Limits...
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.

Show MeSH

Related in: MedlinePlus

Assessment of MEF-2 binding site(s) within the HTLV-1 LTR. (A) HTLV-1 LTR nucleotide sequence with two putative MEF-2 binding sites shown in blue and red. The HTLV-1 LTR comprises U3 (unique 3′), R (Repeated), and U5 (unique 5′) regions. The U3 region regulates viral gene expression via three 21 bp repeats known as Tax-responsive element - 1 (TRE - 1), which confers Tax-based trans-activation. These repeats contain 3 conserved domains labeled A, B and C. The location for ATF/CREB binding and putative MEF-2 binding are illustrated. (B) EMSA was performed with a probe corresponding to the MEF-2 site in the HTLV-1 LTR using nuclear extracts from Jurkat, MT-2, MT-4 and C8166 cells. (C) EMSA competition assay was performed using nuclear extracts from MT-2 cells, with increasing amounts of unlabeled consensus MEF-2 specific probe (200, 300 and 400 fold molar excess respectively) or a mutated MEF-2 specific probe. Oct-1 was used as a loading control. (D) HTLV-1 LTR luciferase assays in 293 T cells transfected with empty vector (EV) or Flag-Tax using LTR luciferase WT plasmid (LTR Luc WT) or a MEF-2-specific binding mutant (LTR Luc MEF-2 Mut). Luciferase values are presented as “fold induction” relative to the control (EV). Two-tailed unpaired t-test was performed with Prism software. Error bars represent the standard deviation of triplicate samples. The level of significance was defined as: ***p < 0.001. Western blots were performed with anti-Flag and anti-β-actin using whole-cell lysates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Assessment of MEF-2 binding site(s) within the HTLV-1 LTR. (A) HTLV-1 LTR nucleotide sequence with two putative MEF-2 binding sites shown in blue and red. The HTLV-1 LTR comprises U3 (unique 3′), R (Repeated), and U5 (unique 5′) regions. The U3 region regulates viral gene expression via three 21 bp repeats known as Tax-responsive element - 1 (TRE - 1), which confers Tax-based trans-activation. These repeats contain 3 conserved domains labeled A, B and C. The location for ATF/CREB binding and putative MEF-2 binding are illustrated. (B) EMSA was performed with a probe corresponding to the MEF-2 site in the HTLV-1 LTR using nuclear extracts from Jurkat, MT-2, MT-4 and C8166 cells. (C) EMSA competition assay was performed using nuclear extracts from MT-2 cells, with increasing amounts of unlabeled consensus MEF-2 specific probe (200, 300 and 400 fold molar excess respectively) or a mutated MEF-2 specific probe. Oct-1 was used as a loading control. (D) HTLV-1 LTR luciferase assays in 293 T cells transfected with empty vector (EV) or Flag-Tax using LTR luciferase WT plasmid (LTR Luc WT) or a MEF-2-specific binding mutant (LTR Luc MEF-2 Mut). Luciferase values are presented as “fold induction” relative to the control (EV). Two-tailed unpaired t-test was performed with Prism software. Error bars represent the standard deviation of triplicate samples. The level of significance was defined as: ***p < 0.001. Western blots were performed with anti-Flag and anti-β-actin using whole-cell lysates.

Mentions: Results from the competitive promoter assays and CHIP assays confirmed that MEF-2 is recruited to the HTLV-1 LTR suggesting the presence of a MEF-2 binding site(s) within the LTR. Therefore, we examined the consensus site for all MEF-2 isoforms CTA(T/A)4TA(G/A)C within the LTR and identified two imperfect MEF-2 sites - one just downstream of repeat III of the Tax-response element (TRE) and upstream of the TATA box (Figure 7A, blue highlight) that matches 7/10 bases of the consensus site; the other overlaps with the TATA box (Figure 7B, red highlight), which is highly unlikely to be functional due to competition with general TFs. To determine if MEF-2 interacted with the more upstream MEF-2 site in the HTLV-1 LTR, a double-stranded DNA probe corresponding to this site was generated and used for EMSA DNA binding assays. EMSA revealed a specific protein/DNA complex with nuclear extracts from the HTLV-1 transformed cell lines MT-2, MT-4 and C8166, but not Jurkat cells (Figure 7B). To demonstrate that this protein complex consisted of MEF-2, competition EMSA assays were performed with 200–400 fold molar excess of unlabeled consensus MEF-2 probe or a mutated MEF-2 probe. As seen in Figure 7C, unlabelled consensus MEF-2, but not the mutant form, effectively competed with the MEF-2 probe derived from the HTLV-1 LTR for protein binding. This result strongly supports the idea that MEF-2 directly binds to the imperfect MEF-2 site in the HTLV-1 LTR, just upstream of the TATA box. To ascertain the functional significance of this MEF-2 site, the first three nucleotides were mutated (CTA- > ACG) in the context of the HTLV-1 LTR luciferase reporter. Luciferase assays were conducted to examine Tax activation of a wild-type HTLV-1 LTR and the MEF-2 mutated LTR. As expected, Tax strongly activated the HTLV-1 LTR; however, Tax-induced activation of the MEF-2 mutant LTR was significantly decreased even though Tax expression was similar (Figure 7D). Together, these results indicate that MEF-2 directly binds to DNA in the HTLV-1 LTR and is important for Tax activation of the LTR.Figure 7


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)

Assessment of MEF-2 binding site(s) within the HTLV-1 LTR. (A) HTLV-1 LTR nucleotide sequence with two putative MEF-2 binding sites shown in blue and red. The HTLV-1 LTR comprises U3 (unique 3′), R (Repeated), and U5 (unique 5′) regions. The U3 region regulates viral gene expression via three 21 bp repeats known as Tax-responsive element - 1 (TRE - 1), which confers Tax-based trans-activation. These repeats contain 3 conserved domains labeled A, B and C. The location for ATF/CREB binding and putative MEF-2 binding are illustrated. (B) EMSA was performed with a probe corresponding to the MEF-2 site in the HTLV-1 LTR using nuclear extracts from Jurkat, MT-2, MT-4 and C8166 cells. (C) EMSA competition assay was performed using nuclear extracts from MT-2 cells, with increasing amounts of unlabeled consensus MEF-2 specific probe (200, 300 and 400 fold molar excess respectively) or a mutated MEF-2 specific probe. Oct-1 was used as a loading control. (D) HTLV-1 LTR luciferase assays in 293 T cells transfected with empty vector (EV) or Flag-Tax using LTR luciferase WT plasmid (LTR Luc WT) or a MEF-2-specific binding mutant (LTR Luc MEF-2 Mut). Luciferase values are presented as “fold induction” relative to the control (EV). Two-tailed unpaired t-test was performed with Prism software. Error bars represent the standard deviation of triplicate samples. The level of significance was defined as: ***p < 0.001. Western blots were performed with anti-Flag and anti-β-actin using whole-cell lysates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Assessment of MEF-2 binding site(s) within the HTLV-1 LTR. (A) HTLV-1 LTR nucleotide sequence with two putative MEF-2 binding sites shown in blue and red. The HTLV-1 LTR comprises U3 (unique 3′), R (Repeated), and U5 (unique 5′) regions. The U3 region regulates viral gene expression via three 21 bp repeats known as Tax-responsive element - 1 (TRE - 1), which confers Tax-based trans-activation. These repeats contain 3 conserved domains labeled A, B and C. The location for ATF/CREB binding and putative MEF-2 binding are illustrated. (B) EMSA was performed with a probe corresponding to the MEF-2 site in the HTLV-1 LTR using nuclear extracts from Jurkat, MT-2, MT-4 and C8166 cells. (C) EMSA competition assay was performed using nuclear extracts from MT-2 cells, with increasing amounts of unlabeled consensus MEF-2 specific probe (200, 300 and 400 fold molar excess respectively) or a mutated MEF-2 specific probe. Oct-1 was used as a loading control. (D) HTLV-1 LTR luciferase assays in 293 T cells transfected with empty vector (EV) or Flag-Tax using LTR luciferase WT plasmid (LTR Luc WT) or a MEF-2-specific binding mutant (LTR Luc MEF-2 Mut). Luciferase values are presented as “fold induction” relative to the control (EV). Two-tailed unpaired t-test was performed with Prism software. Error bars represent the standard deviation of triplicate samples. The level of significance was defined as: ***p < 0.001. Western blots were performed with anti-Flag and anti-β-actin using whole-cell lysates.
Mentions: Results from the competitive promoter assays and CHIP assays confirmed that MEF-2 is recruited to the HTLV-1 LTR suggesting the presence of a MEF-2 binding site(s) within the LTR. Therefore, we examined the consensus site for all MEF-2 isoforms CTA(T/A)4TA(G/A)C within the LTR and identified two imperfect MEF-2 sites - one just downstream of repeat III of the Tax-response element (TRE) and upstream of the TATA box (Figure 7A, blue highlight) that matches 7/10 bases of the consensus site; the other overlaps with the TATA box (Figure 7B, red highlight), which is highly unlikely to be functional due to competition with general TFs. To determine if MEF-2 interacted with the more upstream MEF-2 site in the HTLV-1 LTR, a double-stranded DNA probe corresponding to this site was generated and used for EMSA DNA binding assays. EMSA revealed a specific protein/DNA complex with nuclear extracts from the HTLV-1 transformed cell lines MT-2, MT-4 and C8166, but not Jurkat cells (Figure 7B). To demonstrate that this protein complex consisted of MEF-2, competition EMSA assays were performed with 200–400 fold molar excess of unlabeled consensus MEF-2 probe or a mutated MEF-2 probe. As seen in Figure 7C, unlabelled consensus MEF-2, but not the mutant form, effectively competed with the MEF-2 probe derived from the HTLV-1 LTR for protein binding. This result strongly supports the idea that MEF-2 directly binds to the imperfect MEF-2 site in the HTLV-1 LTR, just upstream of the TATA box. To ascertain the functional significance of this MEF-2 site, the first three nucleotides were mutated (CTA- > ACG) in the context of the HTLV-1 LTR luciferase reporter. Luciferase assays were conducted to examine Tax activation of a wild-type HTLV-1 LTR and the MEF-2 mutated LTR. As expected, Tax strongly activated the HTLV-1 LTR; however, Tax-induced activation of the MEF-2 mutant LTR was significantly decreased even though Tax expression was similar (Figure 7D). Together, these results indicate that MEF-2 directly binds to DNA in the HTLV-1 LTR and is important for Tax activation of the LTR.Figure 7

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