Figure 6.
PHF6 regulates ISG expression in hematopoietic stem and progenitor cells. (A) Barcode enrichment plots showing positive correlation between the reactome IFN α/β signaling pathway and the gene-expression changes in Phf6-deleted vs control HPC-1 cells. The horizontal axis shows t statistics for all genes in the Phf6-deleted dataset, whereas vertical lines represent genes in the reactome IFN α/β signaling pathway. Red and blue shaded areas indicate genes that are upregulated and downregulated, respectively, in the Phf6-deleted cells, and worms show the relative enrichment of the IFN signature. The IFN signature is enriched among upregulated genes on the right of the plot. (B) Heat maps of a subset of IFN-stimulated genes comparing Phf6-deleted HPC-1 cells with control HPC-1 cells. Each column represents 1 animal. n = 4 controls (Phf6+/Y;Tie2-creTg/+), n = 4 Phf6lox/Y;Tie2-creTg/+. (C) Number of HPC-1 cells in the bone marrow. n = 10 Phf6-control;Ifnar1−/− (3 Phf6+/Y;Tie2-creTg/+;Ifnar1−/−, 5 Phf6lox/Y;Ifnar1−/−, 2 Phf6+/Y;Ifnar1−/−), n = 11 controls (6 Phf6+/Y;Tie2-creTg/+;Ifnar1+/+, 1 Phf6+/Y;Ifnar1+/+, 1 Phf6lox/Y;Ifnar1+/+, 3 Phf6+/Y;Tie2-creTg/+;Ifnar1+/−), n = 6 Phf6lox/Y;Tie2-creTg/+;Ifnar1+/+, n = 4 Phf6lox/Y;Tie2-creTg/+;Ifnar1+/−, and n = 7 Phf6lox/Y;Tie2-creTg/+;Ifnar1−/−. (D) Percentage of Ki67+ cells in the indicated populations. n = 8 Phf6-control;Ifnar1−/− (3 Phf6+/Y;Tie2-creTg/+;Ifnar1−/−, 3 Phf6lox/Y;Ifnar1−/−, 2 Phf6+/Y;Ifnar1−/−), n = 10 controls (6 Phf6+/Y;Tie2-creTg/+;Ifnar1+/+, 1 Phf6+/Y;Ifnar1+/+, 1 Phf6lox/Y;Ifnar1+/−, 2 Phf6+/Y;Tie2-creTg/+;Ifnar1+/−), n = 7 Phf6lox/Y;Tie2-creTg/+;Ifnar1+/+, n = 3 Phf6lox/Y;Tie2-creTg/+;Ifnar1+/−, and n = 6 Phf6lox/Y;Tie2-creTg/+;Ifnar1−/−. Genotype colors are shown in panel C. (E) RT-qPCR analysis showing mRNA levels of Irf7, Oas2, and Iigp1 in HPC-1 cells of the indicated genotypes relative to mRNA levels in housekeeping genes (HK; Gapdh, Actb, and Pgk1). n = 4 Phf6+/Y;Tie2-creTg/+;Ifnar1−/−, n = 4 Phf6+/Y;Tie2-creTg/+;Ifnar1+/+, n = 4 Phf6lox/Y;Tie2-creTg/+;Ifnar1+/+, and n = 4 Phf6lox/Y;Tie2-creTg/+;Ifnar1−/−. Genotype colors are shown in panel C. (F) RT-qPCR analysis showing mRNA levels of Irf7 in CD45.1+ and CD45.2+ HPC-1 cells isolated from irradiated CD45.1+ host mice 4 months posttransplantation with 2.5 million whole bone marrow cells of the indicated genotype, along with 2.5 million CD45.1+ wild-type competitor cells. n = 4 Phf6+/Y;Tie2-creTg/+;Ifnar1−/− donors, n = 5 control donors (2 Phf6+/Y;Tie2-creTg/+;Ifnar1+/+, 2 Phf6+/Y;Ifnar1+/+, 1 Phf6lox/Y;Ifnar1+/+), n = 5 Phf6lox/Y;Tie2-creTg/+;Ifnar1+/+ donors, and n = 4 Phf6lox/Y;Tie2-creTg/+;Ifnar1−/− donors. Average expression of donors was calculated prior to statistical analysis. Note that 1 host mouse transplanted with control cells was excluded from the analysis because expression of Irf7 was >3 standard deviations above the values of the other control transplants in CD45.1+ and CD45.2+ cells (values of 7.1 and 5.2 arbitrary units relative to HK, respectively). Thus, the average of the other 2 host mice receiving bone marrow from the same donor was used. (G) Concentration of IFN-α in plasma of untreated mice. n = 6 controls (3 Phf6+/Y;Tie2-creTg/+, 3 Phf6+/Y) and n = 5 Phf6lox/Y;Tie2-creTg/+. (H) Number of pDCs in the bone marrow of control mice (n = 5 Phf6+/Y;Tie2-creTg/+) compared with Phf6lox/Y;Tie2-creTg/+ mice (n = 5), as defined by cell surface expression of PDCA1 and Siglec H. (A-B) See “Materials and methods” for details on RNA sequencing analysis. Data in panels C-F were analyzed by 1-way analysis of variance and are individual data points with mean ± SEM showing significant results from multiple comparisons using the Fisher’s least significant difference test. Data in panels G-H were analyzed by the 2-tailed Student t test and are shown as individual points for each animal with mean ± SEM.

PHF6 regulates ISG expression in hematopoietic stem and progenitor cells. (A) Barcode enrichment plots showing positive correlation between the reactome IFN α/β signaling pathway and the gene-expression changes in Phf6-deleted vs control HPC-1 cells. The horizontal axis shows t statistics for all genes in the Phf6-deleted dataset, whereas vertical lines represent genes in the reactome IFN α/β signaling pathway. Red and blue shaded areas indicate genes that are upregulated and downregulated, respectively, in the Phf6-deleted cells, and worms show the relative enrichment of the IFN signature. The IFN signature is enriched among upregulated genes on the right of the plot. (B) Heat maps of a subset of IFN-stimulated genes comparing Phf6-deleted HPC-1 cells with control HPC-1 cells. Each column represents 1 animal. n = 4 controls (Phf6+/Y;Tie2-creTg/+), n = 4 Phf6lox/Y;Tie2-creTg/+. (C) Number of HPC-1 cells in the bone marrow. n = 10 Phf6-control;Ifnar1−/− (3 Phf6+/Y;Tie2-creTg/+;Ifnar1−/−, 5 Phf6lox/Y;Ifnar1−/−, 2 Phf6+/Y;Ifnar1−/−), n = 11 controls (6 Phf6+/Y;Tie2-creTg/+;Ifnar1+/+, 1 Phf6+/Y;Ifnar1+/+, 1 Phf6lox/Y;Ifnar1+/+, 3 Phf6+/Y;Tie2-creTg/+;Ifnar1+/−), n = 6 Phf6lox/Y;Tie2-creTg/+;Ifnar1+/+, n = 4 Phf6lox/Y;Tie2-creTg/+;Ifnar1+/−, and n = 7 Phf6lox/Y;Tie2-creTg/+;Ifnar1−/−. (D) Percentage of Ki67+ cells in the indicated populations. n = 8 Phf6-control;Ifnar1−/− (3 Phf6+/Y;Tie2-creTg/+;Ifnar1−/−, 3 Phf6lox/Y;Ifnar1−/−, 2 Phf6+/Y;Ifnar1−/−), n = 10 controls (6 Phf6+/Y;Tie2-creTg/+;Ifnar1+/+, 1 Phf6+/Y;Ifnar1+/+, 1 Phf6lox/Y;Ifnar1+/−, 2 Phf6+/Y;Tie2-creTg/+;Ifnar1+/−), n = 7 Phf6lox/Y;Tie2-creTg/+;Ifnar1+/+, n = 3 Phf6lox/Y;Tie2-creTg/+;Ifnar1+/−, and n = 6 Phf6lox/Y;Tie2-creTg/+;Ifnar1−/−. Genotype colors are shown in panel C. (E) RT-qPCR analysis showing mRNA levels of Irf7, Oas2, and Iigp1 in HPC-1 cells of the indicated genotypes relative to mRNA levels in housekeeping genes (HK; Gapdh, Actb, and Pgk1). n = 4 Phf6+/Y;Tie2-creTg/+;Ifnar1−/−, n = 4 Phf6+/Y;Tie2-creTg/+;Ifnar1+/+, n = 4 Phf6lox/Y;Tie2-creTg/+;Ifnar1+/+, and n = 4 Phf6lox/Y;Tie2-creTg/+;Ifnar1−/−. Genotype colors are shown in panel C. (F) RT-qPCR analysis showing mRNA levels of Irf7 in CD45.1+ and CD45.2+ HPC-1 cells isolated from irradiated CD45.1+ host mice 4 months posttransplantation with 2.5 million whole bone marrow cells of the indicated genotype, along with 2.5 million CD45.1+ wild-type competitor cells. n = 4 Phf6+/Y;Tie2-creTg/+;Ifnar1−/− donors, n = 5 control donors (2 Phf6+/Y;Tie2-creTg/+;Ifnar1+/+, 2 Phf6+/Y;Ifnar1+/+, 1 Phf6lox/Y;Ifnar1+/+), n = 5 Phf6lox/Y;Tie2-creTg/+;Ifnar1+/+ donors, and n = 4 Phf6lox/Y;Tie2-creTg/+;Ifnar1−/− donors. Average expression of donors was calculated prior to statistical analysis. Note that 1 host mouse transplanted with control cells was excluded from the analysis because expression of Irf7 was >3 standard deviations above the values of the other control transplants in CD45.1+ and CD45.2+ cells (values of 7.1 and 5.2 arbitrary units relative to HK, respectively). Thus, the average of the other 2 host mice receiving bone marrow from the same donor was used. (G) Concentration of IFN-α in plasma of untreated mice. n = 6 controls (3 Phf6+/Y;Tie2-creTg/+, 3 Phf6+/Y) and n = 5 Phf6lox/Y;Tie2-creTg/+. (H) Number of pDCs in the bone marrow of control mice (n = 5 Phf6+/Y;Tie2-creTg/+) compared with Phf6lox/Y;Tie2-creTg/+ mice (n = 5), as defined by cell surface expression of PDCA1 and Siglec H. (A-B) See “Materials and methods” for details on RNA sequencing analysis. Data in panels C-F were analyzed by 1-way analysis of variance and are individual data points with mean ± SEM showing significant results from multiple comparisons using the Fisher’s least significant difference test. Data in panels G-H were analyzed by the 2-tailed Student t test and are shown as individual points for each animal with mean ± SEM.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal