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Myocyte enhancer factor (MEF)-2 plays essential roles in T-cell transformation associated with HTLV-1 infection by stabilizing complex between Tax and CREB.

Jain P, Lavorgna A, Sehgal M, Gao L, Ginwala R, Sagar D, Harhaj EW, Khan ZK - Retrovirology (2015)

Bottom Line: Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase).MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4 T-cell transformation have yet to be fully delineated. Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.

Results: Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and T-cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was recruited to the viral promoter (LTR, long terminal repeat) in the context of chromatin, and constituted Tax/CREB transcriptional complex via direct binding to the HTLV-1 LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (known Tax-interacting transcription factor), and HATs (p300, CBP, and p/CAF). Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase). MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

Conclusions: We demonstrate the involvement of MEF-2 in Tax-mediated LTR activation, viral replication, and T-cell transformation in correlation with its heightened expression in ATL patients through direct binding to DNA within the HTLV-1 LTR.

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MEF-2 physically interacts with Tax. (A) Control (Jurkat), infected (MT-2) cell lines, control primary CD4+ T cells and HTLV-infected primary CD4+ T cells were lysed, sonicated and protein concentration was determined by Bradford assay. Equal protein quantities were then resolved by SDS-PAGE and transferred to PVDF membrane. Following a 1 hr block membranes were incubated with antibodies against the transcription factors. Western blot shows the expression of transcription factors in control and infected cell lines and primary cells. (B) MEF-2 complex formation with Tax and transcription factors was analyzed using an immunoprecipitation assay. Cells were lysed using an immunoprecipitation lysis buffer and then incubated with MEF-2 antibody overnight at 4°C as described in Methods. Western immunoblot analysis was performed to confirm immunoprecipitation. (C) Control and infected cell lines and primary cells were enriched for Tax and immunoblotted to determine complex formation with MEF-2 and transcription factors. Data is representative of multiple individual experiments.
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Fig4: MEF-2 physically interacts with Tax. (A) Control (Jurkat), infected (MT-2) cell lines, control primary CD4+ T cells and HTLV-infected primary CD4+ T cells were lysed, sonicated and protein concentration was determined by Bradford assay. Equal protein quantities were then resolved by SDS-PAGE and transferred to PVDF membrane. Following a 1 hr block membranes were incubated with antibodies against the transcription factors. Western blot shows the expression of transcription factors in control and infected cell lines and primary cells. (B) MEF-2 complex formation with Tax and transcription factors was analyzed using an immunoprecipitation assay. Cells were lysed using an immunoprecipitation lysis buffer and then incubated with MEF-2 antibody overnight at 4°C as described in Methods. Western immunoblot analysis was performed to confirm immunoprecipitation. (C) Control and infected cell lines and primary cells were enriched for Tax and immunoblotted to determine complex formation with MEF-2 and transcription factors. Data is representative of multiple individual experiments.

Mentions: Prior to protein-protein interaction studies, we examined the expression of MEF-2A and other cellular factors both in cell lines and primary cells without and with HTLV-1 infection. As shown in Figure 4A, we noticed an upregulation of the HATs p300, CBP and p/CAF, as well as TFs, pCREB and MEF-2A upon infection. We also observed the complex formation of MEF-2A with Tax and pCREB, confirming a direct interaction with the Tax/CREB heterodimer complex (Figure 4B). Interestingly, upon infection, the interaction of MEF-2A with HDAC9 was expectedly diminished since HDAC9 binds to the C-terminal TAD domain of MEF-2 to repress its transcriptional activity. MEF-2A direct interaction with Tax was confirmed while enriching for Tax, which coprecipitated both pCREB and MEF-2A (Figure 4C). Tax enriched samples contain some levels of HATs but not HDAC9 suggesting that Tax can directly interact with co-activators of transcription as shown before [68]. It appeared that anti-Tax nonspecifically pulled down some MEF-2 in the absence of Tax; however, the band was greatly increased in the presence of Tax. Nevertheless, we performed an alternative experiment to validate the interaction of these two proteins in C8166 cells, which contain both Tax and MEF-2 proteins in abundance and demonstrated a specific interaction of these two proteins (Additional file 5: Figure S5A).Figure 4


Myocyte enhancer factor (MEF)-2 plays essential roles in T-cell transformation associated with HTLV-1 infection by stabilizing complex between Tax and CREB.

Jain P, Lavorgna A, Sehgal M, Gao L, Ginwala R, Sagar D, Harhaj EW, Khan ZK - Retrovirology (2015)

MEF-2 physically interacts with Tax. (A) Control (Jurkat), infected (MT-2) cell lines, control primary CD4+ T cells and HTLV-infected primary CD4+ T cells were lysed, sonicated and protein concentration was determined by Bradford assay. Equal protein quantities were then resolved by SDS-PAGE and transferred to PVDF membrane. Following a 1 hr block membranes were incubated with antibodies against the transcription factors. Western blot shows the expression of transcription factors in control and infected cell lines and primary cells. (B) MEF-2 complex formation with Tax and transcription factors was analyzed using an immunoprecipitation assay. Cells were lysed using an immunoprecipitation lysis buffer and then incubated with MEF-2 antibody overnight at 4°C as described in Methods. Western immunoblot analysis was performed to confirm immunoprecipitation. (C) Control and infected cell lines and primary cells were enriched for Tax and immunoblotted to determine complex formation with MEF-2 and transcription factors. Data is representative of multiple individual experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374383&req=5

Fig4: MEF-2 physically interacts with Tax. (A) Control (Jurkat), infected (MT-2) cell lines, control primary CD4+ T cells and HTLV-infected primary CD4+ T cells were lysed, sonicated and protein concentration was determined by Bradford assay. Equal protein quantities were then resolved by SDS-PAGE and transferred to PVDF membrane. Following a 1 hr block membranes were incubated with antibodies against the transcription factors. Western blot shows the expression of transcription factors in control and infected cell lines and primary cells. (B) MEF-2 complex formation with Tax and transcription factors was analyzed using an immunoprecipitation assay. Cells were lysed using an immunoprecipitation lysis buffer and then incubated with MEF-2 antibody overnight at 4°C as described in Methods. Western immunoblot analysis was performed to confirm immunoprecipitation. (C) Control and infected cell lines and primary cells were enriched for Tax and immunoblotted to determine complex formation with MEF-2 and transcription factors. Data is representative of multiple individual experiments.
Mentions: Prior to protein-protein interaction studies, we examined the expression of MEF-2A and other cellular factors both in cell lines and primary cells without and with HTLV-1 infection. As shown in Figure 4A, we noticed an upregulation of the HATs p300, CBP and p/CAF, as well as TFs, pCREB and MEF-2A upon infection. We also observed the complex formation of MEF-2A with Tax and pCREB, confirming a direct interaction with the Tax/CREB heterodimer complex (Figure 4B). Interestingly, upon infection, the interaction of MEF-2A with HDAC9 was expectedly diminished since HDAC9 binds to the C-terminal TAD domain of MEF-2 to repress its transcriptional activity. MEF-2A direct interaction with Tax was confirmed while enriching for Tax, which coprecipitated both pCREB and MEF-2A (Figure 4C). Tax enriched samples contain some levels of HATs but not HDAC9 suggesting that Tax can directly interact with co-activators of transcription as shown before [68]. It appeared that anti-Tax nonspecifically pulled down some MEF-2 in the absence of Tax; however, the band was greatly increased in the presence of Tax. Nevertheless, we performed an alternative experiment to validate the interaction of these two proteins in C8166 cells, which contain both Tax and MEF-2 proteins in abundance and demonstrated a specific interaction of these two proteins (Additional file 5: Figure S5A).Figure 4

Bottom Line: Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase).MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4 T-cell transformation have yet to be fully delineated. Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.

Results: Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and T-cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was recruited to the viral promoter (LTR, long terminal repeat) in the context of chromatin, and constituted Tax/CREB transcriptional complex via direct binding to the HTLV-1 LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (known Tax-interacting transcription factor), and HATs (p300, CBP, and p/CAF). Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase). MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

Conclusions: We demonstrate the involvement of MEF-2 in Tax-mediated LTR activation, viral replication, and T-cell transformation in correlation with its heightened expression in ATL patients through direct binding to DNA within the HTLV-1 LTR.

Show MeSH
Related in: MedlinePlus