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. 2014 Dec 22;3(12):e135. doi: 10.1038/oncsis.2014.49

Figure 4.

Figure 4

HoxA9 interacts with the proximal CDX4 promoter cis element in vivo. (a) In vivo interaction of HoxA9 with the proximal CDX4 cis element increases during differentiation of U937 cells. U937 cells were analyzed by chromatin co-immunoprecipitation with antibody to HoxA9, HoxA10 or irrelevant control antibody. Precipitating chromatin was amplified by real-time PCR with primers flanking regions of the CDX4 gene. Cells were analyzed with or without differentiation. Statistically significant differences in co-precipitation of chromatin sequences are indicated by * and *** for HoxA9 antibody versus HoxA10 antibody; ** and # for differentiated versus undifferentiated cells. Differences of P<0.01 were considered statistically significant. (b) In vivo interaction of HoxA9 with the proximal CDX4 cis element increases during ex vivo differentiation of murine bone marrow cells. Bone marrow mononuclear cells were harvested from wild-type mice and cultured in GM-CSF, interleukin 3 (IL3) and Scf followed by separation of CD34+ cells (myeloid progenitor conditions) or differentiation with G-CSF. Chromatin was co-precipitated and analyzed as above. Statistically significant differences in co-precipitation of chromatin sequences are indicated by * and *** for HoxA9 antibody versus HoxA10 antibody, ** and # for G-CSF-differentiated cells versus cells cultured under myeloid progenitor conditions. (c) Constitutively active Shp2 blocks binding of HoxA9 to the CDX4 promoter during myeloid differentiation. Bone marrow mononuclear cells were harvested from mice, transduced with a retroviral vector to express E76K-Shp2 (or control vector) and cultured in GM-CSF, IL3 and Scf followed by separation of CD34+ cells (myeloid progenitor conditions) or differentiation with G-CSF. Chromatin was co-precipitated and analyzed by real-time PCR with primers flanking the proximal, HoxA9-binding CDX4 cis element. Statistically significant differences with versus without expression of E76K-Shp2 are indicated by * or **. (d) Constitutively active Shp2 increases the binding of HoxA10 to the CDX4 promoter during myeloid differentiation. Bone marrow cells were transduced and cultured as described above and co-precipitating chromatin was analyzed for binding to the distal, HoxA10-binding CDX4 cis element. Statistically significant differences with versus without expression of E76K-Shp2 are indicated by *. (e) Constitutive Shp2 activity decreases tyrosine phosphorylation of HoxA9 and HoxA10 in murine bone marrow myeloid progenitor cells undergoing G-CSF-induced differentiation. Murine bone marrow cells were transduced and treated as described above. Cell lysates were immunoprecipitated with an antibody to phosphotyrosine and immunoprecipitates were analyzed by western blots probed for HoxA9 or HoxA10. Equivalence of protein in the immunoprecipitation samples is addressed in f. (f) Shp2 expression is increased in cells transduced with E76K-Shp2 expression vector. Lysates from the cells described in e above were also analyzed for by western blots probed with antibodies to Shp2 and Gapdh (as a loading control).