β-Catenin activates the HOXA10 and CDX4 genes in myeloid progenitor cells

Abstract

HoxA10 is a homeodomain transcription factor that is involved in maintenance of the myeloid progenitor population and implicated in myeloid leukemogenesis. Previously, we found that FGF2 and CDX4 are direct target genes of HoxA10 and that HOXA10 is a Cdx4 target gene. We also found that increased production of fibroblast growth factor 2 (Fgf2) by HoxA10-overexpressing myeloid progenitor cells results in activation of β-catenin in an autocrine manner. In this study, we identify novel cis elements in the CDX4 and HOXA10 genes that are activated by β-catenin in myeloid progenitor cells. We determine that β-catenin interacts with these cis elements, identifying both CDX4 and HOXA10 as β-catenin target genes in this context. We demonstrate that HoxA10-induced CDX4 transcription is influenced by Fgf2-dependent β-catenin activation. Similarly, Cdx4-induced HOXA10 transcription is influenced by β-catenin in an Fgf2-dependent manner. Increased expression of a set of Hox proteins, including HoxA10, is associated with poor prognosis in acute myeloid leukemia. Cdx4 contributes to leukemogenesis in Hox-overexpressing acute myeloid leukemia, and increased β-catenin activity is also associated with poor prognosis. The current studies identify a molecular mechanisms through which increased expression of HoxA10 increases Cdx4 expression by direct CDX4 activation and by Fgf2-induced β-catenin activity. This results in Cdx4-induced HoxA10-expression, creating a positive feedback mechanism.

FIGURE 1.

β-Catenin increases Cdx4 and HoxA10 expression in myeloid progenitor cells. A, increased β-catenin expression increases Cdx4 and HoxA10 mRNA in myeloid cell lines. U937 cells were stably transfected with a vector to overexpress β-catenin, with an empty control vector, or with a vector to express β-catenin-specific shRNAs or scrambled control shRNA. Expression of Cdx4 and HoxA10 mRNA was determined by real-time PCR. *, **, *** and #, ##, or ###, statistically significant differences in gene expression (p < 0.001, n = 6). B, increased β-catenin expression increases Cdx4 and HoxA10 protein in myeloid cell lines. The stable transfectants referred to above were also analyzed for protein expression. Western blots (WB) of total cell lysates were probed with antibodies to β-catenin, Cdx4, HoxA10, and Gapdh (as a loading control). C, increased β-catenin expression increases Cdx4 and HoxA10 mRNA in primary bone marrow cells. Bone marrow cells were isolated from WT mice and transduced with a retroviral vector to express β-catenin or with control vector. Cells were cultured in GM-CSF, IL-3, and SCF, and CD34⁺ cells were separated. Expression of Cdx4, HoxA10, and β-catenin was determined by real-time PCR. *, **, or ***, statistically significant differences in gene expression (p < 0.001, n = 3). D, increased β-catenin expression increases Cdx4 and HoxA10 protein in primary bone marrow cells. The bone marrow cells described above were also analyzed for protein expression. Western blots of lysate proteins were probed with antibodies to Cdx4, HoxA10, β-catenin, and Gapdh (as a loading control). Error bars, S.E.

FIGURE 2.

β-Catenin activates the CDX4 promoter in myeloid progenitor cells. A, sequence homology between the human and murine CDX4 promoters. A β-catenin-Lef DNA-binding consensus sequence in the human CDX4 promoter has a homologous sequence in the murine CDX4 promoter. Human sequence is in black, murine sequence is in blue, and the consensus sequence is in red. B, β-catenin overexpression activates the CDX4 promoter. U937 cells were co-transfected with a series of reporter gene constructs with sequence from the CDX4 5′-flank and a vector to overexpress β-catenin (or control vector). Transfectants were analyzed for luciferase reporter activity. *, **, or ***, statistically significant differences in reporter activity (p < 0.0001, n = 6). C, β-catenin overexpression increased activity of the HoxA10-binding CDX4 cis element in a HoxA10-dependent manner. U937 cells were co-transfected with a construct with three copies of the −139 to −145 bp sequence from the CDX4 promoter linked to a minimal promoter and reporter and a vector to overexpress β-catenin (or control vector) and a HoxA10-specific shRNA (or scrambled shRNA control vector). Transfectants were analyzed for luciferase reporter activity. *, **, or ***, statistically significant differences in reporter activity (p < 0.0001, n = 6). D, HoxA10 knockdown does not influence activation of a β-catenin-binding cis element. U937 cells were co-transfected with a construct with four copies of a β-catenin/Lef binding consensus sequence linked to a minimal promoter and reporter (TopFlash) or non-binding control vector (FopFlash), a vector to express β-catenin (or control vector), and a HoxA10-specific shRNA (or scrambled shRNA control vector). The HoxA10-specific shRNA vector was used at a level that did not alone influence activity of the −139 to −145 bp CDX4 cis element but blocked activation by overexpressed β-catenin. *, statistically significant differences (p < 0.001, n = 4). E, β-catenin activates a specific CDX4 cis element. U937 cells were co-transfected with a construct with three copies of the −1028 to −1035 bp CDX4 promoter sequence linked to a minimal promoter and a reporter and a vector to overexpress β-catenin (or control vector), a β-catenin-specific shRNA, both, or control vectors. Transfectants were analyzed for luciferase reporter activity. * or **, statistically significant differences in reporter activity (p < 0.0001, n = 6). Error bars, S.E.

FIGURE 3.

β-Catenin activates the HOXA10 promoter in myeloid progenitor cells. A, sequence homology between the human and murine HOXA10 promoters. A β-catenin-Lef DNA-binding consensus sequence in the human HOXA10 promoter has a homologous sequence in the murine HOXA10 promoter. The human sequence is in black, the murine sequence is in blue, and the consensus sequence is in red. B, β-catenin overexpression activates the HOXA10 promoter. U937 cells were co-transfected with a series of reporter gene constructs with sequence from the HOXA10 5′-flank and a vector to overexpress β-catenin (or control vector). Transfectants were analyzed for luciferase reporter activity. *, **, or ***, statistically significant differences in reporter activity (p < 0.002, n = 6). C, β-catenin overexpression increased activity of the Cdx4-binding HOXA10 cis element in a Cdx4-dependent manner. U937 cells were co-transfected with a construct with three copies of the −129 to −136 bp sequence from the HOXA10 promoter linked to a minimal promoter and reporter and a vector to overexpress β-catenin (or control vector) and a Cdx4-specific shRNA (or scrambled shRNA control vector). Transfectants were analyzed for luciferase reporter activity. *, **, or ***, statistically significant differences in reporter activity (p < 0.001, n = 6). D, Cdx4 knockdown does not influence activation of a β-catenin-binding cis element. U937 cells were co-transfected with a construct with four copies of a β-catenin/Lef binding consensus sequence linked to a minimal promoter and reporter (TopFlash) or non-binding control vector (FopFlash), a vector to express β-catenin (or control vector), and a Cdx4-specific shRNA (or scrambled shRNA control vector). The Cdx4-specific shRNA vector was used at a level that did not alone influence activity of the −129 to −136 bp HOXA10 cis element but blocked activation by overexpressed β-catenin. *, statistically significant differences (p < 0.001, n = 4). E, β-catenin activates a specific HOXA10 cis element. U937 cells were co-transfected with a construct with two copies of the −800 to −830 bp HOXA10 promoter sequence linked to a minimal promoter and a reporter and a vector to overexpress β-catenin (or control vector), a β-catenin-specific shRNA, both, or control vectors. Transfectants were analyzed for luciferase reporter activity. * or **, statistically significant differences in reporter activity (p < 0.01, n = 6). Error bars, S.E.

FIGURE 4.

β-Catenin interacts with the CDX4 and HOXA10 promoters in myeloid cells. A, β-catenin interacts with the CDX4 promoter in vitro. EMSAs were performed with a double-stranded oligonucleotide probe representing −1020 to −1045 bp sequence from the CDX4 promoter and nuclear proteins from U937 cells. Some binding assays were preincubated with double-stranded oligonucleotide competitors. Lane 1, no competitor; lane 2, homologous oligonucleotide; lane 3, homologous oligonucleotide with mutation in the β-catenin-Lef consensus sequence; lane 4, another oligonucleotide with the β-catenin-Lef consensus sequence; lane 5, a mutant form of this oligonucleotide; lane 6, oligonucleotide representing the HoxA10 binding (−139 to −146 bp) CDX4 sequence; lane 7, irrelevant oligonucleotide from the CDX4 5′-flank; lane 8, β-catenin antibody; lane 9, control irrelevant antibody. B, β-catenin interacts with the HOXA10 promoter in vitro. EMSAs were performed with a double-stranded oligonucleotide probe representing −800 to −835 bp sequence from the HOXA10 promoter and nuclear proteins from U937 cells. Some binding assays were preincubated with double-stranded oligonucleotide competitors. Lane 1, no competitor; lane 2, homologous oligonucleotide; lane 3, homologous oligonucleotide with mutation in the β-catenin-Lef consensus sequence; lane 4, another oligonucleotide with the β-catenin-Lef consensus sequence; lane 5, a mutant form of this oligonucleotide; lane 6, oligonucleotide representing the Cdx4-binding (−129 to −136 bp) HOXA10 sequence; lane 7, irrelevant oligonucleotide from the HOXA10 5′-flank; lane 8, β-catenin antibody; lane 9, control irrelevant antibody. C, β-catenin interacts with the CDX4 and HOXA10 promoters in vivo. Chromatin was immunoprecipitated from U937 lysates with an antibody to β-catenin or control antibody. Co-precipitating chromatin was amplified with primer sets representing sequences from the CXD4 and HOXA10 promoters. * or **, statistically significant differences (p < 0.0001, n = 3). Error bars, S.E.

FIGURE 5.

Expression of HoxA10, Cdx4, and β-catenin is interdependent in myeloid leukemia cells. A, representation of influences of HoxA10, Cdx4, and β-catenin on each other. The schematic represents the interrelationship between HoxA10, Cdx4, and β-catenin. Black arrows, influence on gene transcription; purple arrows, signal transduction pathways. B, HoxA10 influences Cdx4 expression in an Fgf2-dependent manner, and Cdx4 influences HoxA10 expression in an Fgf2-dependent manner. U937 stable transfectants with vectors to express HoxA10 or Cdx4 or with empty vector control were analyzed after treatment with Fgf2-blocking antibody or control antibody. Real-time PCR was used to determine mRNA expression. * or **, statistically significant differences (p < 0.003, n = 3). C, HoxA10 influences Cdx4-expression in a β-catenin-dependent manner, and Cdx4 influences HoxA10 expression in a β-catenin-dependent manner. U937 stable transfectants were generated with vectors to express β-catenin, HoxA10, Cdx4, β-catenin-specific shRNAs, HoxA10 + β-catenin-specific shRNAs, Cdx4 + β-catenin-specific shRNAs, or control vectors. Cells were analyzed for mRNA expression by real-time PCR. *, **, #, or ##, statistically significant differences (p < 0.0001, n = 3). D, both total and serine 552-phosphorylated β-catenin are increased by Fgf2. U937 cells were treated with Fgf2, and cell lysate proteins were analyzed by Western blots serially probed with antibodies to Ser(P)-552 β-catenin, β-catenin, or tubulin (as a loading control). Error bars, S.E.

FIGURE 6.

Expression of HoxA10, Cdx4, and β-catenin is interdependent in primary murine myeloid progenitor cells. A, β-catenin influences Cdx4 expression in a HoxA10-dependent manner in primary bone marrow cells. Bone marrow cells were isolated from WT, HoxA10^+/−, or HoxA10^−/− mice and transduced with a retroviral vector to express β-catenin or with control vector. Cells were cultured in GM-CSF, IL-3, and SCF, and CD34⁺ cells were separated. Expression of Cdx4, HoxA10, and β-catenin was determined by real-time PCR. *, **, or ***, statistically significant differences in gene expression (p < 0.0001, n = 3). B, Fgf2 influences β-catenin-induced HoxA10 and Cdx4 expression in primary bone marrow cells. Some of the cells described above were treated with Fgf2-blocking antibody or control irrelevant antibody. Expression of Cdx4, HoxA10, and β-catenin was determined by real-time PCR. *, **, ***, #, ##, ###, &, &&, or &&&, statistically significant differences in gene expression (p < 0.002, n = 3). Error bars, S.E.

FIGURE 7.

Cytokine hypersensitivity of Cdx4-overexpressing myeloid cells is influenced by Fgf2 and β-catenin. A, cytokine hypersensitivity of Cdx4-overexpressing myeloid cells is influenced by Fgf2. U937 cells were stably transfected with a vector to express Cdx4 or HoxA10 or with control vector. Cells were deprived of all cytokines for 24 h and stimulated with a dose titration of FCS with or without an Fgf2-blocking antibody. Proliferation was determined by incorporation of [³H]thymidine over 24 h. *, **, or ***, statistically significant differences in proliferation at a given FCS concentration. B, cytokine hypersensitivity of Cdx4-overexpressing myeloid cells is influenced by β-catenin. U937 cells were stably transfected with a vector to express Cdx4, HoxA10, or control vector plus a vector to express β-catenin-specific shRNAs or scrambled control shRNA. Cells were deprived of all cytokines for 24 h and stimulated with a dose titration of FCS. Proliferation was determined by incorporation of [³H]thymidine over 24 h. *, **, or ***, statistically significant differences in proliferation at a given FCS concentration. Error bars, S.E.