Identification of Chromatin Remodeling Genes Arid4a and Arid4b as Leukemia Suppressor Genes

Mei-Yi Wu; Karen W. Eldin; Arthur L. Beaudet

Disclosures

J Natl Cancer Inst. 2008;100(17):1247-1259. 

In This Article

Results

A CMML-like Myelodysplastic/Myeloproliferative Disorder in Arid4a-Deficient Mice

To investigate the function of Arid4a and Arid4b, we bred Arid4a null (Arid4a-/-) mice and mice null for Arid4a and heterozygous for the Arid4b deletion (Arid4a-/-Arid4b±); homozygous null mutants for Arid4b (both Arid4a+/+Arid4b-/- and Arid4a-/-Arid4b-/-) were not viable and died before E7.5.[5] Young (2-5 months old) adult Arid4a-deficient mice initially had ineffective blood cell production in all hematopoietic lineages, with mild leukopenia (WBC: mean wild-type count = 7.64 × 106/mL, mean Arid4a -/- count = 4.09 × 106/mL, difference = 3.55 × 106/mL, 95% confidence interval [CI] = 2.72 × 106/mL to 4.38 × 106/mL, P < .001. Lymphocyte: mean wild-type count = 6.29 × 106/mL, mean Arid4a -/- count = 3.21 × 106/mL, difference = 3.08 × 106/mL, 95% CI = 2.22 × 106/mL to 3.94 × 106/mL, P < .001. Neutrophil: mean wild-type count = 0.84 × 106/mL, mean Arid4a -/- count = 0.51 × 106/mL, difference = 0.33 × 106/mL, 95% CI = 0.16 × 106/mL to 0.51 × 106/mL, P = .043) and mild anemia (RBC: mean wild-type count = 8.96 × 109/ml, mean Arid4a -/- count = 7.88 × 109/mL, difference = 1.08 × 109/mL, 95% CI = 0.5 × 109/mL to 1.66 × 109/mL, P = .017; hemoglobin: mean wild-type count = 137.8 g/L, mean Arid4a -/- count = 125.0 g/L, difference = 12.8 g/L, 95% CI = 5.0 g/L to 20.7 g/L, P = .035) with statistically significant thrombocytopenia (platelet: mean wild-type count = 1144.3 × 106/mL, mean Arid4a -/- count = 225.8 × 106/mL, difference = 918.5 × 106/mL, 95% CI = 802.3 × 106/mL to 1034.7 × 106/mL, P < .001) (Figure 1, A). Beyond 5 months of age, the Arid4a -/- mice manifested monocytosis (monocyte: mean wild-type count = 0.2 × 106/mL, mean Arid4a -/- count = 0.9 × 106/mL, difference = 0.7 × 106/mL, 95% CI = 0.55 × 106/mL to 0.85 × 106/mL, P < .001), accompanied with severe anemia (RBC: mean wild-type count = 8.6 × 109/mL, mean Arid4a -/- count = 3.1 × 109/mL, difference = 5.5 × 109/mL, 95% CI = 5.0 × 109/mL to 6.0 × 109/mL, P < .001; hemoglobin: mean wild-type count = 130 g/L, mean Arid4a -/- count = 48 g/L, difference = 82 g/L, 95% CI = 76 g/L to 88 g/L, P < .001) and severe thrombocytopenia (platelet: mean wild-type count = 1169 × 106/mL, mean Arid4a -/- count = 76 × 106/mL, difference = 1093 × 106/mL, 95% CI = 1036 × 106/mL to 1150 × 106/mL, P < .001) (Figure 1, A). Peripheral blood smears from older (more than 5 months old) sickly Arid4a -/- mice showed the presence of teardrop poikilocytes (Figure 1, B, a) and increased numbers of immature erythroid cells (Figure 1, B, b and c). Immature and maturing mononuclear cells morphologically consistent with the monocyte lineage were also noted in the peripheral blood (Figure 1, B, d). Some monocytoid cells that contained phagocytosed RBCs were also present (Figure 1, B, e). The Arid4a -/- mice showed signs of morbidity (eg, ruffled hair, decreased activity, and rapid respiration). Mortality of the Arid4a -/- mice increased sharply from 6 months of age onward, with no Arid4a -/- mice surviving past 22 months of age (Figure 1, C). Increased mortality appeared to be associated with increasing severity of the hematologic abnormalities, especially severe anemia, in Arid4a -/- mice.

Figure 1.

A chronic myelomonocytic leukemia (CMML)-like myelodysplastic/myeloproliferative disorder in Arid4a-deficient mice. A) Complete blood counts (white blood cell, lymphocyte, neutrophil, monocyte, red blood cell [RBC], hemoglobin, platelet) in wild-type mice at 2-5 months of age (n = 35), in Arid4a -/- mice at 2-5 months of age (n = 30), in wild-type mice more than 5 months old (n = 25), and in Arid4a -/- mice more than 5 months old with symptoms of CMML (n = 25). Means (and 95% confidence intervals) for cell concentrations are shown, and P values were calculated using Student t test. B) Wright-Giemsa staining of peripheral blood from an Arid4a -/- mouse with symptoms of CMML, showing teardrop poikilocytes (a, black arrowheads), red cells with Howell-Jolly bodies (b, black arrowhead), and nucleated red cells (c, black arrowhead). Immature (d, white arrowhead) and maturing (d, black arrowhead) mononuclear cells were also observed, together with phagocytosis of RBC by a monocyte (e, arrowhead). Ten separate analyses were performed. Scale bars = 5 µm. C) Survival of Arid4a -/- (n = 25) mice and wild-type (n = 25) mice. D) Reticulin staining of paraffin sections of bone marrow from a wild-type mouse and a sick Arid4a -/- mouse. The Arid4a -/- sample shows fibrous tissue stained with black color. Scale bars = 20 µm. E) Flow cytometric analysis of apoptotic cells in bone marrow from a wild-type and a sick Arid4a -/- mouse. The percentages of cells positive for annexin V are indicated. Five separate cytometric analyses were performed. F) Splenomegaly and G) hepatomegaly in a sick Arid4a -/- mouse. Hematoxylin and eosin-stained sections of H) spleen and I) liver from a wild-type mouse and a sick Arid4a -/- mouse. Extramedullary hematopoiesis was found in the Arid4a -/- spleen and Arid4a -/- liver, which were infiltrated with nucleated elements of blood cells. Ten separate analyses were performed. Scale bars = 20 µm. J) Flow cytometric analysis of cells from spleen in a wild-type mouse and an Arid4a -/- mouse stained with Ter119 surface antigen. The percentages of cells positive for the antigen are indicated. Twenty separate analyses were performed.

Bone marrow from sick Arid4a -/- mice developing monocytosis and severe anemia in the peripheral blood showed reticulin fibrosis and an increased number (average increase = 8%, 95% CI = 6% to 10%, P < .001; the 10.9% increase in Figure 1, E is from one of five separate experiments) of apoptotic cells compared with wild-type mice (Figure 1, D and E). The sick Arid4a -/- mice (n > 50) also developed splenomegaly (Figure 1, F) and hepatomegaly (Figure 1, G) after 5 months of age, with extramedullary hematopoiesis in the spleen (Figure 1, H) and liver (Figure 1, I). A marked increase of erythropoiesis within enlarged spleens of the Arid4a -/- mice was demonstrated by flow cytometry analysis of spleen cells: 65% of spleen cells were erythroid cells (Ter119+) in mutants versus 7% in wild-type mice (difference = 58%, 95% CI = 53% to 63%, P < .001) (Figure 1, J).

In addition to their hematologic abnormalities, female Arid4a -/- mice showed decreased fertility. Litter sizes for the Arid4a -/- females mating with wild-type males were markedly lower than those for wild-type breeding pairs (0.7 vs 7.8, difference = 5.6, 95% CI = 5.2 to 6.0. P < .001). Anemia, hepatosplenomegaly, and systemic illness may have contributed to the decreased fertility, but in addition there was hemorrhage into the ovarian follicles (Supplementary Figure 1, available online), as a result of profound thrombocytopenia (the mean platelet count in these mice was less than 100 × 106/mL, Figure 1, A).

Thus, Arid4a -/- mice initially developed mild cytopenias with substantial thrombocytopenia and later progressed to monocytosis associated with more severe anemia and thrombocytopenia with spontaneous hemorrhage into organs (ie, ovary). Bone marrow failure with myelofibrosis was associated with hepatosplenomegaly due to compensatory extramedullary hematopoiesis. These abnormalities in the Arid4a -/- mice suggest a myelodysplastic/myeloproliferative disorder and are similar to the course of events in humans with CMML derived from myelodysplastic/myeloproliferative diseases.

Development of AML in Arid4a -/- Mice and Arid4a -/- Arid4b +/- Mice

In patients with myelodysplastic/myeloproliferative diseases, progression to AML occurs with a frequency of 5%-30%. Similarly, we found that 5 of 42 (12%) of the Arid4a -/- mice developed AML showing rapid and large increases of WBC counts with an onset between 12 and 22 months of age (data not shown). Hematologic malignancies were more frequent in the Arid4a -/- Arid4b ± mice. Of the 12 Arid4a -/- Arid4b ± mice monitored beyond 5 months of age, 10 (83%) developed AML with an earlier age of onset (7-15 months).

Neither the mice heterozygous for both Arid4a and Arid4b (n = 36) nor wild-type controls (n = 52) developed AML over a 2-year period. littermates (data not shown). Postnatal growth was also delayed (Figure 2, A); body weight of Arid4a -/- Arid4b ± mice was 30% less than that of wild-type mice at 7 weeks of age (Figure 2, B). The Arid4a -/- Arid4b ± mice also had increased postnatal mortality: 25% of mutant mice died before attaining 1 month of age (Figure 2, C), and those that survived exhibited increased mortality at 7 months of age (Figure 2, C) that was attributable to the increasing severity of their hematologic malignancies.

Figure 2.

Growth and survival of the Arid4a -/- Arid4b ± mice. A) Growth retardation in an Arid4a -/- Arid4b ± mouse compared with a wild-type littermate at 12 days of age. B) Growth curve of the Arid4a -/- Arid4b ± (n = 7) and wild-type (n = 10) littermates by mean body weight plotted against age with 95% confidence intervals. C) Survival of Arid4a -/- Arid4b ± (n = 31) and wild-type (n = 25) mice.

Bone marrow smears from mice with AML (two Arid4a -/- mice and three Arid4a -/- Arid4b ± mice) demonstrated the presence of a mixture of immature and dysplastic WBC precursors with more than 20% nonlymphoid immature forms and blasts (Figure 3, A). Flow cytometric analysis of cell populations within the bone marrow from the Arid4a -/- mice with CMML-like phenotype and the Arid4a -/- Arid4b ± mice with AML revealed that the majority of excess leukocytes were granulocytes and monocytes (22% of wild-type mice, 29% of mice with CMML-like phenotype, and 63% of mice with AML were positive for Gr1 and Mac1, difference between wild-type and CMML-like mice = 7%, P = .124; difference between wild-type and AML mice = 41%, P = .002) (Figure 4, A). This increase in granulocytes and monocytes was accompanied by an increase of T lymphoid cells (CD3+, 0.65% T lymphoid cells in wild-type mice, 3.16% in mice with CMML-like phenotype, and 5.99% in AML mice, difference between wild-type and CMML-like mice = 2.15%, P = .029; difference between wild-type and AML mice = 5.34%, P < .001) and by decreases in B lymphoid cells (B220+CD19+, 9.4% in wild-type mice, 1.8% in mice with CMML-like phenotype, and 0.9% in AML mice, difference between wild-type and CMML-like mice = 7.6%, P < .001; difference between wild-type and AML mice = 8.5%, P < .001) and erythroid populations (Ter119+, 60% erythroid cells in wild-type mice, 54% in mice with CMML-like phenotype, and 36% in AML mice, difference between wild-type and CMML-like mice = 6%, P = .378; difference between wild-type and AML mice = 24%, P = .026) (Figure 4, A).

Figure 3.

Acute myeloid leukemia (AML)-like phenotype in Arid4a -/- Arid4b ± mice. A) Bone marrow smears from a wild-type mouse and an Arid4a -/- Arid4b ± mouse with AML were stained with Wright-Giemsa. Black arrowheads indicate blasts. White arrowheads indicate red blood cell precursors in both wild-type and AML bone marrow (for both wild-type and the Arid4a -/- Arid4b ± bone marrow smears, original magnifications are the same). Five separate analyses were performed. Scale bars = 20 µm. B) Wright-Giemsa staining of peripheral blood smears from the AML Arid4a -/- Arid4b ± mice showing blasts (a, black arrowhead), lymphocytes (a, white arrowhead), increased numbers of immature cells (b-d, arrowheads), and phagocytosis of cells by a macrophage (e). Eight separate analyses were performed. Scale bars = 5 µm.

Figure 4.

Hematopoietic lineage analysis of the Arid4a -/- mice and Arid4a -/- Arid4b ± leukemic mice. A) Comparison of cell populations in bone marrow and spleen (HSC, CMP, granulocyte, monocyte, erythroid cell, T cell, and B cell) and cell counts in peripheral blood (neutrophil, monocyte, hemoglobin, RBC, lymphocyte, and platelet) between wild-type mice (n = 5), the Arid4a -/- mice with CMML-like phenotype (n = 5), and the Arid4a -/- Arid4b ± mice with acute myeloid leukemia (AML) (n = 5). Means (and 95% confidence intervals) for all cell populations and cell counts are shown, and P values were calculated using Student t test. B) Hematopoietic lineage tree displaying the combined impact of the Arid4a mutation with or without the Arid4b mutations in bone marrow and peripheral blood of mice with CMML-like or AML phenotype. Increased and decreased cell populations are indicated by red and green, respectively. HSC, hematopoietc stem cell; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; GMP, granulocyte and monocyte progenitor; MkP, megakaryocyte progenitor; EP, erythroid progenitor.

Examination of peripheral blood smears from the Arid4a -/- and Arid4a -/- Arid4b ± mice with AML showed the presence of more than 20% of atypical cells with morphology consistent with blasts or immature myeloid precursors (Figure 3, B, a-d). There was accompanying monocytosis, and hemophagocytosis was often observed (Figure 3, B, e). Serial monitoring of peripheral blood counts in mice developing AML revealed a rapid increase of WBC counts over a 2- to 4-week interval. The elevated WBC count was attributable to increased neutrophilic and monocytic forms (neutrophil: mean wild-type count = 0.75 × 106/mL, mean AML count = 8.50 × 106/mL, difference = 7.75 × 106/mL, 95% CI = 5.85 × 106/mL to 9.65 × 106/mL, P < .001; monocyte: mean wild-type count = 0.16 × 106/mL, mean AML count = 3.90 × 106/mL, difference = 3.74 × 106/mL, 95% CI = 2.54 × 106/mL to 4.94 × 106/mL, P < .001) (Figure 4, A). Soon after the development of leukocytosis, the mice became moribund and were sacrificed. In most of the Arid4a -/- Arid4b ± mice, AML occurred before development of severe anemia (peripheral blood in Figure 4, A; hemoglobin: mean wild-type count = 130 g/L, mean Arid4a -/- Arid4b ± count = 71 g/L, difference = 59 g/L, 95% CI = 40 g/L to 78 g/L, P < .001). Of the few Arid4a -/- mice that progressed to AML, most developed a CMML-like phenotype and became moribund from severe anemia (hemoglobin: mean wild-type count = 130 g/L, mean Arid4a -/- count = 48 g/L, difference = 82 g/L, 95% CI = 76 g/L to 88 g/L, P < .001) (Figures 1, A and 4, A).

Development of Myeloid Sarcoma in Arid4a -/- and Arid4a -/- Arid4b +/- Mice

In the human World Health Organization classification,[29] myeloid sarcoma is considered an alternative presentation of AML. The Arid4a-/- mice and the Arid4a-/-Arid4b± mice with leukemia also developed myeloid sarcoma. Soft tissue tumor nodules were found within the enlarged spleens (Figure 5, A) and livers (Figure 5, B) that were infiltrated with aggressive leukemic (malignant) cells (Figure 5, C). Blood vessels in the lungs of the AML mice showed a marked increase of nucleated elements, indicative of leukemic involvement (Figure 5, D). Similar lesions composed of leukemic cells were also found in lymph nodes and kidneys (data not shown). Flow cytometric analysis of cell populations within the splenic tissue showed an increase of leukocytes of granulocytic and monocytic origin (Gr1+ and Mac1+, 3% in wild-type mice, 13% in mice with CMML-like phenotype, and 26% in AML mice, difference between wild-type and CMML-like mice = 10%, P < .001; difference between wild-type and AML mice = 23%, P < .001) (Figure 4, A). This increase in granulocytes and monocytes was accompanied by an increase of erythroid cells (Ter119+, 6% erythroid cells in wild-type mice, 63% in mice with CMML-like phenotype, and 54% in AML mice, difference between wild-type and CMML-like mice = 57%, P < .001; difference between wild-type and AML mice = 48%, P < .001) and relative decreases of the T lymphoid (CD3+, 26.7% in wild-type mice, 5.5% in mice with CMML-like phenotype, and 7.6% in AML mice, difference between wild-type and CMML-like mice = 21.2%, P < .001; difference between wild-type and AML mice = 19.1%, P < .001) and B lymphoid (B220+CD19+, 58.9% in wild-type mice, 4.8% in mice with CMML-like phenotype, and 6.7% in AML mice, difference between wild-type and CMML-like mice = 54.1%, P < .001; difference between wild-type and AML mice = 52.2%, P < .001) populations (Figure 4, A). These features fulfill the criteria for AML (granulocytic and monocytic) and myeloid (granulocytic) sarcoma in mice (Bethesda proposals).[30]

Figure 5.

Development of myeloid sarcoma in Arid4a -/- Arid4b ± mice. A) Splenomegaly and B) hepatomegaly in the acute myeloid leukemia (AML) Arid4a -/- Arid4b ± mice relative to spleen and liver from wild-type littermates. C) Histologic analysis of spleen and liver from a wild-type mouse and an AML Arid4a -/- Arid4b ± mouse. Paraffin sections were stained with hematoxylin and eosin. Black arrowheads indicate mitotic leukemic cells in spleen and liver from the Arid4a -/- Arid4b ± mouse. White arrowheads indicate hepatocytes. D) Histologic analysis of lungs from a wild-type and an Arid4a -/- mouse. Paraffin sections were stained with hematoxylin and eosin. Blood vessels in the Arid4a -/- lungs showed a marked increase of leukemic cells.

Expansion of HSCs and Downstream Progenitors in Arid4a -/- and Arid4a -/- Arid4b +/- Mice

Because the abnormalities were found in all hematologic lineages, we tested whether deficiency of Arid4a and Arid4b has effects on HSCs and downstream progenitors. Compared with wild-type mice, Arid4a -/- mice had an increased proportion of HSCs (Lin-Sca1+c-Kit+, referred to as L-S+K+, 0.24% in wild-type mice, 0.40% in Arid4a -/- mice, difference = 0.16%, P = .041) in bone marrow (Figure 4, A). In mice that were also heterozygous for the Arid4b mutation, there was greater expansion of the HSCs population in the Arid4a -/- Arid4b ± bone marrow (L-S+K+, 0.24% in wild-type mice, 0.55% in Arid4a -/- Arid4b ± mice, difference = 0.31%, P < .001) (Figure 4, A). Furthermore, the proportions of CMPs (Lin-Sca1-c-Kit+, referred as L-S-K+) in the Arid4a -/- and Arid4a -/- Arid4b ± bone marrow were statistically significantly higher than those from wild-type mice (L-S-K+, 0.4% in wild-type mice, 2.0% in mice with CMML-like phenotype, and 2.1% in AML mice, difference between wild-type and CMML-like mice = 1.6%, P < .001; difference between wild-type and AML mice = 1.7%, P < .001) (Figure 4, A). In the spleen, dramatic increases of the HSC (L-S+K+, 0.03% in wild-type mice, 0.4% in mice with CMML-like phenotype, and 0.55% in AML mice, difference between wild-type and CMML-like mice = 0.37%, P = .02; difference between wild-type and AML mice = 0.52%, P = .004) and CMP (L-S-K+, 0.01% in wild-type mice, 5.13% in mice with CMML-like phenotype, and 5.01% in AML mice, difference between wild-type and CMML-like mice = 5.12%, P = .028; difference between wild-type and AML mice = 5%, P = .014) populations were found in both Arid4a -/- mice and Arid4a -/- Arid4b ± mice (Figure 4, A), suggesting that extramedullary hematopoiesis in spleen might be due to mobilization of HSCs from bone marrow.

Collectively, our data suggested the following disease model for the observed hematologic disorders. Deficiency of Arid4a and Arid4b results in increase of HSCs, CMPs, and Gr1+Mac1+ myeloid cells in bone marrow and spleen, which leads to increases of neutrophils and monocytes in the peripheral blood (Figure 4, B). Despite the compensatory erythropoiesis within the enlarged spleen due to the decrease of erythroid activity in the bone marrow (Figures 1, J and 4, A and B), the enlarged spleen sequestrates RBCs. Although it is not known if the reduction in platelets is due to decreased production or increased destruction, the reduction leads to spontaneous hemorrhage (Supplementary Figure 1, available online). All of these processes contribute to a decrease of RBCs in peripheral blood (Figure 4, B). Although the proportion of T lymphoid cells was increased in the bone marrow, the population of B lymphoid cells was substantially decreased, leading to the lower number of total lymphocytes in the peripheral blood (Figure 4, B). Thus, Arid4a and Arid4b have essential roles in hematopoietic homeostasis and in lineage fate determination.

Disturbed Patterns of Histone Modifications in Arid4a -/- Bone Marrow Cells

The ARID4A and ARID4B proteins contain a chromodomain and a Tudor domain. Both domains have been reported to mediate binding to methylated lysines of histones H3 and H4.[11,12,13,14] Given the bone marrow failure phenotype found in the Arid4a -/- mice, we investigated the expression and methylation status of histones in bone marrow of these mice. By western blot analysis, levels of histones H3 and H4 were elevated by 2.7- and 2.2-fold, respectively (95% CI = 2.3 to 3.1 and 1.9 to 2.5, respectively) in the Arid4a -/- bone marrow constituents compared with the wild-type samples (Figure 6, A). Levels of H2AX were not different. Methylation of lysine in H3 can lead to either repression or activation of gene expression; trimethylation of H3K4 (H3K4me3) is associated with transcriptional activation at euchromatic regions, whereas trimethylation of H3K9 (H3K9me3) is usually associated with repressive states at pericentric heterochromatin. Analysis of H3 lysine methylation revealed very large increases of both H3K4me3 (32-fold, 95% CI = 27 to 37) and H3K9me3 (45-fold, 95% CI = 41 to 49) in the bone marrow cells from the Arid4a -/- mice compared with wild-type mice (Figure 6, A). As detected by immunofluorescence, H3K4me3 was broadly distributed over euchromatic regions but showed speckled patterns in both wild-type and Arid4a -/- bone marrow cells (Figure 6, B). In wild-type bone marrow cells, H3K9me3 was localized in discrete spots in the DAPI-dense regions that correspond to the pericentric heterochromatin structure (Figure 6, B). Arid4a -/- bone marrow cells showed a diffuse pattern of H3K9me3-derived immunofluorescence with small foci spread throughout the nuclei (Figure 6, B). These different patterns of H3K9me3 between wild-type and the Arid4a -/- samples were obvious across the great majority of cells in the marrow even though the cell populations in wild-type and the Arid4a -/- bone marrow samples were different (Figure 4, A). These differences of immunofluorescence staining patterns of H3K9me3 were not seen in primary mouse embryo fibroblasts (pMEFs) (Figure 6, B). Trimethylation of H4K20 (H4K20me3) is another modification usually found on repressed chromatin accumulated at pericentric heterochromatin regions. When analyzed by western blotting, the Arid4a -/- bone marrow cells revealed a slight increase of H4K20me3 (2.2-fold, 95% CI = 1.7 to 2.7), which might reflect increased expression of histone H4 (2.2-fold, 95% CI = 1.9 to 2.5) (Figure 6, A). The fluorescence signals representing H4K20me3 were focally enriched at pericentric heterochromatin in both wild-type and the Arid4a -/- bone marrow cells (Figure 6, B>). Collectively, these results suggest that Arid4a participates in regulating lysine trimethylation of histones H3 and H4 in bone marrow.

Figure 6.

Histone modifications in bone marrow cells of mice lacking Arid4a. A) Western blot analysis of acid-extracted proteins from bone marrow of a wild-type and an Arid4a -/- mouse. Total proteins were transferred to a nitrocellulose membrane and stained with Ponceau S (top), followed by staining with antibodies against histones H3, H4, H2AX, H3K4me3, H3K9me3, and H4K20me3 (bottom). Ratios of histones were quantified by densitometry. Five separate experiments were performed. B) Immunofluorescence analysis of bone marrow cells or primary mouse embryo fibroblasts from wild-type and the Arid4a -/- mice using antibodies against H3K4me3, H3K9me3, and H4K20me3. DNA was counterstained with DAPI. Images were analyzed by deconvolution microscopy. Three separate experiments were performed, all with similar results.

Decreased Expression of the Hox and Fox Genes in Bone Marrow Cells with Arid4a and Arid4b ± Mutations

To investigate the downstream genes regulated by Arid4a and Arid4b, we first compared gene expression of wild-type pMEFs and Arid4a-/-Arid4b± pMEFs using gene expression microarrays. We identified genotype-specific differences in expression of Hox and Fox genes (data not shown). To further elucidate the regulatory mechanisms by which Arid4a and Arid4b are involved in acquisition of the leukemic phenotype, we compared expression of several genes important for normal hematopoiesis and leukemogenesis. These included the homeobox genes in the Hox clusters (Hoxa, Hoxb, and Hoxc),[31,32], homeodomain transcription factors Pbx1,[33]Meis1,[34] and Pitx2,[35]AML1 (acute myeloid leukemia 1),[36]Mll (mixed-lineage leukemia),[37] and the forkhead box gene Foxp3.[38,39] We compared the expression of these genes in bone marrow from wild-type, Arid4a -/-, and Arid4a -/- Arid4b ± mice. To minimize variation arising from differences in cell populations, we collected bone marrow cells from mice before development of illness. We monitored relative gene expression levels using reverse transcription-PCR. The expression of several Hoxb genes, including Hoxb3, Hoxb5, Hoxb6, and Hoxb8, but not Hoxb4, was decreased in both Arid4a -/- and Arid4a -/- Arid4b ± bone marrow compared to wild-type mice. This decrease was specific for Hoxb genes because the expression of Hoxa7, Hoxa9, Hoxc6, Pbx1, and Meis1 was not altered (Figure 7, A). The expression of Pitx2 that is required for normal hematopoiesis was also reduced in Arid4a -/- and Arid4a -/- Arid4b ± bone marrow (Figure 7, A). In contrast, expression of AML1 and Mll, genes involved in leukemogenesis, was similar in all the bone marrow samples (Figure 7, A). These results suggested that Arid4a regulates hematopoiesis by controlling the expression of specific homeodomain genes, such as Pitx2 and a subset of Hoxb genes.

Figure 7.

Gene expression analysis of bone marrow cells from wild-type, Arid4a -/-, and Arid4a -/- Arid4b ± mice. A) Reverse transcriptase-polymerase chain reaction was performed to analyze the genes indicated, with Hprt serving as the control gene. Three separate experiments were performed. B) Pathways by which Arid4a and Arid4b might regulate hematopoiesis through control of the Hox and Fox genes. In the scenario shown, Arid4a controls erythropoiesis, possibly by positively regulating Pitx2 and Hoxb6 genes. Arid4a also controls the expression of Hoxb8, whose product blocks differentiation of hematopoietic stem cells and common myeloid progenitors. Control of B lymphopoiesis by Arid4a may be achieved by increasing expression of Hoxb3. Arid4a, together with Arid4b, increases expression of Foxp3, which acts on regulatory T (TR) cells to suppress conventional T (Tc) cells. Foxp3 also functions as a tumor suppressor gene. However, it is unclear whether Foxp3 suppresses leukemia malignancies.

The FOXP3 protein plays an important role in control of the regulatory T-cell lineage,[40] and Foxp3 was recently identified as a tumor suppressor gene.[41] The expression of Foxp3 was reduced specifically in the Arid4a -/- Arid4b ± bone marrow but not in the Arid4a -/- bone marrow (Figure 7, A), suggesting that downregulation of Foxp3 may be involved in the mechanisms underlying the increased numbers of T cells in the Arid4a -/- Arid4b ± bone marrow (Figure 4, A) and the high frequency of leukemia malignancies in the Arid4a -/- Arid4b ± mice.

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