Chromatin Remodeling Genes Arid4a and Arid4b as Leukemia Suppressor Genes
Chromatin Remodeling Genes Arid4a and Arid4b as Leukemia Suppressor Genes
Background: Leukemia evolves through a multistep process from premalignancy to malignancy. Epigenetic alterations, including histone modifications, have been proposed to play an important role in tumorigenesis. The involvement of two chromatin remodeling genes, retinoblastoma-binding protein 1 (Rbbp1/Arid4a) and Rbbp1-like 1 (Rbbp1l1/Arid4b), in leukemogenesis was not characterized.
Methods: The leukemic phenotype of mice deficient for Arid4a with or without haploinsufficiency for Arid4b was investigated by serially monitoring complete blood counts together with microscopic histologic analysis and flow cytometric analysis of bone marrow and spleen from the Arid4a mice or Arid4aArid4b mice. Regulation in bone marrow cells of downstream genes important for normal hematopoiesis was analyzed by reverse transcription-polymerase chain reaction. Genotypic effects on histone modifications were examined by western blotting and immunofluorescence analysis. All statistical tests were two-sided.
Results: Young (2-5 months old) Arid4a-deficient mice had ineffective blood cell production in all hematopoietic lineages. Beyond 5 months of age, the Arid4a mice manifested monocytosis, accompanied by severe anemia and thrombocytopenia. These sick Arid4a mice showed bone marrow failure with myelofibrosis associated with splenomegaly and hepatomegaly. Five of 42 Arid4a mice and 10 of 12 Arid4aArid4b mice progressed to acute myeloid leukemia (AML) and had rapid further increases of leukocyte counts. Expression of Hox genes (Hoxb3, Hoxb5, Hoxb6, and Hoxb8) was decreased in Arid4a-deficient bone marrow cells with or without Arid4b haploinsufficiency, and FoxP3 expression was reduced in Arid4aArid4b bone marrow. Increases of histone trimethylation of H3K4, H3K9, and H4K20 (fold increases in trimethylation = 32, 95% confidence interval [CI] = 27 to 32; 45, 95% CI = 41 to 49; and 2.2, 95% CI = 1.7 to 2.7, respectively) were observed in the bone marrow of Arid4a-deficient mice.
Conclusions:Arid4a-deficient mice initially display ineffective hematopoiesis, followed by transition to chronic myelomonocytic leukemia (CMML)-like myelodysplastic/myeloproliferative disorder, and then transformation to AML. The disease processes in the Arid4a-deficient mice are very similar to the course of events in humans with CMML and AML. This mouse model has the potential to furnish additional insights into the role of epigenetic alterations in leukemogenesis, and it may be useful in developing novel pharmacological approaches to treatment of preleukemic and leukemic states.
The human ARID4A and ARID4B genes, previously known as retinoblastoma-binding protein 1 (RBBP1, RBP1) and RBBP1-like protein 1 (RBBP1L1), respectively, are homologous members of the AT-rich interaction domain (ARID) gene family. The ARID4A amino acid sequence, but not that of ARID4B, contains an LVCHE sequence as a conserved LXCXE motif known to interact with the pocket of retinoblastoma protein (RB). We reported that ARID4A interacts with ARID4B. Both ARID4A and ARID4B contain a Tudor domain, an ARID domain, and a chromodomain, as well as two repression domains, R1 and R2, with part of the R1 domain overlapping the ARID domain. The ARID domain contains a helix-turn-helix structure with DNA binding activity. Chromodomains are found in a variety of proteins that play roles in the functional organization of chromosome structure through chromatin remodeling. Both chromodomains and Tudor domains mediate binding to methylated lysines of histones H3 and H4. ARID4A recruited by RB has been defined as a repressor of E2F-dependent transcription. Through their R2 regions, ARID4A and ARID4B serve as adapters to recruit the mSin3A-histone deacetylase (HDAC) histone-modifying complex to E2F-dependent promoters. ARID4A and ARID4B also provide a repressive function in an HDAC-independent manner through their R1 regions. Transcriptional repression activity via the R1 region is controlled by SUMOylation, a posttranslational modification by small ubiquitin-related modifier proteins.
Several lines of evidence suggest that ARID4A and ARID4B may be involved in the pathogenesis of breast and other cancers. RB (with which ARID4A interacts and which is inactivated in many tumors) has been shown to be involved in many cellular processes, including control of the cell cycle, cell differentiation, DNA-damage responses, DNA replication, and protection against apoptosis. Repression of E2F-dependent transcription by ARID4A and ARID4B in conjunction with RB leads to cell arrest reminiscent of senescence. Screening a cDNA library from the MCF7 breast cancer cell line with IgG purified from the serum of a breast cancer patient identified ARID4A and ARID4B as tumor-associated antigens, and breast cancer patients have high titers of antibodies against both proteins. Furthermore, human cytotoxic T cells stimulated with ARID4A peptides kill breast cancer cells. ARID4A also interacts with the breast cancer metastasis suppressor 1 (BRMS1) and the BRMS1 homologue p40 in the mSin3-HDAC complex. BRMS1 reduces the metastatic activity of cancer cells without affecting tumorigenicity. Despite these reports, the role of ARID4A and ARID4B in cancer development is unclear.
Preleukemic conditions are relatively common in aging populations, and little is known about their pathogenesis or how they predispose to leukemia. Many physicians and patients face this distressing clinical situation on a chronic basis, and there is little that the physician can offer to prevent the potential transformation to malignancy because the mechanisms by which cancer progresses from premalignancy to malignancy are not fully understood.
Epigenetic alterations such as histone modifications of chromatin structure have been suggested to be involved in the development of leukemia and other cancers. Although an increasing number of chromatin remodeling proteins have been defined biochemically, their biological functions, particularly in the context of an animal model, are poorly characterized, and the molecular events governing chromatin reorganization in cancer cells remain relatively unexplored. Recently, we developed mouse models for Arid4a and Arid4b deficiency that demonstrated the function of Arid4a and Arid4b in the regulation of genomic imprinting through control of epigenetic modifications. Here, we use these mouse models deficient for Arid4a, alone or in combination with haploinsufficiency for Arid4b, to analyze a premalignant hematopoietic disorder and eventual leukemia.
Abstract and Introduction
Abstract
Background: Leukemia evolves through a multistep process from premalignancy to malignancy. Epigenetic alterations, including histone modifications, have been proposed to play an important role in tumorigenesis. The involvement of two chromatin remodeling genes, retinoblastoma-binding protein 1 (Rbbp1/Arid4a) and Rbbp1-like 1 (Rbbp1l1/Arid4b), in leukemogenesis was not characterized.
Methods: The leukemic phenotype of mice deficient for Arid4a with or without haploinsufficiency for Arid4b was investigated by serially monitoring complete blood counts together with microscopic histologic analysis and flow cytometric analysis of bone marrow and spleen from the Arid4a mice or Arid4aArid4b mice. Regulation in bone marrow cells of downstream genes important for normal hematopoiesis was analyzed by reverse transcription-polymerase chain reaction. Genotypic effects on histone modifications were examined by western blotting and immunofluorescence analysis. All statistical tests were two-sided.
Results: Young (2-5 months old) Arid4a-deficient mice had ineffective blood cell production in all hematopoietic lineages. Beyond 5 months of age, the Arid4a mice manifested monocytosis, accompanied by severe anemia and thrombocytopenia. These sick Arid4a mice showed bone marrow failure with myelofibrosis associated with splenomegaly and hepatomegaly. Five of 42 Arid4a mice and 10 of 12 Arid4aArid4b mice progressed to acute myeloid leukemia (AML) and had rapid further increases of leukocyte counts. Expression of Hox genes (Hoxb3, Hoxb5, Hoxb6, and Hoxb8) was decreased in Arid4a-deficient bone marrow cells with or without Arid4b haploinsufficiency, and FoxP3 expression was reduced in Arid4aArid4b bone marrow. Increases of histone trimethylation of H3K4, H3K9, and H4K20 (fold increases in trimethylation = 32, 95% confidence interval [CI] = 27 to 32; 45, 95% CI = 41 to 49; and 2.2, 95% CI = 1.7 to 2.7, respectively) were observed in the bone marrow of Arid4a-deficient mice.
Conclusions:Arid4a-deficient mice initially display ineffective hematopoiesis, followed by transition to chronic myelomonocytic leukemia (CMML)-like myelodysplastic/myeloproliferative disorder, and then transformation to AML. The disease processes in the Arid4a-deficient mice are very similar to the course of events in humans with CMML and AML. This mouse model has the potential to furnish additional insights into the role of epigenetic alterations in leukemogenesis, and it may be useful in developing novel pharmacological approaches to treatment of preleukemic and leukemic states.
Introduction
The human ARID4A and ARID4B genes, previously known as retinoblastoma-binding protein 1 (RBBP1, RBP1) and RBBP1-like protein 1 (RBBP1L1), respectively, are homologous members of the AT-rich interaction domain (ARID) gene family. The ARID4A amino acid sequence, but not that of ARID4B, contains an LVCHE sequence as a conserved LXCXE motif known to interact with the pocket of retinoblastoma protein (RB). We reported that ARID4A interacts with ARID4B. Both ARID4A and ARID4B contain a Tudor domain, an ARID domain, and a chromodomain, as well as two repression domains, R1 and R2, with part of the R1 domain overlapping the ARID domain. The ARID domain contains a helix-turn-helix structure with DNA binding activity. Chromodomains are found in a variety of proteins that play roles in the functional organization of chromosome structure through chromatin remodeling. Both chromodomains and Tudor domains mediate binding to methylated lysines of histones H3 and H4. ARID4A recruited by RB has been defined as a repressor of E2F-dependent transcription. Through their R2 regions, ARID4A and ARID4B serve as adapters to recruit the mSin3A-histone deacetylase (HDAC) histone-modifying complex to E2F-dependent promoters. ARID4A and ARID4B also provide a repressive function in an HDAC-independent manner through their R1 regions. Transcriptional repression activity via the R1 region is controlled by SUMOylation, a posttranslational modification by small ubiquitin-related modifier proteins.
Several lines of evidence suggest that ARID4A and ARID4B may be involved in the pathogenesis of breast and other cancers. RB (with which ARID4A interacts and which is inactivated in many tumors) has been shown to be involved in many cellular processes, including control of the cell cycle, cell differentiation, DNA-damage responses, DNA replication, and protection against apoptosis. Repression of E2F-dependent transcription by ARID4A and ARID4B in conjunction with RB leads to cell arrest reminiscent of senescence. Screening a cDNA library from the MCF7 breast cancer cell line with IgG purified from the serum of a breast cancer patient identified ARID4A and ARID4B as tumor-associated antigens, and breast cancer patients have high titers of antibodies against both proteins. Furthermore, human cytotoxic T cells stimulated with ARID4A peptides kill breast cancer cells. ARID4A also interacts with the breast cancer metastasis suppressor 1 (BRMS1) and the BRMS1 homologue p40 in the mSin3-HDAC complex. BRMS1 reduces the metastatic activity of cancer cells without affecting tumorigenicity. Despite these reports, the role of ARID4A and ARID4B in cancer development is unclear.
Preleukemic conditions are relatively common in aging populations, and little is known about their pathogenesis or how they predispose to leukemia. Many physicians and patients face this distressing clinical situation on a chronic basis, and there is little that the physician can offer to prevent the potential transformation to malignancy because the mechanisms by which cancer progresses from premalignancy to malignancy are not fully understood.
Epigenetic alterations such as histone modifications of chromatin structure have been suggested to be involved in the development of leukemia and other cancers. Although an increasing number of chromatin remodeling proteins have been defined biochemically, their biological functions, particularly in the context of an animal model, are poorly characterized, and the molecular events governing chromatin reorganization in cancer cells remain relatively unexplored. Recently, we developed mouse models for Arid4a and Arid4b deficiency that demonstrated the function of Arid4a and Arid4b in the regulation of genomic imprinting through control of epigenetic modifications. Here, we use these mouse models deficient for Arid4a, alone or in combination with haploinsufficiency for Arid4b, to analyze a premalignant hematopoietic disorder and eventual leukemia.
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