A balanced transcription between telomerase and the telomeric DNA-binding proteins TRF1, TRF2 and Pot1 in resting, activated, HTLV-1-transformed and Tax-expressing human T lymphocytes

Abstract

Background: The functional state of human telomeres is controlled by telomerase and by a protein complex named shelterin, including the telomeric DNA-binding proteins TRF1, TRF2 and Pot1 involved in telomere capping functions. The expression of hTERT, encoding the catalytic subunit of telomerase, plays a crucial role in the control of lymphocyte proliferation by maintaining telomere homeostasis. It has been previously found that hTERT activity is down-regulated by the human T cell leukaemia virus type 1 (HTLV-1) Tax protein in HTLV-1 transformed T lymphocytes. In this study, we have examined the effects of Tax expression on the transcriptional profile of telomerase and of shelterin in human T lymphocytes.

Results: We first provide evidence that the up-regulation of hTERT transcription in activated CD4+ T lymphocytes is associated with a down-regulation of that of TERF1, TERF2 and POT1 genes. Next, the down-regulation of hTERT transcription by Tax in HTLV-1 transformed or in Tax-expressing T lymphocytes is found to correlate with a significant increase of TRF2 and/or Pot1 mRNAs. Finally, ectopic expression of hTERT in one HTLV-1 T cell line induces a marked decrease in the transcription of the POT1 gene. Collectively, these observations predict that the increased transcriptional expression of shelterin genes is minimizing the impact on telomere instability induced by the down-regulation of hTERT by Tax.

Conclusion: These findings support the notion that Tax, telomerase and shelterin play a critical role in the proliferation of HTLV-1 transformed T lymphocytes.

Figure 1

Real time quantitative PCR (qPCR) analysis of hTERT expression in HTLV-1-infected and uninfected T cells. A) ATL cell lines (HTLV-1 infected but negative for Tax expression); B) In vitro transformed cell lines (HTLV-1 infected and positive for Tax expression); C) Jurkat T-cell clones positive for Tax expression (E12, C11 and C50); D) Uninfected primary CD4 T lymphocytes either activated or not (resting); T-cell line (DCH-4) immortalized with a lentivirus vector encoding a Tax-YFP fusion protein; Infected T lymphocytes isolated from patient suffering from TSP/HAM. Cytoplasmic RNAs were isolated, reverse transcribed and cDNA were analyzed by qPCR using primers for hTERT and PBGD. Results are expressed as indicated in legend of table 1. Standard deviations are from at least three determinations performed in duplicate.

Figure 2

Analysis of TERF1, TERF2 and POT1 gene expression in T-cell lines expressing Tax. Cytoplasmic RNAs were isolated, reverse transcribed and cDNA were analyzed by qPCR using specific primers. Results are expressed as the amount of indicated mRNA relative to PBGD. Each quantification was compared to that obtained with Jurkat cells, referred as 1. Standard deviations are from at least three determinations performed in duplicate.

Figure 3

Characterization of C91PL cells over-expressing hTERT gene. Cells were transduced with a lentivirus vector encoding GFP with or without hTERT A) Flow cytometry determination of the expression of GFP and p19_gag. The percentage of cells in each quadrant is indicated. B) Analysis of hTERT, and of telomere protein gene expression in hTERT/GFP transduced C91PL cells (grey bars) and in control GFP transduced C91PL (white bars) was performed and quantified as in Figure 2.

Figure 4

A model for hTERT and shelterin gene expression in T lymphocytes. In the upper panel, CD4+ T lymphocytes were either resting or activated with a cocktail of anti CD3/anti CD28 antibodies. In the lower panel, three types of Tax-expressing T lymphocytes were represented. The model is based on the telomeric transcriptional profile defined as a balance between hTERT on one hand, and shelterin (POT1, TERF1 and TERF2) gene expression on the other hand.