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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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Related in: MedlinePlus

Model explaining MEF-2 activity on Tax-mediated transactivation of HTLV-1 LTR. Type II HDACs (HDAC4/5/7/9) bind to MEF-2A and repress its transcriptional activity. Upon HTLV-1 infection, Tax activates p38 and ERK5, which phosphorylate MEF-2 leading to its dissociation from the MEF-2A: HDAC repressive complex. On the other hand, 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. Tax also activates Calcineurin (a calcium-dependent serine-threonine phosphatase), which dephosphorylates NFAT. Upon dephosphorylation, NFAT translocates to nucleus and is recruited to the HTLV-1 5′ 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. HDAC, Histone deacetylase; MEF-2A, Myocyte-specific enhancer factor 2A; ERK5, Extracellular-signal-regulated kinase 5; Smad, Sma- and Mad-Related Protein; CREB, cAMP response element-binding protein; CBP, CREB-binding protein; NFAT, Nuclear factor of activated T cells.
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Fig9: Model explaining MEF-2 activity on Tax-mediated transactivation of HTLV-1 LTR. Type II HDACs (HDAC4/5/7/9) bind to MEF-2A and repress its transcriptional activity. Upon HTLV-1 infection, Tax activates p38 and ERK5, which phosphorylate MEF-2 leading to its dissociation from the MEF-2A: HDAC repressive complex. On the other hand, 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. Tax also activates Calcineurin (a calcium-dependent serine-threonine phosphatase), which dephosphorylates NFAT. Upon dephosphorylation, NFAT translocates to nucleus and is recruited to the HTLV-1 5′ 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. HDAC, Histone deacetylase; MEF-2A, Myocyte-specific enhancer factor 2A; ERK5, Extracellular-signal-regulated kinase 5; Smad, Sma- and Mad-Related Protein; CREB, cAMP response element-binding protein; CBP, CREB-binding protein; NFAT, Nuclear factor of activated T cells.

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)

Model explaining MEF-2 activity on Tax-mediated transactivation of HTLV-1 LTR. Type II HDACs (HDAC4/5/7/9) bind to MEF-2A and repress its transcriptional activity. Upon HTLV-1 infection, Tax activates p38 and ERK5, which phosphorylate MEF-2 leading to its dissociation from the MEF-2A: HDAC repressive complex. On the other hand, 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. Tax also activates Calcineurin (a calcium-dependent serine-threonine phosphatase), which dephosphorylates NFAT. Upon dephosphorylation, NFAT translocates to nucleus and is recruited to the HTLV-1 5′ 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. HDAC, Histone deacetylase; MEF-2A, Myocyte-specific enhancer factor 2A; ERK5, Extracellular-signal-regulated kinase 5; Smad, Sma- and Mad-Related Protein; CREB, cAMP response element-binding protein; CBP, CREB-binding protein; NFAT, Nuclear factor of activated T cells.
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Fig9: Model explaining MEF-2 activity on Tax-mediated transactivation of HTLV-1 LTR. Type II HDACs (HDAC4/5/7/9) bind to MEF-2A and repress its transcriptional activity. Upon HTLV-1 infection, Tax activates p38 and ERK5, which phosphorylate MEF-2 leading to its dissociation from the MEF-2A: HDAC repressive complex. On the other hand, 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. Tax also activates Calcineurin (a calcium-dependent serine-threonine phosphatase), which dephosphorylates NFAT. Upon dephosphorylation, NFAT translocates to nucleus and is recruited to the HTLV-1 5′ 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. HDAC, Histone deacetylase; MEF-2A, Myocyte-specific enhancer factor 2A; ERK5, Extracellular-signal-regulated kinase 5; Smad, Sma- and Mad-Related Protein; CREB, cAMP response element-binding protein; CBP, CREB-binding protein; NFAT, Nuclear factor of activated T cells.
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