Dual regulation of TERT activity through transcription and splicing by DeltaNP63alpha

Abstract

P53 homolog p63 was shown to play a role in premature ageing phenotype found in mouse models through regulation of the replicative senescence. We previously showed that the forced DeltaNp63alpha expression decreased the SIRT1 protein levels, and induced the replicative senescence of human keratinocytes, while the ectopic SIRT1 expression decreased the senescence. Using the DeltaNp63alpha overexpressing and p63-/+ heterozygous mice, we found that DeltaNp63alpha induced the mTERT promoter activation through the down regulation of the SIRT1 protein levels, inactivation of p53 deacetylation, decrease of the p53/Sp1 protein-protein interaction, and the overall induction of mTERT transcription regulation. In the same time, by a forming of protein-protein complexes with the ABBP1, DeltaNp63alpha induced the mTERT RNA splicing leading to an increasing expression of spliced mTERT isoforms playing a role of dominant-negative inhibitors of mTERT activity and therefore decreasing the levels of TERT activity in mouse epidermal keratinocytes. The overall effect of the DeltaNp63alpha overexpression resulted in decrease in telomerase activity and increase in replicative senescence observed in mouse keratinocytes. This dual molecular mechanism of telomerase regulation might underline the previously shown effect of DeltaNp63alpha on premature ageing phenotype.

Keywords: P63; SIRT1; Sp1; TERT; ageing; mouse; p53; senescence; splicing; transcription.

Figure 1.. ΔNp63α mediates the SIRT1 degradation and p53 deacetylation.

(A) The proteasome-dependent degradation of SIRT1. (B) The deacetylation of p53. (C) The protein complex formation between p53, SIRT1 and Sp1. Mice with heterozygous p63-/+ and ΔNp63αtransgenic expression were sources for epidermal keratinocytes [29,45]. Total lysates (2x10⁵ cells) were used for immunoblotting with indicated antibodies (dilutions: anti-ΔNp63, 1:500; anti-SIRT1, 1:300; anti-β-actin, 1:400; anti-p53, 1:500; anti-acetyl-p53, 1:400; anti-Sp1, 1:300). Cells were also treated with the proteasome inhibitor, MG-132 (20 μg/ml) for 24 h before lysis. For immuno-precipitation (IP) experiments, we used total lysates obtained from 1x10⁶ cells/500 μl and anti-p53 antibodies (10 μg/500μl). Blots were quantitatively scanned using the PhosphorImager and all of the data (mean +SD) were from at least three independent experiments.

Figure 2.. ShRNA silencing of ΔNp63-SIRT1-p53-Sp1 pathway.

Mouse epidermal keratinocytes (2x10⁵ cells) from p63-/+ (samples 1-5) or overexpressing ΔNp63α(samples 6-10) were treated with control media (samples 1 and 6), SIRT1 inhibitor (Sirtinol, 100 μg/ml for 24 h; samples 2 and 7), or transfected with the SIRT1 shRNA (samples 3 and 8), p53 shRNA (samples 4 and 9), and sh-Sp1 RNA (samples 5 and 10). (A) Immunoblotting with indicated antibodies (dilutions: anti-ΔNp63, 1:500; anti-SIRT1, 1:300; anti-Sp1, 1:300; anti-p53, 1:500; anti-acetyl-p53, 1:400; anti-β-actin, 1:400). The vertical lines separate data obtained from independent protein gels. (B) mTERT promoter luciferase reporter assay. Mouse keratinocytes (1.0 x 10⁵) were transfected with the pGL3-347-Luc plasmid (0.5 μg) or the pGL3 control plasmid (0.5 μg) by using FuGENE6 transfection reagent (Roche Diagnostics). 3 ng of the pRL-SV40 (Promega) was used as a normalization control. Measurements were performed by using the Dual Luciferase reporter assay system (Promega) and a BioOrbit 1251 luminometer. The activity of each TERT promoter fragment was expressed as a relative value. All of the data (mean +SD) were from at least three independent experiments.

Figure 3.. ΔNp63α modulates binding of Sp1 to Sp1 DNA-binding region by decreasing the SIRT1 protein levels and deacetylation of p53.

Chromatin immunoprecipitation assay (X-ChIP). Mouse epidermal keratinocytes (5x10⁷ cells) expressing heterozygous p63-/+ (samples 1-5) and overexpressing ΔNp63α(samples 6-10) were treated with control media (samples 1 and 6), SIRT1 inhibitor (Sirtinol, 100 μg/ml for 24 h; samples 2 and 7), SIRT1 shRNA (samples 3 and 8), p53 shRNA (samples 4 and 9), and shSp1 RNA (samples 5 and 10). The protein-DNA complexes were precipitated with a primary antibody for Sp1. As negative controls, we used immunoglobulins (IgG) from rabbit (IgG-R) or mouse (IgG-M) sera. The mTERT-derived Sp1 promoter region using the following primers: sense (SP1), 5'-CTCACTGTCTGTGCAACCACAGCAGCTG-3' (position-363), and antisense (AP2), 5'-AGAGCACCGCGGGGCAACGAGGAGCGCG-3' (position +143) giving raise to a 506 bp PCR product. The PCR products were run on 2% agarose gels and visualized by ethidium bromide staining.

Figure 4.. ΔNp63α increases levels of the mTERT-spliced isoforms via protein interaction with ABBP1.

Mouse epidermal keratinocytes (2x10⁶ cells) expressing heterozygous p63-/+ and ΔNp63α transgene. (A) Cells were tested for the levels of ΔNp63αand ABBP1 by immunoblotting and ABBP1ΔNp63αprotein complexes using immunoprecipitation (IP) with an antibody to ABBP1 followed by immunoblotting with an antibody to ΔNp63α. As a control, the protein level of β-actin was monitored. (B) Cells were examined for the expression of the mTERT and mTR transcripts using RT-PCR. GAPDH was used in RT-PCR assay, as a control. (C) The relative expression of TERT and TR was quantitatively analyzed and plotted as bars using the Microsoft Excel software. All of the data (mean +SD) were from at least three independent experiments. Samples: cells from p63-/+ mice, 1- TERT/GAPDH ratio; 2- TR/GAPDH ratio; cells from the ΔNp63αtransgenic mice, 3- TERT/GAPDH ratio; 4- TR/GAPDH ratio. PCR experiments with the 2164/ 2620 set of primers generated three products that represent the full-length TERT transcript (457 bp), the α-splice transcript (421 bp), and the β-splice transcript (275 bp). Sequence analysis revealed that the longer transcripts were full-length one and the shorter transcripts were α and β- spliced messages of mTERT.

Figure 5.. ΔNp63α overexpression modulated the overall telomerase activity and induced a S-β-gal activity.

The mouse keratinocytes from the p63-/+ mice (samples 1, 3, 5, 7, 9) and ΔNp63αtransgenic mice (samples 2, 4, 6, 8, 10) were treated with the control media (samples 1 and 2) or Sirtinol (100 μg/ml for 24h, samples 3 and 4) or transfected for 72h with shRNA against SIRT1 (samples 5 and 6), p53 (samples 7 and 8) and Sp1 (samples 9 and 10). (A) Telomerase activity. Telomerase activity was determined by the TRAP assay using 1 μg of protein extract obtained from 2x10⁵ cells. Quantitative analysis was done using Molecular Dynamics densitometer and ImageQuant software. The intensity of the positive control lane was taken as 100%. The experiment was repeated three times, and error bars represent mean ± S.D. (B) S-β-gal activity. The S-β-gal activitywas measured using a senescence kit.

Figure 6.. Schematic representation of regulation of TERT transcription and splicing by ΔNp63α.

(A) mTERT transcription. (B) mTERT splicing.