1. TRIM28 is essential for erythroblast differentiation in the mouse
by Tomonori Hosoya, Mary Clifford, Régine Losson, Osamu Tanabe, and James Douglas Engel
Blood
Volume 122(23):
November 28, 2013
©2013 by American Society of Hematology

2. Erythropoiesis in mice inducibly ablated for Trim28.
Erythropoiesis in mice inducibly ablated for Trim28. TMC [Trim28flox/flox:TgMx1Cre (flox/flox:Mx1Cre)] mutant or control [Trim28flox/flox (flox/flox) and Trim28flox/+:TgMx1Cre (flox/+:Mx1Cre)] mice at ages 5 to 6 weeks were injected every 48 hours with poly(I:C) 5 times to transcriptionally activate the cre-expressing transgene.26 Uninjected Trim28flox/+ (flox/+) mice were used as controls. Mice were analyzed 2 weeks (2w) or 4 weeks (4w) after completion of poly(I:C) administration. (A) The absolute number of total bone marrow cells isolated from 2 femurs plus 2 tibias of individual animals (each circle) of various genotypes. The black bar represents the average. * indicates statistically significant, P < .05. NS: not significant, P > .05. (B) Flow cytometric analysis of erythropoiesis in total bone marrow cells from mutant and control mice. The numbers in each of the boxed areas indicate the mean frequency of cells in each hematopoietic population. (C) The abundance of Trim28 floxed allele in CD71+TER119+ immature erythroid cells was analyzed by qPCR. The CD71hiTER119- cells that aberrantly accumulated in the TMC mutant mice (panel B) were also analyzed. An average of the relative amount of the undeleted gene in the control Trim28flox/flox cell was set to 200, which is the copy number of the floxed allele in 100 cells. (D) The expression level of β-type globin genes normalized to β-actin in CD71+TER119+ immature erythroid cells was analyzed by qRT-PCR. The average of the relative amount in the control Trim28flox/flox cell was set to 1. (E) The absolute number of cells in each of the analyzed populations was calculated from the data shown in (A) and (B). Average with standard error of the mean (SEM). (F) Flow cytometric analysis of TER119+ cells in total bone marrow cells (separated by forward scatter and CD44 antigen according to Chen et al34). The numbers closest to the boxed areas indicate the mean percentage of cells in each population within parental TER119+ cells. (G) The absolute number of basophilic erythroblasts (r2), polychromatic erythroblasts (r3), orthochromatic erythroblasts (r4a), reticulocytes (r4b), and RBCs (r5) in the bone marrow was calculated from the data shown in panels (A) and (F). Error bars represent the SEM. (H) Hematologic parameters of peripheral blood cells. Data represent either a summary (A,C,D,E,G,H) or representative individuals (B,F) of more than 3 mice of each genotype from 2 to 4 independent experiments.
Tomonori Hosoya et al. Blood 2013;122:
©2013 by American Society of Hematology

3. Abnormal development of hematopoietic progenitor cells in Trim28 mutant mice.
Abnormal development of hematopoietic progenitor cells in Trim28 mutant mice. Adult TMC [Trim28flox/flox:TgMx1Cre (flox/flox:Mx1Cre)] mutant or control [Trim28flox/flox (flox/flox) and Trim28flox/+:TgMx1Cre (flox/+:Mx1Cre)] mice at ages 5 to 6 weeks were injected with poly(I:C). (A) Flow cytometric analysis of total bone marrow cells and division into individual early progenitor compartments (shown on the right) according to cell surface expression of cKit, Sca1, FcγR, CD34, and lineage (Lin) markers. Numbers in the boxed areas indicate the mean percentages of cells in the gated area calculated by dividing the number of cells in that subpopulation by the number of cells in its immediate ancestor population (Frequency of Parent in FlowJo software). (B) The absolute number of HSC/MPP, CMP, MEP, and GMP was calculated from the data shown in panel (A) and total bone marrow cell number. Averages are indicated with SEM. * indicates statistically significant, P < .05. NS: not significant, P > .05. (C) The absolute number of Mac1+ cells in the bone marrow of mutant and control mice. (D) The absolute number of cKithi Lin- and cKithiLin+ cells in the bone marrow of control and conditionally mutant mice. (E) The absolute number of cKithiCD71hiTER119-, cKithiCD71-TER119-, and cKithiTER119+ cells in the bone marrow. The lineage mixture used for panels (A, B, and D) includes antibodies that recognize TER119, Mac1, Gr1, B220, CD19, CD5, CD4, and CD8a. Mice were analyzed for 2 weeks (A, B, D, E) or for 2 and 4 weeks (C) after completion of poly(I:C) administration. The data represent a summary (B-E) or are representative (A) of more than 3 mice of each genotype from 2 to 3 independent experiments. Each circle represents an individual mouse, and the black bars represent averages.
Tomonori Hosoya et al. Blood 2013;122:
©2013 by American Society of Hematology

4. Detailed characterization of the aberrant CD71hiTER119- cell population that accumulates in Trim28 mutant bone marrow.
Detailed characterization of the aberrant CD71hiTER119-cell population that accumulates in Trim28 mutant bone marrow. TMC [Trim28flox/flox:TgMx1Cre (flox/flox:Mx1Cre)] mutant or control [Trim28flox/flox (flox/flox) and Trim28flox/+:TgMx1Cre (flox/+:Mx1Cre)] mice at ages 5 to 6 weeks were injected with poly(I:C). Mice were analyzed 2 weeks after the final poly(I:C) administration. (A) Color of cells separated on the basis of CD71 and TER119 expression. Between 4 and 10 × 105 cells were flow sorted and suspended in PBS supplemented with 2% FBS in a 1.5-mL tube and centrifuged to form a pellet. Images were acquired using a Zeiss Stemi SV11 Apo stereomicroscope, AxioCam digital camera, and AxioVision LE software. (B) Flow cytometric analysis of forward scatter (FSC), CD45, cKit, Mac1, and B220 in the CD71hiTER119- bone marrow cells of control mice and Trim28 mutant mice. Numbers in the boxed areas indicate the mean percentages of cells in the boxed area calculated by dividing the number of cells in that subpopulation by the number of cells in its immediate ancestor population. (C) The absolute number of different subpopulations in the CD71hiTER119- cells was calculated from panels (B) and normalized for total bone marrow cell number. Average with SEM. *indicates statistically significant, P < .05. NS: not significant, P > .05. (D) Neutral benzidine staining of individual cells recovered from flow-sorted populations of different genotype mice treated with poly(I:C). (E) Wright-giemsa staining. Images (D and E) were acquired using an Olympus BX-51 upright light microscope with the 100× oil immersion objective lens, DP-70 high-resolution digital camera, and DP Controller software. (F) Flow cytometric analysis of FSC and CD44 in CD71hiTER119- and CD71hiTER119+ cells. Numbers in or near the boxed areas indicate the mean percentage of cells in the boxed area calculated by dividing the number of cells in that subpopulation by the number of cells in its immediate ancestor population. The data represent a summary (C) or representative individuals (A, B, D-F) of more than 3 mice of each genotype examined in 2 to 3 independent experiments.
Tomonori Hosoya et al. Blood 2013;122:
©2013 by American Society of Hematology

5. The contribution of TRIM28 to erythropoiesis is erythroid cell autonomous.
The contribution of TRIM28 to erythropoiesis is erythroid cell autonomous. (A) Experimental design. Total bone marrow donor cells isolated from Trim28flox/flox:TgMx1Cre (flox/flox:Mx1Cre) or control Trim28flox/flox (flox/flox) mice (CD45.2) were coinjected with an approximately equal number of wild-type competitive donor cells (CD45.1) into lethally irradiated wild-type recipient mice (CD45.1). (B) CD45.2 and CD45.1 expression in each population in peripheral blood (before Cre induction) or in bone marrow (2 weeks post Cre activation) were analyzed by flow cytometry (left panel). Peripheral blood cells were analyzed 6 weeks after transplantation for CD45.2+ donor cell contribution in the Mac1+Gr1+ myeloid (PB_M) and B220+CD19+ B lymphoid (PB_B) populations. Total bone marrow cells were analyzed 2 weeks after completing poly(I:C) treatment for CD45.2+ donor cell contribution to the HSC/MPP, CMP, MEP, and GMP compartments. Each circle represents the average of control (black circle) or TMC mutant cells (open circle) with SEM (left panel). Data represent the summary of 8 recipient mice of each genotype from 2 independent experiments. Because erythroblasts and later-stage erythroid cells are negative for CD45,35 the contribution of CD45.2+ (male) cells to the immature erythroid cell fraction was analyzed by qPCR, quantifying the abundance of Sry genomic DNA (Y chromosome; right panel). The average of Trim28flox/flox mice was set to 1. Male CD45.2 and female CD45.1 cells were used in the first experiment. Each circle represents an individual mouse, and black bars represent the average (right panel). The summary of the first experiment with 4 recipients of each donor genotype is shown. In the second set of experiments, female CD45.2 and male CD45.1 cells were used; an (statistically insignificant) increased contribution of coinjected competitive CD45.1 cells was observed (data not shown). (C) The absolute number of different stages of erythroid cells in the bone marrow (2 femurs and 2 tibias). Mixtures of CD45.2 and CD45.1 cells are depicted. Average with SEM. * indicates statistically significant, P < .05. NS: not significant, P > .05.
Tomonori Hosoya et al. Blood 2013;122:
©2013 by American Society of Hematology

6. Expression profile of erythroid related genes in immature erythroid cells of Trim28 mutant mice.
Expression profile of erythroid related genes in immature erythroid cells of Trim28 mutant mice. TMC [Trim28flox/flox:TgMx1Cre (flox/flox:Mx1Cre)] mutant or control [Trim28flox/flox (flox/flox)] mice at ages 5 to 6 weeks were injected with poly(I:C) 5 times. Mice were analyzed 2 weeks after completing the poly(I:C) injections. CD71hiTER119+ immature erythroid cells (closed circle) were isolated from control Trim28flox/flox (flox/flox) mouse. CD71hiTER119+ (open circle) and CD71hiTER119- (open triangle) immature erythroid populations were isolated from mutant Trim28flox/flox:TgMx1Cre (flox/flox:Mx1Cre) mouse. Cells were isolated from 3 animals of each genotype and were processed individually. RNA-Seq analysis was performed as described in “Methods.” Fragments per kilobase of exon per million mapped fragments for each animal are shown. Each circle represents an individual animal, whereas the black bars represent the averages. Gene names are shown at the top. Statistically significant changes in transcript expression analyzed in Cuffdiff analysis is depicted by a *. NS: not significant.
Tomonori Hosoya et al. Blood 2013;122:
©2013 by American Society of Hematology

7. Genomic transcriptional alterations in immature Trim28 mutant erythroid cells.
Genomic transcriptional alterations in immature Trim28 mutant erythroid cells. (A) Gene ontology analysis. Genes that exhibit statistically significant differences between control mice and Trim28 mutant mice in the RNA-Seq experiment were analyzed by DAVID bioinformatics resources,37 and the top 10 categories for each list are shown. (B) Fragments per kilobase of exon per million mapped fragments from RNA-Seq for mitophagy and apoptosis-related genes. (C) Apoptosis analysis. Cells were isolated from bone marrow 2 weeks after poly(I:C) treatment, stained with antibodies and FITC-conjugated annexin V, and analyzed by flow cytometry. The frequency of annexin V+ DAPI- early apoptotic cells in each erythroid progenitor population is shown. CD71hiTER119+ or CD71hiTER119- cells were further subdivided by forward scatter and CD44 expression. Each circle represents an individual animal, whereas the black bars represent the averages. Data represent a summary from 2 independent experiments. * indicates statistically significant P < .05. NS: not significant, P > .05. (D) Summary of the phenotypes observed in the TMC mutant mouse.
Tomonori Hosoya et al. Blood 2013;122:
©2013 by American Society of Hematology