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. 2006 Sep 30;353(2):247-57.
doi: 10.1016/j.virol.2006.06.005. Epub 2006 Jul 14.

Calcium-dependent enhancement of transcription of p300 by human T-lymphotropic type 1 p12I

Amrithraj M Nair  1 , Bindhu Michael, Antara Datta, Soledad Fernandez, Michael D Lairmore
Affiliations

Calcium-dependent enhancement of transcription of p300 by human T-lymphotropic type 1 p12I

Amrithraj M Nair et al. Virology. .

Abstract

Human T-lymphotropic virus type 1 (HTLV-1) p12I localizes to the endoplasmic reticulum and Golgi causing sustained release of calcium, T cell activation, and enhanced expression of several calcium-regulated genes. In recent microarray studies, p300 mRNA was increased in T cells expressing p12I. The co-activator p300 is a key regulator of cellular and viral transcription; however, factors that influence its transcriptional regulation are less well studied. We hypothesized that the transcription of p300 is calcium dependent and that sustained low magnitude increases in intracellular calcium may enhance the transcription of p300. Herein, we report enhanced expression of p300 in T cells by p12I in a calcium-dependent, but calcineurin-independent manner. Sustained low magnitude calcium release induced by ionomycin in T cells was sufficient to increased mRNA and protein levels of p300 resulting in enhanced transcription from a p300-dependent promoter. Promoter analysis of the p300 gene was used to predict calcium-responsive transcription factor binding sites. Using mutant forms of p12I, we demonstrate that ER localization of the viral protein is required to increase p300. In addition, p12I reversed the repression of HTLV-1 LTR-driven transcription by HTLV-1 p30II, a p300-binding protein. HTLV-1 p12I-mediated enhancement of p300 expression represents a novel mechanism of regulation of cellular gene expression by viral proteins. By targeting a ubiquitous second messenger such as calcium, HTLV-1 p12I may regulate the expression of the cellular transcriptional co-activator p300 to modulate viral gene expression and promote lymphocyte survival.

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Figures

Fig. 1
Fig. 1
HTLV-1 p12I enhances p300 protein levels in Jurkat T lymphocytes. (A) Jurkat T lymphocytes (2 × 106) were spin infected with lentiviral vectors expressing p12I at an MOI of 5. Total protein was extracted and western immunoblot analysis for the detection of p300 was performed. Jurkat T cells expressing p12I demonstrated increased protein levels of p300. (B) Densitometric analysis of radiograph was performed using Gel pro analyzer software and normalized to GapDH. Jurkat T cells expressing p12I showed ~2.1-fold increased expression of p300. Statistical analysis was performed using Student’s t test, P < 0.05.
Fig. 2
Fig. 2
Sustained low magnitude increase in intracellular calcium concentration enhances transcription of p300. (A) Jurkat T cells (1 × 106 per ml) were stimulated with varying concentrations of ionomycin for 36 h. Total cellular RNA was extracted and semi-quantitative RT-PCR was performed to identify the mRNA levels of p300. There was a dose-dependent increase in p300 expression in Jurkat T cells stimulated with low concentrations of ionomycin (25, 50, and 100 nM). (B) Densitometric analysis was performed using alpha imager software and normalized to GAPDH. Dose-dependent increases (up to 2.4-fold) in p300 mRNA were observed in Jurkat T cells stimulated with 100 nM ionomycin. Statistical analysis was performed using Student’s t test, P < 0.05.
Fig. 3
Fig. 3
Enhanced mRNA levels of p300 correlates with increased protein levels of p300. (A) Jurkat T cells (1 × 106 per ml) were stimulated with varying concentrations of ionomycin for 60 h. Total protein was extracted and western immunoblot analysis for the detection of p300 protein. There was a dose-dependent increase in p300 expression in Jurkat T cells stimulated with low concentrations of ionomycin (25, 50, and 100 nM). (B) Densitometric analysis of radiograph was performed using Gel pro analyzer software and normalized to GAPDH. Dose-dependent increases (up to 2.5-fold) of p300 were observed in Jurkat T cells stimulated with 100 nM ionomycin. The protein levels were in parallel with mRNA levels observed at similar concentrations of ionomycin. Statistical analysis was performed using Student’s t test, P < 0.05.
Fig. 4
Fig. 4
Ionomycin-mediated increase in p300 influences transcription. (A) 2 × 106 Jurkat T cells were transfected with 5xGT-Luc and pM-VP16. The transfected cells were maintained in cRPMI medium containing increasing concentrations of ionomycin. The luciferase activity was tested 60 h post-transfection. A dose-dependent increase in VP16-mediated p300-dependent luciferase activity was observed with increasing concentrations of ionomycin. (B) The increase in VP16-mediated luciferase activity was confirmed to be p300 dependent by transfecting wild-type and mutants of adenoviral E1A protein. Two million Jurkat T cells were transfected with 5xGT-Luc, pM-VP16, and increasing concentrations of wild-type or mutants of adenoviral E1A protein. The transfected cells were incubated in cRPMI medium supplemented with 100 nM ionomycin. ΔCR1 mutant of E1A is incapable of binding p300 whereas ΔCR2 mutant is capable of binding p300 but not retinoblastoma protein. VP16-mediated p300-dependent luciferase activity was inhibited in a dose-dependent fashion in the presence of wild-type as well as ΔCR2 mutant of E1A. No effect on luciferase activity occurred in the presence of ΔCR1 mutant of E1A.
Fig. 5
Fig. 5
Enhanced expression of p300 is calcium dependent, but calcineurin independent. (A) Jurkat T cells (2 × 106) were transfected with 5xGT-Luc and pM-VP16 and incubated in cRPMI medium supplemented with 100 nM ionomycin. The cells were treated with increasing concentrations of BAPTA-AM or CsA. A dose-dependent reduction in VP16-mediated p300-dependent luciferase activity was noticed in the presence of BAPTA-AM whereas no significant difference in luciferase activity was observed in the presence of CsA. (B) Jurkat T cells (2 × 106) were transfected with 5xGT-Luc, pM-VP16, and pME-p12I and incubated in cRPMI medium. The cells were treated with increasing concentrations of BAPTA-AM or CsA. A dose-dependent reduction in VP16-mediated p300-dependent luciferase activity was evident in the presence of BAPTA-AM, whereas no significant difference in luciferase activity was observed in the presence of CsA.
Fig. 6
Fig. 6
Accumulation of p12I to ER is required for enhanced expression of p300. Jurkat T cells (2 × 106) were transfected with 5xGT-Luc, pM-VP16, along with either of the following plasmids—pME control vector, pME-p12I, pME-15-47, or pME-15-47KKLL, and then incubated in cRPMI medium. pME-15-47, which accumulates in the nucleus of transfected cells (Ding et al., 2002), exhibits a marked reduction in the VP16-mediated p300-dependent luciferase activity. The activity was partially restored by redirecting the protein to the ER.
Fig. 7
Fig. 7
HTLV-1 p12I partially inhibits the transcriptional repression of p30II on HTLV-1 LTR. (A) Jurkat T cells (2 × 106) were transfected with pTRE-Luc, pME-p30II and increasing concentrations of pME-p12I and then incubated in cRPMI medium. A dose-dependent increase in luciferase activity was observed with increasing concentrations of p12I. (B) To confirm that the increased luciferase activity from pTRE-Luc was dependent on increased levels of p300, we transfected adenoviral E1A protein with the above-described plasmids. These data indicated a marked reduction in luciferase activity in the presence of E1A.
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