A germline point mutation in Runx1 uncouples its role in definitive hematopoiesis from differentiation

Abstract

Definitive hematopoiesis requires the master hematopoietic transcription factor Runx1, which is a frequent target of leukemia-related chromosomal translocations. Several of the translocation-generated fusion proteins retain the DNA binding activity of Runx1, but lose subnuclear targeting and associated transactivation potential. Complete loss of these functions in vivo resembles Runx1 ablation, which causes embryonic lethality. We developed a knock-in mouse that expresses full-length Runx1 with a mutation in the subnuclear targeting cofactor interaction domain, Runx1(HTY350-352AAA). Mutant mice survive to adulthood, and hematopoietic stem cell emergence appears to be unaltered. However, defects are observed in multiple differentiated hematopoietic lineages at stages where Runx1 is known to play key roles. Thus, a germline mutation in Runx1 reveals uncoupling of its functions during developmental hematopoiesis from subsequent differentiation across multiple hematopoietic lineages in the adult. These findings indicate that subnuclear targeting and cofactor interactions with Runx1 are important in many compartments throughout hematopoietic differentiation.

Figure 1. Generation of the Runx1^{HTY350-352AAA} mouse

(A) Targeting vector for the knock-in mouse contained a floxed Neo cassette as a positive selection marker and a TK cassette for negative selection to ensure homologous recombination in ES cells. (B) Successful targeting to the native Runx1 locus results in a diagnostic SacII site that allows for genotyping by PCR and enzyme digest. From left to right are genotyping results showing the change in digest fragment sizes from a wildtype, heterozygous and homozygous Runx1^{HTY350-352AAA} mouse. (C) Runx1^{HTY350-352AAA} animals were born at Mendelian ratios. (D) Runx1^{HTY350-352AAA} embryos are healthy at embryonic day 12.5 and are comparable to wildtype littermates (3 litters, n=8 WT, 7 HTY). (E) Adult wildtype and Runx1^{HTY350-352AAA} mice at 12 weeks or 12 months are equivalent in weight (n=16 WT, 19 HTY for 12 weeks and 15 WT, 9 HTY for 12 months). (F) qRT-PCR for Runx1 in bone marrow cells shows no difference in expression levels (p=0.8 by Student’s T test). Each point represents the average of technical replicates from one animal, normalized to mCox (n=9 WT, 9 HTY). (G) Whole cell and nuclear matrix-intermediate filament (NMIF) preparations of bone marrow from wildtype and Runx1^{HTY350-352AAA} animals shows that Runx1 is still present within the nuclear matrix (n=3 WT, 3 HTY). All of the images were acquired using same settings of the microscope and the associated MetaMorph Software for accurate comparison. All error bars are SEM.

Figure 2. Increased ex vivo growth and diminished colony forming ability of Runx1^{HTY350-352AAA} marrow

(A) Ex vivo cultures of bone marrow cells from wildtype or Runx1^{HTY350-352AAA} animals (each point represents the mean cell number of technical replicates from one animal; n=3 WT, 5 HTY). (B) Myeloid colony forming unit assays performed with wildtype or Runx1^{HTY350-352AAA} bone marrow cells (n=28 WT, 34 HTY). Because there was no discernable difference in colony size, only total colony number was counted. (C) Cells from the experiment reported in panel A were harvested at day 7 and the percentage of cells expressing the myeloid antigen CD11b was determined using flow cytometry (n=3 WT, 3 HTY). (D) Ex vivo cultures as in panel A, but with the addition of TGF beta to a final concentration of 10ng per ml (each point represents technical replicates from one animal; n=3 WT, 5 HTY). (E) Colony forming unit assays were performed as in panel B, but with the addition of TGF beta to some cultures to a final concentration of 10ng per ml (n=3 WT, 5 HTY). * p<0.05, *** p<0.001, calculated by Student’s T test. All error bars are SEM.

Figure 3. Progenitors in Runx1^{HTY350-352AAA} bone marrow

(A) Bone marrow cells from wildtype or Runx1^{HTY350-352AAA} animals were stained using antibodies to c-Kit, Sca1, CD16/32, CD34, IL7Ralpha, AA4.1 and Lineage markers (CD3, CD11b, B220, Ter119, Gr1) and analyzed by flow cytometry. Lineage negative cells were gated and are plotted in panel A to highlight the Lin-Sca1+Kit+ (LSK), myeloid potential L-S-K+ and lymphoid potential L-S+K− fractions. (B) Quantification of multiple experiments performed as depicted in A, normalizing to the wildtype average of each experiment (n=13 WT, 13 HTY). (C) Lineage negative and IL7R alpha positive bone marrow cells were gated and c-Kit versus AA4.1 was plotted to measure CLPs. (D) Quantification of multiple experiments in C (n=6 WT, 6 HTY). (E) Lineage negative, c-Kit positive, Sca1 negative (L-S-K+) cells from panel A were plotted as CD16/32 versus CD34 to measure the CMP, GMP and MEP progenitor populations. (F) Quantification of multiple experiments as in E, normalizing to the wildtype average of each experiment (n=10 WT, 10 HTY). * p<0.05, calculated by Student’s T test. All error bars are SEM.

Figure 4. B and T lymphopoiesis in Runx1^{HTY350-352AAA} animals

(A) At sacrifice, animals were weighed then the spleen and/or thymus was removed and weighed. Displayed is the spleen weight normalized to total animal weight. Each point represents one animal (n=14 WT, 15 HTY). (B) Spleens were formalin fixed, sectioned and then stained by H & E. Representative fields are shown (n=3 WT, 3 HTY). (C) Bone marrow cells from wildtype or Runx1^{HTY350-352AAA} animals were stained for B220, IgM, AA4.1 and CD43. B220 positive and IgM positive cells were gated to show expression of AA4.1 to separate Immature and Mature B cells. (D) Quantification of multiple experiments performed as in C (n=12 WT, 12 HTY). (E) Bone marrow cells from wildtype or Runx1^{HTY350-352AAA} animals were stained for B220, IgM, AA4.1, Ly6C, CD49b, CD19 and CD43. Cells positive for Ly6C, CD49b and IgM were excluded by gating, and the CD43+ B220+ subset is shown to measure AA4.1+CD19− pre-Pro-B cells and AA4.1+ CD19+ pro-B cells. (F) Quantification of multiple experiments as in E (n=6 WT, 6 HTY). (G) Spleen or thymus cells were isolated and stained with CD4 and CD8. Spleen cells were gated and plotted as CD4 versus CD8 to measure CD4+ and CD8+ T cell populations. (H) Quantification of multiple experiments as shown in G (n=12 WT, 14 HTY; age-matched animals were used). (I) Displayed is the thymus weight normalized to total animal weight (n=18 WT, 19 HTY). * p<0.05, ** p<0.01, *** p<0.001, calculated by Student’s T test. All error bars are SEM.

Figure 5. Expansion of splenic myeloid compartment and delay in megakaryocytic maturation in Runx1^{HTY350-352AAA} animals

(A) Spleen cells were stained with antibodies to Gr1 and CD11b to measure the Gr1 high, CD11b positive granulocyte and the Gr1 intermediate CD11b positive monocyte / macrophage populations. (B) Quantification of multiple experiments performed as shown in A (n=7 WT, 9 HTY) (C) Bone marrow or spleen cells were stained for CD41 to measure the megakaryocyte population. (D) Quantification of multiple experiments shown in C (n=12 WT, 14 HTY). (E) Bone marrow cells were stained for CD41, fixed with ethanol and then stained with propidium iodide to detect DNA content. Displayed is the CD41 positive fraction, with normal hyperploidy of a representative wildtype sample plotted in black, with several Runx1^{HTY350-352AAA} samples plotted in red to show the reduction in 8N and 16N cells. (F) Histological sections of bone marrow were H & E stained. Megakaryocytes that appear smaller and less hyperploid in the Runx1^{HTY350-352AAA} animals are shown. *** p<0.001, calculated by Student’s T test. All error bars are SEM.

Figure 6. Delayed red blood cell maturation in Runx1^{HTY350-352AAA} animals

(A) Bone marrow cells were stained for CD71 and Ter119 and plotted to measure the CD71 positive, Ter119 intermediate ProE population. Ter119 high cells were plotted CD71 versus forward scatter (FSC) to measure the EryA, EryB and EryC progenitor populations. (B) Spleen cells were stained and analyzed as in A. (C) Quantification of several experiments shown in A (n=12 WT, 14 HTY). (D) Quantification of several experiments shown in B (n=12 WT, 14 HTY). (E) qRT-PCR of bone marrow cells for hemoglobin genes (F) qRT-PCR for factors important for erythroid maturation and an important antagonist of erythropoiesis. Each point represents the average of technical replicates from one animal (n=3 to 11). * p<0.05, ** p<0.01, calculated by Student’s T test. All error bars are SEM.