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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-associated signaling pathways are triggered during HTLV-1 infection. (A) A protein-DNA array was used to determine the activation of the various eukaryotic transcription factors. Nuclear extracts from control and HTLV-infected primary cells were mixed with biotinylated DNA binding oligonucleotides for the formation of protein-DNA complexes. These probes were then hybridized to pretreated array membranes and the bound probe was detected as described in Methods. Densitometric analysis was used to quantify the spots and data was normalized to their respective controls after background subtraction. Fold change in expression of selected transcription factors from the array data compared to relevant uninfected cells. Significance was determined using the Student’s t-test (*P ≤ 0.05). (B) Cells were lysed and Western blotting was performed to confirm the array data.
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Fig8: MEF-2-associated signaling pathways are triggered during HTLV-1 infection. (A) A protein-DNA array was used to determine the activation of the various eukaryotic transcription factors. Nuclear extracts from control and HTLV-infected primary cells were mixed with biotinylated DNA binding oligonucleotides for the formation of protein-DNA complexes. These probes were then hybridized to pretreated array membranes and the bound probe was detected as described in Methods. Densitometric analysis was used to quantify the spots and data was normalized to their respective controls after background subtraction. Fold change in expression of selected transcription factors from the array data compared to relevant uninfected cells. Significance was determined using the Student’s t-test (*P ≤ 0.05). (B) Cells were lysed and Western blotting was performed to confirm the array data.

Mentions: The transcriptional activity of MEF-2 is regulated by protein modifications including phosphorylation, acetylation, sumoylation, etc. In addition, MEF-2 is known to integrate a number of signaling pathways, including PI3K/Akt, NF-κB, MAPK, TGF-β and JAK/STAT signaling [33,38,76]. While many of these signaling pathways are known to be activated by Tax, we obtained a global perspective on these signaling events upon HTLV-1 infection of CD4 T cells by a DNA-protein array. This type of interactome profiling of primary CD4 T cells upon HTLV-1 infection has not been reported yet. In general, an upregulated transcriptome profile was seen in both MT-2 and primary infected CD4+ T cells, emphasizing the highly active and dynamic process of viral infection (Additional file 8: Figure S7A and B). For analysis, cellular factors included in the array were grouped according to their association with relevant signaling pathways in Table 1 and the fold-change in protein expression of the key cellular factors from the MEF-2-integrated signaling cascades is given in Figure 8A. Upon validation of array data by Western blotting, the phosphorylated form of MEF-2A as well as of p38, ERK5, Smad2 and Akt were found to be upregulated upon infection in both MT-2 and primary cells (Figure 8B). Thus, our model (Figure 9) suggests that Tax-mediated activation of cellular signaling pathways contribute to the phosphorylation and activation of MEF-2, which is then dissociated from class II HDACs and interacts with Tax at the viral promoter to boost Tax-mediated transactivation, viral replication as well as T-cell transformation. Tax also binds to Smad2/3/4 to prevent their constitutive binding to transcription co-activators CBP/p300. This leads to increased availability of CBP/p300 to bind Tax/pCREB complex bound to the 5′ LTR region of the provirus. Along with Tax/pCREB/CBP/p300 complex, recruitment of MEF-2A to the 5′ LTR promotes viral gene expression. On the other hand, Tax also activates Calcineurin (a calcium-dependent serine-threonine phosphatase), which dephosphorylates NFAT. Upon dephosphorylation, NFAT translocates to the nucleus and is recruited to the HTLV-1 LTR along with the Tax/pCREB/CBP/p300 complex. NFAT is also recruited to the MEF-2A gene promoter where it binds to MEF-2A and turns on transcription resulting in upregulation of MEF-2A expression. Interestingly, in Figure 4A we showed that MEF-2 in its native form was also upregulated upon HTLV-1 infection making more MEF-2 available for interaction with Tax. This heightened expression of MEF-2 could be a result of Tax-induced calcineurin activity that in turn results in NFAT-mediated MEF-2 transcription via binding to its own promoter.Table 1


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-associated signaling pathways are triggered during HTLV-1 infection. (A) A protein-DNA array was used to determine the activation of the various eukaryotic transcription factors. Nuclear extracts from control and HTLV-infected primary cells were mixed with biotinylated DNA binding oligonucleotides for the formation of protein-DNA complexes. These probes were then hybridized to pretreated array membranes and the bound probe was detected as described in Methods. Densitometric analysis was used to quantify the spots and data was normalized to their respective controls after background subtraction. Fold change in expression of selected transcription factors from the array data compared to relevant uninfected cells. Significance was determined using the Student’s t-test (*P ≤ 0.05). (B) Cells were lysed and Western blotting was performed to confirm the array data.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374383&req=5

Fig8: MEF-2-associated signaling pathways are triggered during HTLV-1 infection. (A) A protein-DNA array was used to determine the activation of the various eukaryotic transcription factors. Nuclear extracts from control and HTLV-infected primary cells were mixed with biotinylated DNA binding oligonucleotides for the formation of protein-DNA complexes. These probes were then hybridized to pretreated array membranes and the bound probe was detected as described in Methods. Densitometric analysis was used to quantify the spots and data was normalized to their respective controls after background subtraction. Fold change in expression of selected transcription factors from the array data compared to relevant uninfected cells. Significance was determined using the Student’s t-test (*P ≤ 0.05). (B) Cells were lysed and Western blotting was performed to confirm the array data.
Mentions: The transcriptional activity of MEF-2 is regulated by protein modifications including phosphorylation, acetylation, sumoylation, etc. In addition, MEF-2 is known to integrate a number of signaling pathways, including PI3K/Akt, NF-κB, MAPK, TGF-β and JAK/STAT signaling [33,38,76]. While many of these signaling pathways are known to be activated by Tax, we obtained a global perspective on these signaling events upon HTLV-1 infection of CD4 T cells by a DNA-protein array. This type of interactome profiling of primary CD4 T cells upon HTLV-1 infection has not been reported yet. In general, an upregulated transcriptome profile was seen in both MT-2 and primary infected CD4+ T cells, emphasizing the highly active and dynamic process of viral infection (Additional file 8: Figure S7A and B). For analysis, cellular factors included in the array were grouped according to their association with relevant signaling pathways in Table 1 and the fold-change in protein expression of the key cellular factors from the MEF-2-integrated signaling cascades is given in Figure 8A. Upon validation of array data by Western blotting, the phosphorylated form of MEF-2A as well as of p38, ERK5, Smad2 and Akt were found to be upregulated upon infection in both MT-2 and primary cells (Figure 8B). Thus, our model (Figure 9) suggests that Tax-mediated activation of cellular signaling pathways contribute to the phosphorylation and activation of MEF-2, which is then dissociated from class II HDACs and interacts with Tax at the viral promoter to boost Tax-mediated transactivation, viral replication as well as T-cell transformation. Tax also binds to Smad2/3/4 to prevent their constitutive binding to transcription co-activators CBP/p300. This leads to increased availability of CBP/p300 to bind Tax/pCREB complex bound to the 5′ LTR region of the provirus. Along with Tax/pCREB/CBP/p300 complex, recruitment of MEF-2A to the 5′ LTR promotes viral gene expression. On the other hand, Tax also activates Calcineurin (a calcium-dependent serine-threonine phosphatase), which dephosphorylates NFAT. Upon dephosphorylation, NFAT translocates to the nucleus and is recruited to the HTLV-1 LTR along with the Tax/pCREB/CBP/p300 complex. NFAT is also recruited to the MEF-2A gene promoter where it binds to MEF-2A and turns on transcription resulting in upregulation of MEF-2A expression. Interestingly, in Figure 4A we showed that MEF-2 in its native form was also upregulated upon HTLV-1 infection making more MEF-2 available for interaction with Tax. This heightened expression of MEF-2 could be a result of Tax-induced calcineurin activity that in turn results in NFAT-mediated MEF-2 transcription via binding to its own promoter.Table 1

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