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EVI1 promotes tumor growth via transcriptional repression of MS4A3.

Heller G, Rommer A, Steinleitner K, Etzler J, Hackl H, Heffeter P, Tomasich E, Filipits M, Steinmetz B, Topakian T, Klingenbrunner S, Ziegler B, Spittler A, Zöchbauer-Müller S, Berger W, Wieser R - J Hematol Oncol (2015)

Bottom Line: U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1.The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems.MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. gerwin.heller@meduniwien.ac.at.

ABSTRACT

Background: The transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells.

Methods: U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice.

Results: Gene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis.

Conclusions: Our data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.

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EVI1 regulatesMS4A3by directly binding to a proximal element in its promoter. A) Luciferase assays with MS4A3 promoter deletion constructs. The MS4A3 5′ region, starting from -3213 relative to the transcription start site, and several 5′ deletion variants thereof were cloned into the promoterless Gaussia luciferase reporter vector, pGluc basic. Reporter plasmids and either an EVI1 expression vector (+EVI1; black bars) or empty vector as a control (-EVI1; grey bars) were transfected into U937 cells, and luciferase activity was measured from cell supernatants two days later. pGluc basic without any MS4A3 5′ sequences was used as negative control. B) Similar experiments were performed using some of the above described reporter plasmids with the HSV tk basal promoter inserted between the MS4A3 5′ regions and the luciferase gene of pGluc basic. Data in A) and B) represent means + SEMs from three independent biological replicate experiments. C) ChIP assays were performed on U937_EVI1 and U937_vec cells using two different EVI1 antibodies (AB1, sc-8707X, Santa Cruz; AB2, C50E12, Cell Signaling). Primers used for ChIP PCR amplified a region in the proximal MS4A3 promoter as indicated by the arrows in the upper panel. IgG, negative control using nonspecific IgG; no AB, negative control without antibody; +, input DNA (positive control); -, H2O (negative) PCR control.
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Fig2: EVI1 regulatesMS4A3by directly binding to a proximal element in its promoter. A) Luciferase assays with MS4A3 promoter deletion constructs. The MS4A3 5′ region, starting from -3213 relative to the transcription start site, and several 5′ deletion variants thereof were cloned into the promoterless Gaussia luciferase reporter vector, pGluc basic. Reporter plasmids and either an EVI1 expression vector (+EVI1; black bars) or empty vector as a control (-EVI1; grey bars) were transfected into U937 cells, and luciferase activity was measured from cell supernatants two days later. pGluc basic without any MS4A3 5′ sequences was used as negative control. B) Similar experiments were performed using some of the above described reporter plasmids with the HSV tk basal promoter inserted between the MS4A3 5′ regions and the luciferase gene of pGluc basic. Data in A) and B) represent means + SEMs from three independent biological replicate experiments. C) ChIP assays were performed on U937_EVI1 and U937_vec cells using two different EVI1 antibodies (AB1, sc-8707X, Santa Cruz; AB2, C50E12, Cell Signaling). Primers used for ChIP PCR amplified a region in the proximal MS4A3 promoter as indicated by the arrows in the upper panel. IgG, negative control using nonspecific IgG; no AB, negative control without antibody; +, input DNA (positive control); -, H2O (negative) PCR control.

Mentions: To test whether EVI1 would affect the MS4A3 promoter in a direct manner, and to identify potential EVI1-responsive regions, reporter gene assays were performed. An approximately 3.2 kb fragment representing the upstream region of the human MS4A3 gene was cloned into the promoterless Gaussia luciferase reporter vector, pGluc basic, to yield pMS4A3(-3213/+11)/pGluc. 5′ deletion variants of this vector were prepared in an analogous manner. The reporter plasmids were transfected into U937 cells, along with an EVI1 expression vector or empty vector as a control. As shown in Figure 2A, all MS4A3 reporter constructs, including pMS4A3(-268/-1)/pGluc, were repressed by EVI1, suggesting that EVI1 acted directly on the MS4A3 promoter, and that the 268 proximal base pairs were sufficient to mediate this effect. Deletion of this region from the full length reporter vector yielded pMS4A3(-3213/-279)/pGluc. The absence of repression by EVI1 indicated that the proximal region of the MS4A3 promoter was not only sufficient, but also necessary for the response to EVI1 (Figure 2A). To ensure that the loss of regulation by EVI1 was not simply a consequence of the removal of basal promoter elements, and therefore to a general expression defect in pMS4A3(-3213/-279)/pGluc, equivalent vectors were generated, but with the HSV tk promoter inserted between the MS4A3 upstream regions and the pGluc sequences. As shown in Figure 2B, pMS4A3(-3213/+11)/tk/pGluc, containing the 3.2 kb MS4A3 upstream sequence in front of the tk promoter, was repressed by EVI1. An even stronger effect was observed with pMS4A3(-268/-1)/tk/pGluc, which comprised only the EVI1-responsive proximal promoter region, whereas removal of this region in pMS4A3(-3213/-279)/tk/pGluc reduced the repression by EVI1 to basal levels.Figure 2


EVI1 promotes tumor growth via transcriptional repression of MS4A3.

Heller G, Rommer A, Steinleitner K, Etzler J, Hackl H, Heffeter P, Tomasich E, Filipits M, Steinmetz B, Topakian T, Klingenbrunner S, Ziegler B, Spittler A, Zöchbauer-Müller S, Berger W, Wieser R - J Hematol Oncol (2015)

EVI1 regulatesMS4A3by directly binding to a proximal element in its promoter. A) Luciferase assays with MS4A3 promoter deletion constructs. The MS4A3 5′ region, starting from -3213 relative to the transcription start site, and several 5′ deletion variants thereof were cloned into the promoterless Gaussia luciferase reporter vector, pGluc basic. Reporter plasmids and either an EVI1 expression vector (+EVI1; black bars) or empty vector as a control (-EVI1; grey bars) were transfected into U937 cells, and luciferase activity was measured from cell supernatants two days later. pGluc basic without any MS4A3 5′ sequences was used as negative control. B) Similar experiments were performed using some of the above described reporter plasmids with the HSV tk basal promoter inserted between the MS4A3 5′ regions and the luciferase gene of pGluc basic. Data in A) and B) represent means + SEMs from three independent biological replicate experiments. C) ChIP assays were performed on U937_EVI1 and U937_vec cells using two different EVI1 antibodies (AB1, sc-8707X, Santa Cruz; AB2, C50E12, Cell Signaling). Primers used for ChIP PCR amplified a region in the proximal MS4A3 promoter as indicated by the arrows in the upper panel. IgG, negative control using nonspecific IgG; no AB, negative control without antibody; +, input DNA (positive control); -, H2O (negative) PCR control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389965&req=5

Fig2: EVI1 regulatesMS4A3by directly binding to a proximal element in its promoter. A) Luciferase assays with MS4A3 promoter deletion constructs. The MS4A3 5′ region, starting from -3213 relative to the transcription start site, and several 5′ deletion variants thereof were cloned into the promoterless Gaussia luciferase reporter vector, pGluc basic. Reporter plasmids and either an EVI1 expression vector (+EVI1; black bars) or empty vector as a control (-EVI1; grey bars) were transfected into U937 cells, and luciferase activity was measured from cell supernatants two days later. pGluc basic without any MS4A3 5′ sequences was used as negative control. B) Similar experiments were performed using some of the above described reporter plasmids with the HSV tk basal promoter inserted between the MS4A3 5′ regions and the luciferase gene of pGluc basic. Data in A) and B) represent means + SEMs from three independent biological replicate experiments. C) ChIP assays were performed on U937_EVI1 and U937_vec cells using two different EVI1 antibodies (AB1, sc-8707X, Santa Cruz; AB2, C50E12, Cell Signaling). Primers used for ChIP PCR amplified a region in the proximal MS4A3 promoter as indicated by the arrows in the upper panel. IgG, negative control using nonspecific IgG; no AB, negative control without antibody; +, input DNA (positive control); -, H2O (negative) PCR control.
Mentions: To test whether EVI1 would affect the MS4A3 promoter in a direct manner, and to identify potential EVI1-responsive regions, reporter gene assays were performed. An approximately 3.2 kb fragment representing the upstream region of the human MS4A3 gene was cloned into the promoterless Gaussia luciferase reporter vector, pGluc basic, to yield pMS4A3(-3213/+11)/pGluc. 5′ deletion variants of this vector were prepared in an analogous manner. The reporter plasmids were transfected into U937 cells, along with an EVI1 expression vector or empty vector as a control. As shown in Figure 2A, all MS4A3 reporter constructs, including pMS4A3(-268/-1)/pGluc, were repressed by EVI1, suggesting that EVI1 acted directly on the MS4A3 promoter, and that the 268 proximal base pairs were sufficient to mediate this effect. Deletion of this region from the full length reporter vector yielded pMS4A3(-3213/-279)/pGluc. The absence of repression by EVI1 indicated that the proximal region of the MS4A3 promoter was not only sufficient, but also necessary for the response to EVI1 (Figure 2A). To ensure that the loss of regulation by EVI1 was not simply a consequence of the removal of basal promoter elements, and therefore to a general expression defect in pMS4A3(-3213/-279)/pGluc, equivalent vectors were generated, but with the HSV tk promoter inserted between the MS4A3 upstream regions and the pGluc sequences. As shown in Figure 2B, pMS4A3(-3213/+11)/tk/pGluc, containing the 3.2 kb MS4A3 upstream sequence in front of the tk promoter, was repressed by EVI1. An even stronger effect was observed with pMS4A3(-268/-1)/tk/pGluc, which comprised only the EVI1-responsive proximal promoter region, whereas removal of this region in pMS4A3(-3213/-279)/tk/pGluc reduced the repression by EVI1 to basal levels.Figure 2

Bottom Line: U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1.The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems.MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. gerwin.heller@meduniwien.ac.at.

ABSTRACT

Background: The transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells.

Methods: U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice.

Results: Gene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis.

Conclusions: Our data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.

Show MeSH
Related in: MedlinePlus