriers with other WT haplotypes (P = 0.019). No effect on age of cancer onset was observed in subjects without a TP53 mutation. The G4 in intron 3 has been shown to regulate alternative p53 messenger RNA splicing, whereas the biological roles of predicted G4s in the 3' flanking region remain to be elucidated. In conclusion, this study demonstrates that G4 polymorphisms in haplotypes of the WT TP53 allele have an impact on LFS/LFL penetrance in germline TP53 mutation carriers.

G-quadruplex (G4) structures in intron 3 of the p53 pre-mRNA modulate intron 2 splicing, altering the balance between the fully spliced p53 transcript (FSp53, encoding full-length p53) and an incompletely spliced transcript retaining intron 2 (p53I2 encoding the N-terminally truncated Δ40p53 isoform). The nucleotides forming G4s overlap the polymorphism rs17878362 (A1 wild-type allele, A2 16-base pair insertion) which is in linkage disequilibrium with rs1642785 in intron 2 (c.74+38 G>C). Biophysical and biochemical analyses show rs17878362 A2 alleles form similar G4 structures as A1 alleles although their position is shifted with respect to the intron 2 splice acceptor site. In addition basal FSp53 and p53I2 levels showed allele specific differences in both p53-null cells transfected with reporter constructs or lymphoblastoid cell lines. The highest FSp53 and p53I2 levels were associated with combined rs1642785-GG/rs17878362-A1A1 alleles, whereas the presence of rs1642785-C with either rs17878362 allele was associated with lower p53 pre-mRNA, total TP53, FSp53 and p53I2 levels, due to the lower stability of transcripts containing rs1642785-C. Treatment of lymphoblastoid cell with the G4 binding ligands 360A or PhenDC3 or with ionizing radiation increased FSp53 levels only in cells with rs17878362 A1 alleles, suggesting that under this G4 configuration full splicing is favoured. These results demonstrate the complex effects of intronic TP53 polymorphisms on G4 formation and identify a new role for rs1642785 on mRNA splicing and stability, and thus on the differential expression of isoform-specific transcripts of the TP53 gene.