Mitogen-Activated Protein Kinase Cascade-Mediated Histone H3 Phosphorylation Is Critical for Telomerase Reverse Transcriptase Expression/Telomerase Activation Induced by Proliferation

Cells and reagents.

Normal human T lymphocytes were isolated from buffy coats of healthy individuals, and HDFs including lung fetal (LF1) (37) and dermal-derived fibroblasts (19) were kindly provided by J. Sedivy (Brown University) and Z. Yan (Karolinska Institutet). The above cells as well as Jurkat cells, a T-cell lymphoma line, were all maintained in RPMI 1640 medium (Life Technologies, Paisley, Scotland) supplemented with 10% of fetal calf serum (FCS), 2 mM l-glutamine, and antibiotics. Epidermal growth factor (EGF), concanavalin A (ConA), anisomycin, CHX, PD98095, 12-O-tetradecanoylphorbol-13-acetate (TPA), and TSA were purchased from Sigma, and H89 was from Alexis Biochemicals. Prior to the treatment of HDFs with the above reagents, the cells were cultured in 0.5% FCS-containing RPMI 1640 medium for 48 to 72 h to induce quiescence. T lymphocytes were incubated with 15 μg/ml of ConA to stimulate their proliferation or activation.

RNA extraction and RT-PCR.

Total cellular RNA was extracted using the ULTRASPEC-II RNA kit (Biotecx Laboratories, Houston, TX). cDNA was synthesized using random primers (N6) (Pharmacia, Uppsala, Sweden) and Moloney murine leukemia virus reverse transcriptase. The reverse transcription-PCR (RT-PCR) primers and conditions for hTERT mRNA were as described previously (32), and the PCR cycles were 32 and 37 for T or Jurkat cells and fibroblasts, respectively. β₂-microglobulin (β₂-M) expression was used as a control for RNA loading and RT efficiency and was amplified with its specific primers for 25 cycles. With the above amplification conditions, PCR for both hTERT and β₂-M mRNA was in a linear phase, which allowed a semiquantitative evaluation for the level of hTERT transcript, as shown in our previous study (38).

Chromatin immunoprecipitation (ChIP).

The ChIP assay was carried out according to the protocol from Upstate Biotechnology (Lake Placid, NY). All the ChIP reagents, including antibodies against phospho-ser10 H3 and acetylated (K14) H3, were from Upstate Biotechnology. Briefly, the cells with different treatments were cross-linked by incubating them in 1% (vol/vol) formaldehyde-containing medium for 7 to 8 min at 37°C and then sonicated (four times for 8 s each) to make soluble chromatin. With these conditions, the produced DNA fragments were between 200 and 800 bp (data not shown). The antibodies against phospho-ser10 H3 and acetylated (K14) H3 were used to precipitate DNA fragments bound by phosphorylated and acetylated histone H3, respectively. The protein-DNA complex was collected with protein A Sepharose beads, eluted, and reverse cross-linked. The samples were then extracted with phenol-chloroform and precipitated with ethanol. The recovered DNA was resuspended in double-distilled H₂O and used for the PCR amplification. The PCR primer (TERT-p) sequences for the hTERT promoter (see Fig. 2B) were 5′ CCA GGC CGG GCT CCC AGT GGA T 3′ (forward) and 5′ GGC TTC CCA CGT GCG CAG CAG GA 3′ (reverse), and for the downstream region of the hTERT gene (TERT-np) they were 5′ GCT TGC AGA GGT GGC TCT AA 3′ (forward) and 5′ GCT GTG GTT TGG GAG ACT AAA 3′ (reverse). The GAPDH gene was amplified with the primers 5′ AAA GGG CCC TGA CAA CTC TT 3′ (forward) and 5′ GGT GGT CCA GGG GTC TTA CT 3′ (reverse), which resulted in the production of a 117-bp DNA fragment.

Histone and nuclear protein extraction, Western blot, and immunofluorescence staining.

We isolated histone and nuclear proteins and performed Western blot and immunofluorescence according to the protocol described previously (15, 19). The specific primary antibodies against phospho-H3 (Upstate Biotechnology) and hTERT (Santa Cruz Biotechnology, Santa Cruz, CA) and secondary antibodies conjugated with horseradish peroxidase or fluorescein isothiocyanate (Dako Cytomation, Glostrup, Denmark) were used for the analyses.

Telomerase activity assay.

Telomerase activity was determined by using a TRAPEZE kit (Chemicon International, Temecula, CA) according to the protocol provided by the manufacturer. One microgram of protein was used for each reaction, and 31 PCR cycles were carried out. The telomeric repeat amplification protocol (TRAP) result was visualized by staining the PCR product with SYBR Green I (Roche Diagnostics Scandinavia AB, Stockholm, Sweden) after polyacrylamide gel electrophoresis gel separation.

Rapid accumulation of ser10-phosphorylated histone H3 in T cells treated with ConA.

It is unclear whether ConA-stimulated T-cell proliferation through the ERK-MAPK cascade would lead to H3 phosphorylation, as seen in fibroblasts at G₀/G₁ transition. Thus, we first examined the H3 phosphorylation status in these cells by using both Western blotting and immunofluorescence staining. The amount of phosphorylated H3 was negligible in untreated resting T cells, whereas it accumulated promptly following exposure of the cells to ConA, as shown by Western blot (Fig. 1A). The peak level was observed approximately 4 h post-ConA stimulation, and a significant decrease occurred at 8 h. The immunofluorescence result exhibited a similar pattern, with a weak staining of the ser10-phosphorylated H3 signal in the control cells but bright signals in the ConA-treated cells (Fig. 1B). Moreover, in these activated T cells, the phosphorylated H3 was not distributed evenly; rather, in many foci scattered throughout the nuclei, there was a pattern in accordance with that observed in fibroblasts treated with growth factors (4) (Fig. 1B).

Accumulation of histone H3 ser10 phosphorylation at the hTERT promoter preceding hTERT mRNA induction and telomerase activation in ConA-treated T cells.

Previous studies have shown that activated T cells undergo up-regulation of hTERT mRNA expression and of telomerase activity (3, 16, 19, 39). Consistent with earlier reports, we observed robust increases in hTERT expression in normal T cells treated with ConA (Fig. 2A). Enhanced levels of hTERT mRNA readily occurred 2 to 3 h after ConA exposure, and a maximal induction was seen at 16 to 24 h, which was concomitant with up-regulation of hTERT protein expression and of telomerase activity (Fig. 2A).

Having demonstrated the sequential onset of histone H3 ser10 phosphorylation and hTERT induction in activated T cells, we examined whether histone H3 at the hTERT locus was specifically targeted for ser10 phosphorylation. For this purpose, ChIP was performed and the primers (TERT-p) spanning the proximal promoter (Fig. 2B) were used for PCR amplification of DNA fragments precipitated by an antibody against ser10-phosphorylated H3. In resting T cells, there was no detectable histone H3 ser10 phosphorylation associated with the hTERT promoter, whereas ConA treatment within 30 min led to the accumulation of phosphorylated H3 at this promoter, and a maximal level was reached after 1 to 3 h, when hTERT mRNA was starting to appear (Fig. 2B). Thereafter, the H3 phosphorylation on the hTERT promoter decreased but persisted. To determine whether phosphorylation of H3 ser10 spreads throughout the hTERT locus, the same set of precipitated DNA was further analyzed for the presence of the downstream sequences of the hTERT gene by using another pair of PCR primers (TERT-np) indicated in Fig. 2B. These primers did not lead to detectable PCR signals (Fig. 2B), which indicate that H3 ser10 phosphorylation is likely limited to the hTERT promoter region. T lymphocytes from more than 10 healthy individuals were analyzed, and similar results were obtained. No-antibody controls were always performed to exclude nonspecific bindings (data not shown).

One of the important characteristics of H3 ser10 phosphorylation is that it is especially susceptible to hyperacetylation at lys14, because of a stronger preference of histone acetyltransferases (HATs) for ser10-phosphorylated H3 than for the unmodified form (6, 8). To see whether this was true for the hTERT chromatin in activated T cells, ChIP was performed with the antibody against lys14-acetylated histone H3. As shown in Fig. 2B, H3 lys14 acetylation concomitantly occurred at the same region of the hTERT promoter with slightly different kinetics, including persistent increases up to 16 h.

Defective induction of the hTERT expression by inhibiting H3 ser10 phosphorylation.

Having shown that the onset of H3 ser10 phosphorylation at the hTERT promoter was followed by transcriptional activation of this gene and induction of telomerase activity once T cells were entering the cell cycle in response to ConA stimulation, we sought to address whether these two events are causally linked by interfering with H3 ser10 phosphorylation in T cells. First, we incubated T cells with ConA in the presence of 10 μM H89, a compound that specifically inhibits MSK activities at this concentration, thereby abrogating histone H3 ser10 phosphorylation (35). ConA-mediated accumulation of H3 ser10 phosphorylation at the hTERT promoter and induction of hTERT mRNA/telomerase activity was significantly reduced by pretreatment of the cells with H89 (Fig. 2C). Furthermore, lys14 acetylation of H3 was also abrogated, likely secondary to diminished recruitment of HATs (Fig. 2C). Secondly, PD98095, a specific ERK inhibitor that blocks the MAPK signaling pathway (35), when incubated with T cells prior to the addition of ConA similarly led to a reduction in both hTERT promoter-associated H3 phosphorylation/acetylation and hTERT mRNA/telomerase expression (Fig. 2C). The above results support a mechanistic link between histone H3 phosphorylation and transcriptional hTERT activation in proliferating T cells.

Concomitant H3 ser10 phosphorylation at the hTERT promoter and up-regulation of hTERT expression in serum-starved quiescent malignant T cells at reentry into cell cycle.

Holt et al. and others have demonstrated that immortalized and tumor cells exhibit growth-regulated telomerase/hTERT expression in a similar manner (13, 17). Serum withdrawal and contact inhibition induce a G₀ quiescence of these cells coupled with transcriptional repression of hTERT and diminished telomerase activity; once the cells reenter the cell cycle pool, a rapid recovery of hTERT and telomerase expression occurs. To determine a potential association between H3 phosphorylation and reversible hTERT expression in malignant cells, T-cell lymphoma-derived Jurkat cells, after 72 h of serum depletion, were re-fed with 20% FCS and then analyzed for hTERT mRNA expression, telomerase activity, and H3 phosphorylation at the hTERT promoter. As depicted in Fig. 3, the result clearly showed a concomitant accumulation of the hTERT promoter-associated H3 phosphorylation and hTERT mRNA/telomerase induction in the treated Jurkat cells. Furthermore, significantly increased H3 lys14 acetylation was simultaneously identified at the hTERT promoter in these cells. This result is almost identical to that seen in ConA-treated T cells, which suggests that the H3 phosphorylation signaling governs the proliferation-regulated telomerase activity in both normal and malignant T cells.

Transcriptional activation of the hTERT gene in normal fibroblasts by histone H3 ser10 phosphorylation modulators.

It has been well established that H3 ser10 phosphorylation is induced in normal fibroblasts through the MAPK cascade by either mitogen-stimulated ERK or stress-activated p38 signaling (10). Moreover, we previously observed induction of hTERT mRNA in HDFs treated with CHX (data not shown). Given a recent report that normal human fibroblasts transiently express hTERT mRNA in response to mitogen stimulation (31), we hypothesized that the H3 phosphorylation event is likely responsible for transcriptional regulation of the hTERT gene in this type of cell. To test this, dermal and lung HDFs under serum starvation for 48 to 72 h were exposed to different stimuli known to result in H3 phosphorylation and then analyzed for hTERT mRNA expression. As hypothesized, but still surprisingly, all the tested reagents, including EGF, 20% FCS, TPA, and the protein synthesis inhibitors anisomycin and CHX, induced detectable hTERT mRNA expression in two different strains of HDFs (Fig. 4A and data not shown). The induction of hTERT expression by these stimuli could be efficiently abolished in the presence of H89 (Fig. 4C). Compared to activated T cells, the abundance of hTERT transcripts in the treated HDFs was around 50-fold lower. Telomerase activity was undetectable in the FCS-treated cells using TRAP (Fig. 4C), probably explained by a technical limitation (31). The ChIP assay revealed the occupancy of phosphorylated ser10 at the hTERT promoter region in the treated but not control HDFs (Fig. 4B), which occurred prior to hTERT mRNA expression. However, H3 lys14 acetylation at the hTERT promoter was absent in both treated and untreated cells, as shown in Fig. 4B, which is in striking contrast to that seen in activated T cells. Taken together, histone H3 phosphorylation at ser10 via the MAPK cascade is a critical event that triggers transient hTERT transcription in HDFs.

Synergistic trans-activation of the hTERT gene in normal human fibroblasts by histone H3 phosphorylation and acetylation.

We and others have previously shown that inhibition of HDACs by TSA leads to the transcriptional activation of the hTERT gene in normal and malignant cells (9, 11, 19, 25, 36, 39). In these studies, however, TSA-treated cells were exclusively maintained in 10% FCS, and it was unclear whether the observed hTERT induction was totally attributed to the HDAC inhibition. Moreover, we indeed documented a superinduction of hTERT mRNA in both T cells and HDFs treated with TSA in the presence of CHX (19). Given the present finding that CHX or serum alone was capable of stimulating hTERT expression through H3 phosphorylation, it is likely that histone H3 phosphorylation and acetylation cooperate to synergistically trans-activate the hTERT gene. To address this issue, we incubated 48-h serum-starved HDFs with either 20% FCS, anisomycin, TSA alone, or a combination of FCS or anisomycin with TSA for 16 h. Remarkably, TSA only induced a weak expression of hTERT mRNA in the absence of serum, with a level comparable to that seen in the cells treated with FCS or anisomycin alone. The combined treatment of the cells with TSA and serum or anisomycin led to much higher levels of hTERT mRNA than those in the cells incubated with any single reagent (Fig. 4C). Moreover, telomerase activity was only detectable in the cells treated with TSA plus FCS (Fig. 4C). Thus, both histone H3 phosphorylation and acetylation are required for the optimal induction of hTERT expression and telomerase activity. This set of data also suggest that TSA-induced high levels of hTERT mRNA observed in earlier studies virtually reflect a combined trans-activating effect of histone H3 phosphorylation and acetylation on the hTERT gene.