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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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Related in: MedlinePlus

MS4A3is strongly repressed by EVI1 in human myeloid cells. A) Heatmap summarizing expression changes of 56 genes affected by induction of EVI1 in U937T_EVI1-HA cells (clones E10 and E14) as determined by microarray analyses at different time points after transfer to tetracycline (tet) free media. Parental U937T cells and U937T_vec (clone P2) cells incubated with or without tet for 48 h were used as controls. Log2 transformed expression changes relative to cultures maintained in the presence of tet (red, upregulated; blue, downregulated) are shown in descending order. B) qRT-PCR confirmed repression of MS4A3 in U937T_EVI1-HA, but not U937T_vec cells after tet withdrawal. C, D) qRT-PCR showing EVI1-mediated down-regulation of MS4A3 in U937 (C) or HL-60 (D) cells constitutively expressing ectopic EVI1. E) qRT-PCR showing induction of MS4A3 after siRNA mediated down-regulation of EVI1 in UCSD-AML1 cells. Data in B-E represent means + SEMs from at least three independent biological replicate experiments. F) MS4A3 mRNA levels in a panel of 12 human myeloid cell lines (8 with low and 4 with high EVI1 expression) represented in GEO data set GSE35159 [54]. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test, two-tailed). The induction of MS4A3 after knock-down of EVI1 in UCSD-AML1 cells was not significant, but an at least 1.8-fold up-regulation was observed in four out of four independent biological replicate experiments.
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Fig1: MS4A3is strongly repressed by EVI1 in human myeloid cells. A) Heatmap summarizing expression changes of 56 genes affected by induction of EVI1 in U937T_EVI1-HA cells (clones E10 and E14) as determined by microarray analyses at different time points after transfer to tetracycline (tet) free media. Parental U937T cells and U937T_vec (clone P2) cells incubated with or without tet for 48 h were used as controls. Log2 transformed expression changes relative to cultures maintained in the presence of tet (red, upregulated; blue, downregulated) are shown in descending order. B) qRT-PCR confirmed repression of MS4A3 in U937T_EVI1-HA, but not U937T_vec cells after tet withdrawal. C, D) qRT-PCR showing EVI1-mediated down-regulation of MS4A3 in U937 (C) or HL-60 (D) cells constitutively expressing ectopic EVI1. E) qRT-PCR showing induction of MS4A3 after siRNA mediated down-regulation of EVI1 in UCSD-AML1 cells. Data in B-E represent means + SEMs from at least three independent biological replicate experiments. F) MS4A3 mRNA levels in a panel of 12 human myeloid cell lines (8 with low and 4 with high EVI1 expression) represented in GEO data set GSE35159 [54]. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test, two-tailed). The induction of MS4A3 after knock-down of EVI1 in UCSD-AML1 cells was not significant, but an at least 1.8-fold up-regulation was observed in four out of four independent biological replicate experiments.

Mentions: We have previously established U937T_EVI1-HA clones E10 and E14, which express an HA epitope-tagged version of the human EVI1 cDNA in a tetracycline (tet) repressible manner in the background of the human myeloid cell line U937 [34]. In E10 and E14 cells, expression of the EVI1 protein is strongly induced as early as 12 h after tet withdrawal, is sustained for at least 120 h, and its peak levels are comparable to those in HNT-34 cells [34], which express endogenous EVI1 due to a rearrangement of its gene locus at 3q26 [52]. In order to identify genes whose mRNA levels were altered rapidly in response to induction of EVI1, E10 and E14 cells were cultured in the absence or presence of tet for 6, 12, 24, and 48 h, RNA was extracted, converted to cRNA, and hybridized to Human Genome U133 Plus 2.0 arrays (Affymetrix). As controls, parental U937T cells and empty vector transfected U937T_vec (clone P2) cells that had been incubated with or without tet for 48 h were processed in the same manner. Tet withdrawal affected gene expression patterns not only in EVI1-expressing, but also in control cells [53]. Consequently, only those genes were considered to be regulated by EVI1 whose mRNA levels changed at least 2-fold 48 h after tet withdrawal in both E10 and E14 cells, and whose induction or repression at this time point exceeded any background effects observed in either U937T or P2 cells as described in the Methods section. According to these criteria, 56 unique genes were found to be responsive to EVI1: 27 genes were up-, and 29 genes were down-regulated subsequent to the induction of this transcription factor (Figure 1A). Gene ontology (GO) analysis revealed significant enrichment of the terms “cytokine biosynthetic process” and “regulation of apoptosis” among the EVI1-regulated genes (Additional file 1: Table S1). The gene most strongly induced by EVI1 in this system was CD52, which has previously been shown to be up-regulated by EVI1 and proposed as an immunotherapeutic target for EVI1-positive leukemia [54]. On the other hand, the most strongly (~16-fold) repressed gene was MS4A3, which has been reported as a negative regulator of the mitotic cycle of hematopoietic cells [50], and was also strongly repressed in response to inducible ectopic expression of Evi1 in primary murine hematopoietic cells [29]. Even though a region ~4 kb from the transcriptional start site of the murine Ms4a3 gene was found to be bound by EVI1 in a ChiP-seq screen [39], the precise mechanistic basis and biological consequences of the repression of MS4A3 by EVI1 have so far not been investigated.Figure 1


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)

MS4A3is strongly repressed by EVI1 in human myeloid cells. A) Heatmap summarizing expression changes of 56 genes affected by induction of EVI1 in U937T_EVI1-HA cells (clones E10 and E14) as determined by microarray analyses at different time points after transfer to tetracycline (tet) free media. Parental U937T cells and U937T_vec (clone P2) cells incubated with or without tet for 48 h were used as controls. Log2 transformed expression changes relative to cultures maintained in the presence of tet (red, upregulated; blue, downregulated) are shown in descending order. B) qRT-PCR confirmed repression of MS4A3 in U937T_EVI1-HA, but not U937T_vec cells after tet withdrawal. C, D) qRT-PCR showing EVI1-mediated down-regulation of MS4A3 in U937 (C) or HL-60 (D) cells constitutively expressing ectopic EVI1. E) qRT-PCR showing induction of MS4A3 after siRNA mediated down-regulation of EVI1 in UCSD-AML1 cells. Data in B-E represent means + SEMs from at least three independent biological replicate experiments. F) MS4A3 mRNA levels in a panel of 12 human myeloid cell lines (8 with low and 4 with high EVI1 expression) represented in GEO data set GSE35159 [54]. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test, two-tailed). The induction of MS4A3 after knock-down of EVI1 in UCSD-AML1 cells was not significant, but an at least 1.8-fold up-regulation was observed in four out of four independent biological replicate experiments.
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Related In: Results  -  Collection

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Fig1: MS4A3is strongly repressed by EVI1 in human myeloid cells. A) Heatmap summarizing expression changes of 56 genes affected by induction of EVI1 in U937T_EVI1-HA cells (clones E10 and E14) as determined by microarray analyses at different time points after transfer to tetracycline (tet) free media. Parental U937T cells and U937T_vec (clone P2) cells incubated with or without tet for 48 h were used as controls. Log2 transformed expression changes relative to cultures maintained in the presence of tet (red, upregulated; blue, downregulated) are shown in descending order. B) qRT-PCR confirmed repression of MS4A3 in U937T_EVI1-HA, but not U937T_vec cells after tet withdrawal. C, D) qRT-PCR showing EVI1-mediated down-regulation of MS4A3 in U937 (C) or HL-60 (D) cells constitutively expressing ectopic EVI1. E) qRT-PCR showing induction of MS4A3 after siRNA mediated down-regulation of EVI1 in UCSD-AML1 cells. Data in B-E represent means + SEMs from at least three independent biological replicate experiments. F) MS4A3 mRNA levels in a panel of 12 human myeloid cell lines (8 with low and 4 with high EVI1 expression) represented in GEO data set GSE35159 [54]. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test, two-tailed). The induction of MS4A3 after knock-down of EVI1 in UCSD-AML1 cells was not significant, but an at least 1.8-fold up-regulation was observed in four out of four independent biological replicate experiments.
Mentions: We have previously established U937T_EVI1-HA clones E10 and E14, which express an HA epitope-tagged version of the human EVI1 cDNA in a tetracycline (tet) repressible manner in the background of the human myeloid cell line U937 [34]. In E10 and E14 cells, expression of the EVI1 protein is strongly induced as early as 12 h after tet withdrawal, is sustained for at least 120 h, and its peak levels are comparable to those in HNT-34 cells [34], which express endogenous EVI1 due to a rearrangement of its gene locus at 3q26 [52]. In order to identify genes whose mRNA levels were altered rapidly in response to induction of EVI1, E10 and E14 cells were cultured in the absence or presence of tet for 6, 12, 24, and 48 h, RNA was extracted, converted to cRNA, and hybridized to Human Genome U133 Plus 2.0 arrays (Affymetrix). As controls, parental U937T cells and empty vector transfected U937T_vec (clone P2) cells that had been incubated with or without tet for 48 h were processed in the same manner. Tet withdrawal affected gene expression patterns not only in EVI1-expressing, but also in control cells [53]. Consequently, only those genes were considered to be regulated by EVI1 whose mRNA levels changed at least 2-fold 48 h after tet withdrawal in both E10 and E14 cells, and whose induction or repression at this time point exceeded any background effects observed in either U937T or P2 cells as described in the Methods section. According to these criteria, 56 unique genes were found to be responsive to EVI1: 27 genes were up-, and 29 genes were down-regulated subsequent to the induction of this transcription factor (Figure 1A). Gene ontology (GO) analysis revealed significant enrichment of the terms “cytokine biosynthetic process” and “regulation of apoptosis” among the EVI1-regulated genes (Additional file 1: Table S1). The gene most strongly induced by EVI1 in this system was CD52, which has previously been shown to be up-regulated by EVI1 and proposed as an immunotherapeutic target for EVI1-positive leukemia [54]. On the other hand, the most strongly (~16-fold) repressed gene was MS4A3, which has been reported as a negative regulator of the mitotic cycle of hematopoietic cells [50], and was also strongly repressed in response to inducible ectopic expression of Evi1 in primary murine hematopoietic cells [29]. Even though a region ~4 kb from the transcriptional start site of the murine Ms4a3 gene was found to be bound by EVI1 in a ChiP-seq screen [39], the precise mechanistic basis and biological consequences of the repression of MS4A3 by EVI1 have so far not been investigated.Figure 1

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