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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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Tax and MEF-2 are recruited to the HTLV-1 LTR. Chromatin immunoprecipitation of Tax protein and transcription factors bound to cellular and viral promoters during HTLV-1 infection in (A) cell lines, (B) primary CD4+ T cells, and (C) primary CD4+CD25+ T cells was performed using the ChIP-IT Express kit. Cells were lysed in a dounce homogenizer to obtain sheared chromatin following formaldehyde fixation. The sheared chromatin was immunoprecipitated at 4°C overnight using 2 μg of antibodies against the Tax protein, indicated cellular factors and controls. The immunoprecipitated chromatin was then subjected to PCR using primers for HTLV-1 LTR and human GAPDH. Data is presented as average fold change over control IgG immunoprecipitation, and is representative of three independent experiments.
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Fig3: Tax and MEF-2 are recruited to the HTLV-1 LTR. Chromatin immunoprecipitation of Tax protein and transcription factors bound to cellular and viral promoters during HTLV-1 infection in (A) cell lines, (B) primary CD4+ T cells, and (C) primary CD4+CD25+ T cells was performed using the ChIP-IT Express kit. Cells were lysed in a dounce homogenizer to obtain sheared chromatin following formaldehyde fixation. The sheared chromatin was immunoprecipitated at 4°C overnight using 2 μg of antibodies against the Tax protein, indicated cellular factors and controls. The immunoprecipitated chromatin was then subjected to PCR using primers for HTLV-1 LTR and human GAPDH. Data is presented as average fold change over control IgG immunoprecipitation, and is representative of three independent experiments.

Mentions: Having generated confidence in MEF-2 involvement in HTLV-1 pathogenesis, we proceeded to understand the underlying molecular interactions in the context of primary CD4+ T cells and viral infection. We infected primary CD4+ T cells with HTLV-1 as previously described [65,66], and confirmed intracellular Tax expression by flow cytometry as well as by Western blotting (Additional file 2: Figure S2). Upon confirmation of infection, cells were subjected to ChIP analyses. In both cell lines and primary cells, we noted strong binding of CBP, pCREB, p300, p/CAF, and MEF-2A but not Tax to the GAPDH promoter (Figure 3, left panels). This was not surprising since the amplified region of GAPDH contained binding sites for these TFs. Although recruitment of some of these factors to the GAPDH promoter was more efficient in infected cells, we did not see any increase in GAPDH expression upon HTLV-1 infection (Additional file 3: Figure S3). We also observed efficient recruitment of TFs and Tax to the viral LTR in MT-2 cells (Figure 3A, right panel) and infected CD4+ cells (Figure 3B, right panel), but not in uninfected control cells. CD4+CD25+ T cells were also included in our analysis, as they are the primary subset of CD4+ T cells targeted by HTLV-1 [67]. These cells showed efficient recruitment of MEF-2A and other cellular factors to the LTR upon infection (Figure 3C, right panel). As a control, we enriched cells for viral core protein p19 and as expected did not detect recruitment of any factors analyzed to GAPDH or LTR promoters (Additional file 4: Figure S4). Altogether, these results confirmed that MEF-2A is recruited to the HTLV-1 LTR in association with Tax and co-activators of transcription including p300, CBP, and p/CAF.Figure 3


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)

Tax and MEF-2 are recruited to the HTLV-1 LTR. Chromatin immunoprecipitation of Tax protein and transcription factors bound to cellular and viral promoters during HTLV-1 infection in (A) cell lines, (B) primary CD4+ T cells, and (C) primary CD4+CD25+ T cells was performed using the ChIP-IT Express kit. Cells were lysed in a dounce homogenizer to obtain sheared chromatin following formaldehyde fixation. The sheared chromatin was immunoprecipitated at 4°C overnight using 2 μg of antibodies against the Tax protein, indicated cellular factors and controls. The immunoprecipitated chromatin was then subjected to PCR using primers for HTLV-1 LTR and human GAPDH. Data is presented as average fold change over control IgG immunoprecipitation, and is representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374383&req=5

Fig3: Tax and MEF-2 are recruited to the HTLV-1 LTR. Chromatin immunoprecipitation of Tax protein and transcription factors bound to cellular and viral promoters during HTLV-1 infection in (A) cell lines, (B) primary CD4+ T cells, and (C) primary CD4+CD25+ T cells was performed using the ChIP-IT Express kit. Cells were lysed in a dounce homogenizer to obtain sheared chromatin following formaldehyde fixation. The sheared chromatin was immunoprecipitated at 4°C overnight using 2 μg of antibodies against the Tax protein, indicated cellular factors and controls. The immunoprecipitated chromatin was then subjected to PCR using primers for HTLV-1 LTR and human GAPDH. Data is presented as average fold change over control IgG immunoprecipitation, and is representative of three independent experiments.
Mentions: Having generated confidence in MEF-2 involvement in HTLV-1 pathogenesis, we proceeded to understand the underlying molecular interactions in the context of primary CD4+ T cells and viral infection. We infected primary CD4+ T cells with HTLV-1 as previously described [65,66], and confirmed intracellular Tax expression by flow cytometry as well as by Western blotting (Additional file 2: Figure S2). Upon confirmation of infection, cells were subjected to ChIP analyses. In both cell lines and primary cells, we noted strong binding of CBP, pCREB, p300, p/CAF, and MEF-2A but not Tax to the GAPDH promoter (Figure 3, left panels). This was not surprising since the amplified region of GAPDH contained binding sites for these TFs. Although recruitment of some of these factors to the GAPDH promoter was more efficient in infected cells, we did not see any increase in GAPDH expression upon HTLV-1 infection (Additional file 3: Figure S3). We also observed efficient recruitment of TFs and Tax to the viral LTR in MT-2 cells (Figure 3A, right panel) and infected CD4+ cells (Figure 3B, right panel), but not in uninfected control cells. CD4+CD25+ T cells were also included in our analysis, as they are the primary subset of CD4+ T cells targeted by HTLV-1 [67]. These cells showed efficient recruitment of MEF-2A and other cellular factors to the LTR upon infection (Figure 3C, right panel). As a control, we enriched cells for viral core protein p19 and as expected did not detect recruitment of any factors analyzed to GAPDH or LTR promoters (Additional file 4: Figure S4). Altogether, these results confirmed that MEF-2A is recruited to the HTLV-1 LTR in association with Tax and co-activators of transcription including p300, CBP, and p/CAF.Figure 3

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