Involvement of TORC2, a CREB co-activator, in the in vivo-specific transcriptional control of HTLV-1
© Jiang et al; licensee BioMed Central Ltd. 2009
Received: 31 March 2009
Accepted: 11 August 2009
Published: 11 August 2009
Human T-cell leukemia virus type 1 (HTLV-1) causes adult T -cell leukemia (ATL) but the expression of HTLV-1 is strongly suppressed in the peripheral blood of infected people. However, such suppression, which may explain the long latency in the development of ATL, is readily reversible, and viral expression resumes quickly with ex vivo culture of infected T -cells. To investigate the mechanism of in vivo -specific transcriptional suppression, we established a mouse model in which mice were intraperitoneally administered syngeneic EL4 T -lymphoma cells transduced with a recombinant retrovirus expressing a GFP-Tax fusion protein, Gax, under the control of the HTLV-1 enhancer (EL4-Gax).
Gax gene transcription was silenced in vivo but quickly up-regulated in ex vivo culture. Analysis of integrated Gax reporter gene demonstrated that neither CpG methylation of the promoter DNA nor histone modification was associated with the reversible suppression. ChIP-analysis of LTR under suppression revealed reduced promoter binding of TFIIB and Pol-II, but no change in the binding of CREB or CBP/p300 to the viral enhancer sequence. However, the expression of TORC2, a co-activator of CREB, decreased substantially in the EL4-Gax cells in vivo, and this returned to normal levels in ex vivo culture. The reduced expression of TORC2 was associated with translocation from the nucleus to the cytoplasm. A knock-down experiment with siRNA confirmed that TORC2 was the major functional protein of the three TORC-family proteins (TORC1, 2, 3) in EL4-Gax cells.
These results suggest that the TORC2 may play an important role in the in vivo -specific transcriptional control of HTLV-1. This study provides a new model for the reversible mechanism that suppresses HTLV-1 expression in vivo without the DNA methylation or hypoacetylated histones that is observed in the primary cells of most HTLV-1 -infected carriers and a substantial number of ATL cases.
Human T-cell leukemia virus type 1 (HTLV-1), a life-long persistent CD4+T -lymphotropic retrovirus, causes an aggressive mature T -cell malignancy termed "adult T-cell leukaemia" (ATL) [1, 2] and an inflammatory disease of the central nervous system known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) [3, 4]. HTLV-1 infects 10–20 million people worldwide; 2–3% of infected individuals develop ATL, and a further 0.25–3% develop HAM/TSP.
Tax protein, encoded by the HTLV-1 pX region , is closely associated with the development of these diseases by triggering in a pleiotropic manner viral transcription [6–9] and by deregulating the expression of cellular genes [10, 11]. However, the expression of viral genes, including Tax, is almost completely suppressed in the peripheral blood of infected people . This may explain the long latency in the development of ATL and other HTLV-1 related diseases. It has been assumed that there is a specific mechanism for this in vivo -specific suppression, because gene expression of HTLV-1 in peripheral blood cells from infected people, with the exception of two-thirds of ATL patients , resumes quickly when the infected cells are moved to in vitro conditions, without any stimulation . Such reversible control of the gene expression should benefit HTLV-1 because Tax protein harbors several strong epitopes for cytotoxic T -cells . Thus, the transient expression of Tax is essential for the propagation of viral infection and/or infected cells under strict surveillance by the host immune system , the efficiency of which may vary among individuals . In contrast, evading the suppressed state leading to the reactivation of viral gene expression may be a key step in the development of HTLV-1 associated diseases.
DNA methylation accumulated in HTLV-1 5'-LTR silences viral gene transcription in leukemic cells [13, 17]. However, further analysis revealed that viral gene transcription is silenced in most carriers, and in about 20% of ATL cases, despite no or only partial methylation of the 5'-LTR . Furthermore, in the case of ATL, transcriptional silencing was observed regardless of the acetylation of histones H3 and H4, markers of active transcription, in the 5'-LTR . Thus, a reversible mechanism that suppresses viral gene transcription without DNA methylation or hypoacetylated histones in 5'-LTR has been postulated but remains to be clarified.
As observed in other retroviruses, transcription of HTLV-1 is under the control of an enhancer/promoter located in its LTR. The U3 region in the HTLV-1 LTR harbors an enhancer element consisting of three 21 -bp direct repeats that are activated exclusively in the presence of Tax. In the center of each 21 -bp enhancer sequence there are Tax-responsive elements (TRE) or viral cyclic AMP response elements (CRE) [9, 19], to which a variety of enhancer binding proteins, including members of the CREB/ATF family, bind, with or without Tax protein . Among them, CREB has been implicated as the primary player in both basal and Tax-activated HTLV-1 transcription [21, 22]. CREB stimulates HTLV-1 viral transcription by binding to the viral CRE and interacts with Tax, which is also associated with the GC-rich sequences immediately flanking the viral CRE, and recruits CBP/p300 to form a Tax/CREB/CBP/p300/DNA quaternary complex [23, 24].
In contrast, proteins belonging to another recently identified family of CREB cofactors, termed "transducers of regulated CREB activity" (TORCs) [25, 26] have been suggested to enhance HTLV-1 transcription, alone or in combination with Tax, in a CREB-dependent manner in vitro [27, 28]. TORCs were originally found in the CREB -dependent, but pCREB(phosphor-CREB)-independent, activation of cellular genes . The recruitment of TORCs to the promoter does not appear to modulate CREB DNA binding activity, but rather enhances the interaction of CREB with the TAFII130 component of TFIID . Among three members of the TORC-family protein, the activity of TORC2 is tightly regulated by phosphorylation at Ser 171, which promotes the export of the protein into the cytoplasm and its degradation .
To gain insights into the mechanism of this in vivo -specific transcriptional suppression, we established a mouse model in which mice were intraperitoneally administered syngeneic EL4 T -lymphoma cells transduced with a recombinant retrovirus expressing GFP-Tax fusion protein under the control of the HTLV-1 enhancer (EL4-Gax). Gax protein retains the properties of Tax as a transcriptional transactivator and also as an antigen, providing epitopes for CTL . Furthermore, Gax expression in EL4-Gax cells is suppressed in vivo but is quickly up-regulated in ex vivo culture, thus modeling the activity of HTLV-1 -infected cells in asymptomatic carriers . The present study analyzed epigenetic modifications and factors in the integrated HTLV-1 promoter/enhancer in EL4-Gax cells in vivo as well as ex vivo. We found that reduced expression of TORC2, but not of CREB or its phosphorylated form (pCREB), was responsible for the suppression of viral gene expression in vivo.
Gax expression in vivowas suppressed at the level of transcription
Immunoblot analysis confirmed that in vivo protein expression of Gax was abolished in cells, and the expression was reactivated in ex vivo culture (Fig. 1C, Gax). The reduction of Gax protein is not simply due to a severe growth condition inducing cell death since the proteolytic cleavage of poly(ADP-ribose) polymerase, which is known to be a sensitive marker of apoptosis  and necrosis , was not observed (Fig. 1C, PARP).
The expression of Gax mRNA was analyzed using quantitative reverse transcription polymerase chain reaction (RT-PCR) to determine whether the suppression of Tax expression was controlled at the level of transcription (Fig. 1D). The transcriptional suppression in vivo is specific for the Gax gene because no suppression was observed in the expression of cellular genes such as EF1-a, GAPDH, β-actin, 18S-ribosomal RNA and endogenous retrovirus. On the contrary, gene expression of CD4 was upregulated in vivo, while it was silenced in EL4-Gax cells grown in vitro. Real-time PCR analysis of Gax cDNA prepared from total RNA in EL4-Gax cells demonstrated that the expression of Gax mRNA was reduced in vivo and recovered after ex vivo culturing to a level comparable with that before peritoneal inoculation of the cells. Thus, Gax expression in vivo was suppressed transcriptionally.
CpG methylation is not associated with the suppression of the Gaxgene
Binding of CREB and pCREB to the HTLV-1 enhancer
CREB has been implicated as the primary player in both basal and Tax-activated HTLV-1 transcription . CRE-dependent transcription is generally explained by the recruitment of histone acetylating proteins, CBP/p300, to the enhancer region of genes through an interaction with CREB protein, which binds to the CRE sequence, and acetylation of histones, opening the chromatin and providing access to basic transcriptional factors including RNA polymerase. Thus, since the reduction of recruitment of either factor to the promoter region might result in the suppression of transcription, a chromatin immunoprecipitation (ChIP) assay was used to analyze the binding of these factors to the U3 region of the 5'-LTR.
As Gax is expressed in EL4-Gax cells in vitro, it was of interest whether Gax is associated with the enhancer DNA. ChIP assay was performed with antibody against GFP, which recognizes the Gax protein. Consistent with the protein expression, Gax was associated with the enhancer DNA in EL4-Gax cells grown in vitro (lane 15) but not in in vivo cells (lane 16), where the expression of Gax protein was decreased. Tax recruits CBP to the HTLV-1 enhancer by tethering with CREB at the CRE sequence; however, the enhancer binding of CBP remained unchanged in the absence of Tax (Fig 3B, lane 11, 12). In this respect, it is noteworthy that phosphorylation of CREB protein, which leads to a complex formation between CREB and CBP, is increased in EL4-Gax cells grown in vivo (Fig 3C). Thus, pCREB seems to be involved in the sustained enhancer binding of CBP in the absence of Tax.
Although the amount of pCREB was increased in EL4-Gax cells grown in vivo, no significant difference was observed in the amount of pCREB binding to the enhancer DNA in cells either in vivo or under in vitro culture conditions. Since pCREB has been demonstrated to preferentially bind to the enhancer sequence of HTLV-1 in a complex with Tax , Tax might have selectively incorporated pCREB in the complex.
Modifications of histones H3 and H4
Activated transcription is associated with histone acetylation in the chromatin of the respective genes; thus, histone acetylation at the promoter region of the provirus was analyzed using a ChIP assay, with antibodies against acetylated histones H3 and H4. Unexpectedly, this analysis revealed that histones at the LTR of the HTLV-1 provirus were equally acetylated in EL4-Gax cells (Fig. 3D, lanes 11–14), either in vivo (b) and in vitro (a), whereas RNA expression from the HTLV-1 promoter in these cells differed substantially (Fig. 1). Methylation of histone H3 at the lysine residue was also analyzed, because this methylation is closely linked with transcriptional activation. However, no change was observed in the methylation of histone H3 in the promoter region of the provirus (Fig. 3D, lanes 15–16). Thus, the in vivo -specific transcriptional repression of the HTLV-1 promoter was not associated with an altered level of chromatin modification. These results are consistent with the previous finding that gene silencing of HTLV-1 in an ATL case was observed regardless of hyperacetylation of histones H3 and H4 in the promoter .
Recruitment of basal transcription machinery to the proviral promoter
The recruitment of RNA polymerase II (Pol-II) and TFIIB, a key general transcription factor in forming and stabilizing the early initiation complex , was analyzed to determine whether suppression was present in the formation of the transcription initiation complex in the 5'-LTR promoter. Although binding of TFIIB (Fig. 3D, lanes 9, 10) and Pol-II (Fig. 3D, lanes 7,8) to the constitutive promoter of the EF-1a gene as positive controls was observed equally in the ChIP assay, a substantial reduction in the binding of these factors to the provirus promoter sequence was detected under condition of suppressed Gax expression in comparison with EL4 -Gax cells in the in vitro culture (37 ± 5% for TFIIB and 47 ± 5% for Pol-II). These results suggest that the loss of recruitment of basal transcription factors is associated, at least in part, with the suppression of Gax expression in vivo, regardless of the constitutive binding of CREB-CBP/p300 to the enhancer DNA.
Expression of TORC1 and TORC2 is repressed in EL4-Gax cells in vivo
In addition to the CREB-CBP/p300 pathway, another family of CREB cofactors, TORCs, has been recently identified as activating CREB-dependent, but pCREB-independent, transcription [25, 26], including that of HTLV-1, with or without Tax [27, 28]. Thus, we next examined the involvement of TORCs in transcriptional control in vivo.
When expression of the TORC proteins in EL4-Gax cells grown in vitro, in vivo, and ex vivo was compared, the amounts of TORC1 and TORC2 were reduced significantly under in vivo growth conditions, and they recovered to some extent upon their ex vivo culturing (Fig. 4A, B). In contrast, the expression of TORC3 increased little, if any, in in vivo or ex vivo conditions (Fig. 4C). Because a previous report demonstrated that the suppression of TORC1, TORC2 or TORC3 expression by siRNA resulted in reduced transcription from the HTLV-1 LTR , it seems likely that reduced expression of TORC1 and/or TORC2 is involved in the suppression of Gax gene expression in in vivo conditions. It is noteworthy that the reduction of the unphosphorylated TORC2 protein was more significant than that of the phosphorylated form, because the former is an active form of TORC2 retained in the nucleus.
Binding of TORC proteins to the HTLV-1 enhancer
As TORC proteins are recruited to enhancer DNA in combination with CREB protein to activate CRE-dependent transcription of HTLV-1, a ChIP assay was used to analyze whether these proteins are associated with the U3 region of the 5'-LTR in EL4-Gax cells grown in vitro and in vivo. As shown in Figure 4E, recruitment of TORC2 and TORC3 proteins to the enhancer sequence was demonstrated in EL4-Gax cells in vitro and both of the bindings were substantially reduced in in vivo cells, where little or no binding of TORC1 to enhancer DNA was observed. As judged by densitometoric analysis, TORC2 appears to be the main TORC protein that is associated with the enhancer sequence of HTLV-1 in EL4-Gax cells, and the reduced enhancer binding of TORC2 in cells grown in vivo was in good agreement with the transcriptional suppression of Gax in vivo.
TORC2 is primarily involved in the transcriptional control of the HTLV-1 promoter in EL4-Gax cells
Nuclear expression of TORC2 protein was reduced in EL4-Gax cells in vivo
Phosphorylation of TORC2 protein by cellular kinases, such as AMPK (AMP-activated protein kinase) kinase, induces the translocation of TORC2 from the nucleus to the cytoplasm, thereby suppressing CREB-dependent transcription. In fact, the unphosphorylated form of the TORC2 protein in vivo appeared to be reduced more significantly than the phosphorylated form, when compared in vitro or ex vivo by Western blotting (Fig. 4B). Therefore, activity of AMPK was examined by measuring the phosphorylation at Thr172, which is required for AMPK activation . The results shown in Figure 6C clearly demonstrate the activation of AMPK activity in EL4-Gax cells in vivo and its reduction in cells cultured ex vivo.
Because cytoplasmic retention of TORC2 results in its degradation by proteasomes, it appears that some in vivo -specific cellular signal(s) may induce the cytoplasmic translocation, and thereby the degradation of the TORC2 protein, resulting in the suppression of HTLV-1 transcription in EL4-Gax cells.
Tax protein plays a key role in the development of ATL and other HTLV-1-related diseases through pleiotropic actions, that include transactivation of the NF-κB , CREB [22, 21, 24], and SRF pathways [37, 38]; transrepression of lck , p18 , DNA polymerase β , and histone gene transcription ; and functional inactivation of p53  and MAD1 . However, the expression of viral genes, including Tax, is strongly suppressed in the peripheral blood of patients infected with HTLV-1 , mainly because the Tax protein harbors several strong epitopes for cytotoxic T -cells . Such suppression is readily reversible, because gene expression of HTLV-1 in peripheral blood cells from infected people, with the exception of two-thirds of ATL patients , quickly resumes when the infected cells are moved to in vitro conditions, without any additional stimulation . This indicates that the transient expression of Tax is essential for the propagation of viral infection, and/or the infected cells are under strict surveillance by the host immune system .
DNA methylation is a host defense mechanism for inactivating transposable elements, such as retroviruses, to inhibit their transcription and their generation of new viruses. Thus, the transcriptional silencing of the Tax gene has been studied extensively in terms of DNA methylation of the 5'-LTR, which is the promoter of viral transcription [45, 17, 13, 18]. In ATL-derived cell lines, complete- or hypermethylation of CpG DNA in 5'-LTR is closely associated with the suppression of viral gene transcription, and partial methylation is not sufficient to silence transcription of the Tax gene [13, 17]. In contrast, the 5'-LTR of the provirus genome was found to be unmethylated or partially methylated in about 70% of ATL leukemic cells with intact provirus sequences , whereas Tax gene expression was mostly silenced in vivo. Furthermore, lack of DNA methylation in the 5'-LTR was more frequently observed in HTLV-1 carriers than in primary ATL cells . These data indicate that a mechanism other than DNA methylation is involved in the silencing of Tax gene transcription in the peripheral blood of HTLV-1 -infected individuals in vivo. The mouse model system studied in this report models gene control in HTLV-1 -infected cells in ATL patients and carriers, where reversible silencing of Tax gene transcription is observed, without extensive DNA methylation of the 5'-LTR.
The transcription of HTLV-1 is controlled by CREB binding to the enhancer sequence, which consists of three 21-bp direct repeats containing CREs in the middle. The viral transactivator, Tax, interacts with both CREB and CBP/p300 in a manner independent of the phosphorylation of CREB and activates the HTLV-1 enhancer. In the current study, approximately equivalent binding of CREB and CBP on the enhancer DNA was observed in the Gax expressing EL4-Gax cells cultured in vitro or EL4-Gax cells grown in vivo, where Gax expression was substantially suppressed (Fig. 3B and Fig. 1D). In addition, although histone acetylation, a downstream effect of CREB-CBP/p300 association, remained constant, recruitment of the basic transcription factor (TFIIB) and RNA polymerase II to the promoter was diminished in cells grown in vivo (Fig. 3C). These observations suggest that activation machinery other than the CREB-CBP/p300 pathway might be involved in the transcriptional control of HTLV-1 in vivo, whereby constitutive association of CREB-CBP/p300 and acetylated histones keeps the promoter in an inducible condition. In this respect, it is noteworthy that reversible silencing was observed in an ATL case regardless of hyperacetylation of histones H3 and H4 in the 5'-LTR .
CREB-dependent promoters have been thought to respond to various intracellular and extracellular signals via stimulus-dependent phosphorylation of CREB at Ser-133 and the resulting recruitment of the co-activator paralogues, CBP and p300 . Although a number of growth factors and hormones trigger CREB phosphorylation with comparable stoichiometry and kinetics, they are often ineffective in promoting transcription through the CREB binding site. Furthermore, knock-in mice with mutations in CBP/p300 that blocked the interaction with phosphor-CREB showed only modest changes in cAMP-dependent transcription. Thus, the involvement of additional CREB co-activators was postulated, and, subsequently, a new family of proteins has been identified as co-activators independent of CREB phosphorylation using high-throughput transformation assays [25, 26]. The novel co-activators, TORC 1–3, interact with the bZIP of CREB in a phospho-Ser133-independent manner through their N-terminal coiled-coil structure, leading to the activation of CREB -mediated transcription.
Accordingly, we examined the involvement of TORCs in the regulation of HTLV-1 transcription in vivo and found that expression of TORC1 and TORC2 in EL4-Gax cells coincided with the suppressed expression of Gax in vivo and its up-regulation in vitro or ex vivo. Previous reports have demonstrated that the forced expression of human TORC proteins activates the transcription of the HTLV-1 LTR, whereas depletion of TORCs by siRNA-mediated knock-down impairs Tax-dependent and Tax-independent transcription [27, 28]. We also observed the activation of a luciferase reporter gene under control of HTLV-1 LTR in association with the over-expression of TORC1, TORC2, or TORC3 of mouse origin (data not shown). It was recently reported that TORC3 repressed the transcription of HTLV-1 by interacting with Bcl3, which, in turn, recruits HDAC1 to deacetylate histones. However, such a mechanism is likely not involved in the current system because no significant change in the promoter association of acetylated histones H3 and H4 was observed in cells grown in vitro or in vivo, although the expression of TORC3 appeared to increase slightly in vivo. In contrast, depletion of TORC1 or TORC3 expression in EL4-Gax cells by siRNA did not affect the expression of Gax, whereas TORC2 knock-down resulted in a significant reduction in Gax expression, although expression of siRNA to any of the TORC genes reduced the amount of the respective TORC proteins in EL4-Gax cells to about the same extent. Consistently, the analysis of enhancer binding of TORC proteins by using a ChIP assay revealed that TORC2 is the major binding factor among the three TORC-family proteins in EL4-Gax cells (Fig. 4E). These results indicate that TORC2 is primarily responsible for the transcriptional control of HTLV-1 LTR in EL4-Gax cells in vivo.
TORC2 activity is regulated by phosphorylation of the protein, which induces the cytoplasmic translocation of TORC2 from the nucleus, leading to its degradation by the 26S proteasome. In fact, we demonstrated that the activity of AMPK, a family of Ser/Thr kinases, which phosphorylates TORC2 [29, 35], was activated in EL4-Gax cells in vivo (Fig. 6C). In EL4-Gax cells cultured in vitro, most TORC2 protein was localized in the nucleus where it would be involved in the transcription of the provirus. When the cells were grown in the peritoneal cavity of mice, the nuclear expression of TORC2 was markedly diminished, whereas its cytoplasmic expression remained relatively constant. This result is consistent with the Western blotting data, which showed that the amount of the unphosphorylated form of TORC2 protein was more significantly reduced in vivo than that of the phosphorylated form (Fig. 4B). Together, these findings suggest that some signal(s) specific for in vivo growth induces the phosphorylation of TORC2 protein, leading to the cytoplasmic translocation and degradation of the protein. In this respect, it is noteworthy that the activity of AMPK, is modulated by various pathological stresses and physiological stimuli, including glucose deprivation, hyperosmotic stress, heat shock, hypoxia, and ischemia . In addition, calcium influx after hormonal stimulation or antigen stimulation of lymphocytes induces calcineurin activity, which in turn dephosphorylates TORC2. Thus, various physiological and immunological signals can induce the reactivation of provirus, even in vivo, through modulation of TORC2 activity.
Using a mouse model system in which the transcriptional control was quite similar to that in asymptomatic HTLV-1 -infected carriers , we demonstrated that TORC2, a recently identified co-activator of CREB, is a key determinant of HTLV-1 gene expression in vivo. An analysis of promoter binding by transcriptional factors and chromatin modifications in EL4-Gax cells revealed that neither CpG methylation of the HTLV-1 LTR nor reduced association of enhancer binding factors such as the CREB-CBP/p300 complex coincided with the in vivo -specific suppression of HTLV-1 transcription. Instead, the expression of TORC2 was coordinated with suppression in vivo and the reactivation ex vivo of provirus transcription. In addition, the transcriptional activities of the provirus gene in vivo and in vitro were associated with the nuclear accumulation of TORC2 protein, which is tightly controlled by phosphorylation of the protein in response to various physiological signals.
Cell lines and animal model
The establishment of the EL4-Gax mouse model system was previously reported . Briefly, 1 × 106 EL4-Gax cells were injected into the peritoneal cavity of 6-week-old male C57BL/6J mice (Charles River Laboratories Japan, Kanagawa, Japan) in 0.1 mL phosphate -buffered saline (PBS). After three weeks, cells in ascitic fluids (in vivo cells) were recovered and cultured in culture medium for 48 h (ex vivo culture). Gax expression was monitored using a FACS (Becton Dickinson, NJ) as the intensity of GFP fluorescence. The mice were maintained under standard pathogen-free conditions in the animal facility of Kansai Medical University, and the protocol for the experiments was approved by the Institutional Animal Care and Use Committee of Kansai Medical University.
Quantitative real-time RT-PCR analysis of Gaxgene transcripts
Total RNA was extracted from cells using the Trizol reagent (Invitrogen, Carlsbad, CA) and treated with RNase-free DNase I (GIBCO BRL) at 37°C for 30 min, followed by phenol-chloroform extraction and ethanol precipitation. Total RNA (1 μg) was subjected to reverse transcription using RivaTra Ace (Toyobo, Osaka, Japan) with random 9 mer oligonucleotides (Takara, Kyoto, Japan) as a primer.
Real-time PCR was performed in a 25-μL reaction mixture consisting of 2-fold SYBR Green mastermix (Applied Biosystems, UK); 50 pg or 10 ng cDNA for 18S-rRNA or Gax, respectively; 0.4 μM primer; and the BioRad-iQ-Real-Time PCR Detection System. The cycling conditions for all amplifications were 95°C for 15 min; 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, with a single fluorescence measurement melting curve program (60–95°C with a heating rate of 0.2°C per second and a continuous fluorescence measurement); and finally a cooling step to 24°C. Each individual sample was run in triplicate wells; the cycle threshold (Ct) of each well was recorded at the end of the reaction. The Ct was manually set up at the level that reflected the best kinetic PCR parameters, and melting curves were analyzed. The average and standard deviation (SD) of the three Cts was calculated, and the average value was accepted if the SD was less than 0.38. The 2ΔΔCt method was used to analyze the relative changes in Gax gene expression in in vivo or ex vivo samples with those in vitro.
The primers used for PCR were as follows: for the Gax gene, 5'-ACGCCTATGATTTCCGGGCC-3' and 5'-GGATATTTGGGGCTCATGGTCA-3'; for CD4, 5'-CAGAGCCTGACCCTGACCTTG-3' and 5'-CATCACCACCAGGTTCACTTCC-3'; for endogenouse mouse retroviral env, 5'-GAAGGTCCAGCGTTCTCAAAAT-3' and 5'-CACGTGATTTCACTTCTTCTGG-3'; for elongation factor (EF)-1α, 5'-TCTGGTTGGAATGGTGACAACATGC-3' and 5'-CCAGGAAGAGCTTCACTCAAAGCTT-3'; for GAPDH, 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'; for β-actin: 5'-GAGATCTGCCGATCCGCCGCCCG-3', and 5'-GCTCGAGGTGTGCACTTTTATTCAACTGG-3'; for 18S-rRNA, 5'-CTCGATTCCGTGGGTGGTGGTG-3' and 5'-GGCCGATCCGAGGGCCTCAC-3'.
Western blot analysis
Cells were lysed and sonicated in cell lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1%Triton X-100, Cell Signaling Technology, MA) plus complete proteinase inhibitor cocktail and phosphatase inhibitor mixture (Roche Applied Science, Indianapolis, IN). Aliquots containing 5–30 μg of protein were separated by SDS/polyacrylamide gel electrophoresis; transferred onto PVDF membrane (Immobilon-P, Millipore, Billerica, MA); and detected by the ECL or ECL plus system (GE Healthcare, Buckinghamshire, UK). Used antibodies were TORC2 rabbit polyclonal antibody (#ST1099, Calbiochem, La Jolla, CA), TORC1 rabbit polyclonal antibody (#2501), TORC3 rabbit polyclonal antibody (#2768), phospho-AMPKα (Thr172) rabbit monoclonal antibody (40H9, #2535), AMPKα rabbit monoclonal antibody (23A3, #2603, Cell Signaling Technology); CREB1 (#06–863), phospho-CREB (#06–519, Upstate Technology, NY); PARP-1 mouse monoclonal antibody (#611038, BD. Biosciences, San Jose, CA) or β-actin mouse monoclonal antibody (clone AC-5, Sigma-Aldrich, MO) and HRP-conjugated protein A for rabbit primary antibodies or anti-mouse IgG-HRP (GE Healthcare) for mouse monoclonal primary antibodies.
Phosphatase treatment of protein from EL4-Gax
1 × 106 EL4-Gax cells were lysed in 250 μl of cell lysis buffer (mentioned above, Cell Signaling Technology) and 1× complete protease inhibitor cocktail (Roche) with or without phosphatase inhibitor cocktail (2.5 mM sodium pyrophosphate; 1 mM β-glycerophosphate; 1 mM Na3VO4; 50 mM sodium fluoride), plugged into liquid nitrogen and thawed on ice immediately. Lysate was clarified by centrifugation at 14,000 rpm for 10 minutes at 4°C. After measuring protein concentration, 20 μg protein was dispended to 1× rAPid Alkaline Phosphatase buffer with or without phosphatase inibitor cocktail, treated with or without 20 units of rAPid Alkaline Phosphatase (Cat No.04898133001, Roche) for 60 minutes at 37°C in a 100 μl reaction volumes. The 1/3 volume of samples was then separated by SDS-PAGE.
Analysis of CpG methylation
Methylation of the cytosine residue at CpG sites was analyzed using the bisulfite genomic sequencing method  with minor modifications. Briefly, 1 μg genomic DNA was used for bisulfite treatment. The DNA sample in 0.2 N NaOH was denatured at 37°C for 10 min, followed by incubation at 50°C overnight in 2.6 M sodium bisulfite and 0.5 mM hydroquinone (Sigma-Aldrich) solution. The solution was then sealed with 200 μL mineral oil and kept in the dark. Sample DNA was purified using the Wizard DNA Clean-Up system (Promega, Madison, WI) and treated with 0.3 N NaOH. DNA was precipitated with ethanol and dissolved in 50 μL H2O. Then 30% of this solution was subjected to PCR.
PCR was performed in 50-μL reaction mixtures containing 1 μL Acuprime Taq DNA polymerase and its buffer (Invitrogen) and a primer set at 0.2 μM. The primer sequences to amplify the 5'-LTR HTLV-1 U3 region were sense primer (complementary to the modified cellular flanking region), 5'-CCTCCTATCAAAACTAACTTTAAC-3', and antisense primer (complementary to the modified HTLV-1 U3 region), 5'-AAGTTTTTTGAGGTGAGGGGT-3'.
The cycling conditions for all amplifications were 94°C for 2 min; 40 cycles of 94°C for 30 s, 56°C for 30 s, and 68°C for 30 s; and a final extension at 68°C for 5 min. The amplified PCR products, purified with the QIA quick PCR kit (QIAGEN, Germantown, MD), were subcloned into a pGEM-T easy vector (Promega), and the nucleotide sequences of at least 13 clones were determined.
ChIP assays were performed according to the manufacturer's protocol (Upstate Biotechnology, Lake Placid, NY) with some modifications. Briefly, 2 × 106 cells were fixed with 1% formaldehyde for 5 min at room temperature and sonicated (Biorupter UCD-200T; CosmoBio, Tokyo, Japan) to obtain on average 600 -bp length of soluble chromatin. The chromatin solutions were pre-cleared with 80 μL 50% Protein G Sepharose slurry (Amersham) preabsorbed with 0.2 mg/mL sonicated salmon sperm DNA and 0.05% BSA. After a 10-fold dilution with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl), 2 mL pre-cleared complex was combined with 2–15 μg antibodies against CREB1 (06–863), phosphor-CREB (06–519), acetylated H3 at Lys-9, 14 (06–599), acetylated H4 at Lys-5, 8, 12, 16 (06–598, Upstate Technology), CBP (sc369), TFIIB (sc-225), Pol-II (sc899, Santa Cruz Biotechnology, CA), methylated H3 Lys-4 (AB8580, Abcam Ltd., Cambridge, UK), GFP (mouse monoclonal antibody, clones 7.1 and 13.1, Boehringer Mannheim), or normal mouse IgG (I-5381, Sigma), rabbit IgG (011-000-002, Jackson immunoresearch Laboratories, West Grove, PA) and rotated at 4°C overnight. The immune complexes were collected by incubating with 80 μL of protein G slurry. DNA-protein cross-linking was reversed by incubation at 65°C overnight, and the samples were treated with RNase A and then with proteinase K. After phenol extraction and ethanol precipitation, the DNA was finally dissolved into 30 μL of water.
The primers used for PCR were as follows. For the 5'-LTR enhancer, sense primer, complementary to the 5'flanking cellular region, 5'-AAGCACAGAAGACACCTTGTGCAC-3' and antisense primer, complementary to 5'-LTR HTLV-1 U3 region, 5'-AAGTTTTTTGAGGTGAGGGGTTGTCG-3'. 5'-LTR promoter region, sense primer, complementary to HTLV-1 U3/MLV U3 junction region, 5'-CATGGCACGCATATGTCTAGAGAACC-3', antisense primer, complementary to MLV gag region, 5'-TCTCCCGATCCCGGACGAGCC-3'. The following sequences of primers were used for other control gene promoters: for EF1a, f (-20): 5'-CGAGGGTGGGGGAGAACGGTAT-3' and r (152): 5'-AGCTAATCCCCGCCGACGACAG-3'; for β-globin, f (-117): 5'-ACCGAAGCCTGATTCCGTAGAGC-3' and r (+133): 5'-CTCACCACCAACTTCATCGGAGTT-3'.
All PCRs were performed in 20-μL reaction mixes consisting of 1–10 μL of the ChIP product, 1 U Ex Taq polymerase (Takara), 200 mM each of deoxynucleoside triphosphates, and a primer set at 0.4 μM. The cycling conditions for all amplifications were 94°C for 5 min; 33–35 cycles of 94°C for 30 s, 62–68°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min.
Cells were grown in suspension or harvested from ascites and were then fixed by adding an equal volume of 4% paraformaldehyde diluted in PEM/PBS(-) buffer (1/5 volume of PEM [see below] and 4/5 volume of PBS(-)) for 30 min at 4°C, collected by centrifugation (300g, 3 min), washed and resuspended in PBS(-), and then cytospun (Shandon Cytospin3; 20,000 cells/slide, 700 rpm, 1 min) onto 1 N HCl and 95% ethanol-washed slide glasses (Matsunami, Osaka, Japan). The cells were washed twice with PEM buffer (80 mM potassium PIPES [piperazine-N, N'-bis(2-ethanesulfonic acid)] [pH 6.8], 5 mM EGTA [pH 7.0], 2 mM MgCl2) and permeabilized by incubation in PEM/PBS(-) buffer containing 0.1% Triton X-100 for 30 min at room temperature and blocked with goat serum (1:20 dilution in TBS containing 0.1% Tween-20) for 30 min at room temperature. After blocking, the cells were incubated with anti-TORC2 antiserum ((#ST1099, Calbiochem) or normal rabbit IgG for 3 h at room temperature or overnight at 4°C in TBS containing 0.1% Tween-20 (TBS-T). Thereafter, the cells were washed three times with TBS-T and incubated with Cy5-conjugated AffiniPure donkey anti-rabbit IgG (#711-175-152, Jackson Immuno Research Laboratories) in TBS-T. After 1 h, the cells were washed three times in TBS-T. To visualize the nucleus, we counterstained the cells with DAPI (4', 6'-diamidino-2-phenylindole; Nacalai Tesque, Kyoto, Japan; final concentration 0.4 μg/mL, 5 min incubation at room temperature) and washed them once with PBS(-), then mounted them in mounting media and examined them with a Zeiss LSM510 META microscope.
TORC siRNA hairpin expression vectors (shTORC1 retroviral vector, #NM-001004062; shTORC3 retroviral vector, #XM-344915; shTORC2 lentiviral vector, #NM-02881) were purchased from Open Biosystems (Huntsville, AL). Recombinant retroviruses for shTORC1 and shTORC3 were generated by transfecting respective retroviral vector plasmids into BOSC23 cells, and recombinant lentivirus expressing shTORC2 was generated in HEK293T cells by the cotransfection of the lentiviral vector plasmid with expression vectors for Gag/Pol (pGag/Pol), Rev (pRev), and VSV-G protein (pVSV-G) using the Lipofectamine 2000 (Invitrogen) transfection reagent. Viral supernatant was collected 48 h after transfection and added to EL4-Gax cells for infection in the presence of polybrene (8 μg/mL). Infected cells were selected with puromycin (4 μg/mL), and mixtures of 40–200 clones of each virus were used for further analysis.
We thank Dr. R. Tatsumi for her helpful discussions. This study was partly supported by a Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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