Figure 6.
Figure 6. Phf6 activates downstream effectors of TNFα signaling. (A) RT-qPCR analysis of Nr4a1 in control (pMYs) or Phf6-transduced LSK cells. (B) ChIP qPCR analysis using the PHF6 antibody in K562 cells. The relative amount of immunoprecipitated DNA is depicted as a percentage of input DNA. Data are shown as the mean plus or minus SEM of triplicate PCRs. Significance is shown relative to the immunoglobulin G (IgG) control. (C) Gain-of-function analysis of the Nr4a1 gene. HSCs were transduced with a control retrovirus (pMIG) or a retrovirus expressing Nr4a1 (Nr4a1). After GFP sorting, GFP+ cells were subjected to a RT-qPCR analysis (left panel) and proliferation assay (right panel). In the proliferation assay, GFP+ cells were cultured under HSC culture conditions (SCF+TPO) for 12 days. (D) Loss-of-function analysis of the Nr4a1 gene. HSCs were transduced with lentiviruses generating shRNAs against Luciferase (Luc) or Nr4a1, and GFP+ cells were then processed as shown in panel C. (E) Effects of Nr4a1 expression on VC;Phf6Δ/y HSCs in vivo. HSCs were sorted from VC or VC;Phf6Δ/y mice, transduced with a control retrovirus (pMIG), or a retrovirus expressing Nr4a1 (Nr4a1), and transplanted into irradiated CD45.1 recipients together with CD45.1 rescue BM cells. The chimerism of GFP+CD45.2+ donor-derived cells in PB (left panel) and BM HSCs 8 weeks after transplantation (right panel) is shown (n = 5 or 6). The chimerism at 0 weeks shows the transduction efficiency of HSCs examined just before transplantation. (F) Pie chart showing the distribution of 75 230 ATAC peaks in HSCs. (G) Scatterplot showing gene-expression changes in Phf6Δ/y HSCs relative to Phf6W/y HSCs and changes in ATAC signals at the proximal promoter regions in VC;Phf6Δ/y HSCs relative to VC HSCs. The correlation coefficient between changes in expression and ATAC signals as well as the linear regression are shown in red. (H) Gene tracks showing ATAC peaks in VC and VC;Phf6Δ/y HSCs at the Nr4a1 locus. The x-axis indicates the linear sequence of genomic DNA, and the y-axis represents the total number of mapped reads. rDNA, ribosomal DNA; TTS, transcription termination site; UTR, untranslated region.

Phf6 activates downstream effectors of TNFα signaling. (A) RT-qPCR analysis of Nr4a1 in control (pMYs) or Phf6-transduced LSK cells. (B) ChIP qPCR analysis using the PHF6 antibody in K562 cells. The relative amount of immunoprecipitated DNA is depicted as a percentage of input DNA. Data are shown as the mean plus or minus SEM of triplicate PCRs. Significance is shown relative to the immunoglobulin G (IgG) control. (C) Gain-of-function analysis of the Nr4a1 gene. HSCs were transduced with a control retrovirus (pMIG) or a retrovirus expressing Nr4a1 (Nr4a1). After GFP sorting, GFP+ cells were subjected to a RT-qPCR analysis (left panel) and proliferation assay (right panel). In the proliferation assay, GFP+ cells were cultured under HSC culture conditions (SCF+TPO) for 12 days. (D) Loss-of-function analysis of the Nr4a1 gene. HSCs were transduced with lentiviruses generating shRNAs against Luciferase (Luc) or Nr4a1, and GFP+ cells were then processed as shown in panel C. (E) Effects of Nr4a1 expression on VC;Phf6Δ/y HSCs in vivo. HSCs were sorted from VC or VC;Phf6Δ/y mice, transduced with a control retrovirus (pMIG), or a retrovirus expressing Nr4a1 (Nr4a1), and transplanted into irradiated CD45.1 recipients together with CD45.1 rescue BM cells. The chimerism of GFP+CD45.2+ donor-derived cells in PB (left panel) and BM HSCs 8 weeks after transplantation (right panel) is shown (n = 5 or 6). The chimerism at 0 weeks shows the transduction efficiency of HSCs examined just before transplantation. (F) Pie chart showing the distribution of 75 230 ATAC peaks in HSCs. (G) Scatterplot showing gene-expression changes in Phf6Δ/y HSCs relative to Phf6W/y HSCs and changes in ATAC signals at the proximal promoter regions in VC;Phf6Δ/y HSCs relative to VC HSCs. The correlation coefficient between changes in expression and ATAC signals as well as the linear regression are shown in red. (H) Gene tracks showing ATAC peaks in VC and VC;Phf6Δ/y HSCs at the Nr4a1 locus. The x-axis indicates the linear sequence of genomic DNA, and the y-axis represents the total number of mapped reads. rDNA, ribosomal DNA; TTS, transcription termination site; UTR, untranslated region.

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