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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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HTLV-1 LTR transcriptional activation complexes contain MEF-2. Binding of various transcription factors present in the nuclear extract isolated from MT-2 cells to the HTLV-1 LTR was assessed using the Promoter-Binding Transcription Factor Profiling Array I upon knocking down MEF-2A (A) and Tax (B) as described in Methods. The nuclear extract was incubated with oligonucleotide probe mix with the HTLV-1 LTR or a control DNA. The binding of each transcription factor to LTR was indicated by average reduction in chemiluminescence of transcription factor-specific oligonucleotide probe specific to each factor from triplicate samples.
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Fig6: HTLV-1 LTR transcriptional activation complexes contain MEF-2. Binding of various transcription factors present in the nuclear extract isolated from MT-2 cells to the HTLV-1 LTR was assessed using the Promoter-Binding Transcription Factor Profiling Array I upon knocking down MEF-2A (A) and Tax (B) as described in Methods. The nuclear extract was incubated with oligonucleotide probe mix with the HTLV-1 LTR or a control DNA. The binding of each transcription factor to LTR was indicated by average reduction in chemiluminescence of transcription factor-specific oligonucleotide probe specific to each factor from triplicate samples.

Mentions: In order to confirm that HTLV-1 LTR transcriptional complexes contain MEF-2, we performed a promoter-binding TF profiling assay. Use of this assay offers the advantage of analyzing multiple TFs at once as opposed to electrophoretic mobility shift assay, which enables characterization of only a single TF at a time. This assay is based upon the fact that if a TF binds the LTR, then the binding of that factor with its oligonucleotide is reduced due to competition between the probe and LTR. Of the 50 total TFs, only 19 including MEF-2 showed an ability to compete for binding to the LTR at significant levels (Figure 6). Observed binding of CREB validated the assay, since it is an established factor that interacts with Tax and facilitates transactivation process [29,69-71]. Some other cellular factors were identified (i.e. AR, Brn-3, Pbx1, etc.) whose roles remain to be tested in HTLV-1 pathogenesis. Similarly, 31 cellular factors (i.e. ATF2, CAR, EGR, etc.) did not show significant competitive inhibition of LTR binding within MT-2 cells. These observations were made in the setting of active infection where both Tax and MEF-2 were readily available to mediate the process.Figure 6


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)

HTLV-1 LTR transcriptional activation complexes contain MEF-2. Binding of various transcription factors present in the nuclear extract isolated from MT-2 cells to the HTLV-1 LTR was assessed using the Promoter-Binding Transcription Factor Profiling Array I upon knocking down MEF-2A (A) and Tax (B) as described in Methods. The nuclear extract was incubated with oligonucleotide probe mix with the HTLV-1 LTR or a control DNA. The binding of each transcription factor to LTR was indicated by average reduction in chemiluminescence of transcription factor-specific oligonucleotide probe specific to each factor from triplicate samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: HTLV-1 LTR transcriptional activation complexes contain MEF-2. Binding of various transcription factors present in the nuclear extract isolated from MT-2 cells to the HTLV-1 LTR was assessed using the Promoter-Binding Transcription Factor Profiling Array I upon knocking down MEF-2A (A) and Tax (B) as described in Methods. The nuclear extract was incubated with oligonucleotide probe mix with the HTLV-1 LTR or a control DNA. The binding of each transcription factor to LTR was indicated by average reduction in chemiluminescence of transcription factor-specific oligonucleotide probe specific to each factor from triplicate samples.
Mentions: In order to confirm that HTLV-1 LTR transcriptional complexes contain MEF-2, we performed a promoter-binding TF profiling assay. Use of this assay offers the advantage of analyzing multiple TFs at once as opposed to electrophoretic mobility shift assay, which enables characterization of only a single TF at a time. This assay is based upon the fact that if a TF binds the LTR, then the binding of that factor with its oligonucleotide is reduced due to competition between the probe and LTR. Of the 50 total TFs, only 19 including MEF-2 showed an ability to compete for binding to the LTR at significant levels (Figure 6). Observed binding of CREB validated the assay, since it is an established factor that interacts with Tax and facilitates transactivation process [29,69-71]. Some other cellular factors were identified (i.e. AR, Brn-3, Pbx1, etc.) whose roles remain to be tested in HTLV-1 pathogenesis. Similarly, 31 cellular factors (i.e. ATF2, CAR, EGR, etc.) did not show significant competitive inhibition of LTR binding within MT-2 cells. These observations were made in the setting of active infection where both Tax and MEF-2 were readily available to mediate the process.Figure 6

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