ABSTRACT
Recent studies suggest that individual subunits of chromatin-remodeling complexes produce biologically specific meaning in different cell types through combinatorial assembly. Here we show that granulocyte development requires SMARCD2, a subunit of ATP-dependent SWI/SNF (BAF) chromatin-remodeling complexes. Smarcd2-deficient mice fail to generate functionally mature neutrophils and eosinophils, a phenotype reminiscent of neutrophil-specific granule deficiency (SGD) in humans, for which loss-of-function mutations in CEBPE (encoding CEBPÉ) have been reported. SMARCD2-containing SWI/SNF complexes are necessary for CEBPÉ transcription factor recruitment to the promoter of neutrophilic secondary granule genes and for granulocyte differentiation. The homologous SMARCD1 protein (63% identical at the amino acid level) cannot replace the role of SMARCD2 in granulocyte development. We find that SMARCD2 functional specificity is conferred by its divergent coiled-coil 1 and SWIB domains. Strikingly, both CEBPE and SMARCD2 loss-of-function mutations identified in patients with SGD abolish the interaction with SWI/SNF and thereby secondary granule gene expression, thus providing a molecular basis for this disease.
Subject(s)
CCAAT-Enhancer-Binding Proteins/genetics , Chromatin/genetics , Granulocytes/metabolism , Hematopoiesis/genetics , Mutation , Transcription Factors/genetics , Animals , Animals, Newborn , CCAAT-Enhancer-Binding Proteins/metabolism , Chromatin/metabolism , Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Gene Expression Regulation , Granulocytes/cytology , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Muscle Proteins/genetics , Muscle Proteins/metabolism , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/metabolismABSTRACT
The ability of hemopoietic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that establish and maintain stage-specific patterns of gene expression. However, the epigenetic factors responsible for mediating these regulatory events remain poorly defined. Here we provide evidence that BAF45a/PHF10, a subunit of SWI/SNF-like chromatin remodeling complexes, is essential for adult hemopoietic stem cell maintenance and myeloid lineage development. Deletion of BAF45a in the mouse is embryonic lethal. Acute deletion of BAF45a in the adult hemopoietic system causes a dose-dependent decrease in the frequency of long-term repopulating hemopoietic stem cells and committed myeloid progenitors without affecting their rate of proliferation. BAF45a-deficient hemopoietic stem cells and myeloid progenitors are selectively lost from mixed bone marrow chimeras, indicating their impaired function even in an intact microenvironment. Together, these studies suggest that the BAF45a subunit of SWI/SNF-like chromatin remodeling complexes plays nonredundant and specialized roles within the developing hemopoietic tissue.
Subject(s)
Cell Self Renewal , Chromosomal Proteins, Non-Histone/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Homeodomain Proteins/metabolism , Animals , Biomarkers , Cell Differentiation/genetics , Cell Self Renewal/genetics , Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Gene Expression , Gene Expression Regulation, Developmental , Gene Order , Genes, Lethal , Genetic Loci , Hematopoietic Stem Cell Transplantation , Homeodomain Proteins/genetics , Mice , Mice, Knockout , Multiprotein Complexes/metabolism , Myeloid Cells/cytology , Myeloid Cells/metabolism , Phenotype , Protein Binding , Protein Interaction Domains and Motifs , Protein Subunits , Transcriptional Activation , Zinc FingersABSTRACT
In mammals, combinatorial assembly of alternative families of subunits confers functional specificity to adenosine triphosphate (ATP)-dependent SWI/SNF-like Brg/Brm-associated factor (BAF) chromatin remodeling complexes by creating distinct polymorphic surfaces for interaction with regulatory elements and DNA-binding factors. Although redundant in terms of biochemical activity, the core ATPase subunits, BRG/SMARCA4 and BRM/SMARCA2, are functionally distinct and may contribute to complex specificity. Here we show using quantitative proteomics that BAF complexes expressed in leukemia are specifically assembled around the BRG ATPase. Moreover, using a mouse model of acute myeloid leukemia, we demonstrate that BRG is essential for leukemia maintenance, as leukemic cells lacking BRG rapidly undergo cell-cycle arrest and apoptosis. Most importantly, we show that BRG is dispensable for the maintenance of immunophenotypic long-term repopulating hematopoietic stem cells, suggesting that adroit targeting of BRG in leukemia may have potent and specific therapeutic effects.
Subject(s)
Chromatin Assembly and Disassembly , DNA Helicases/metabolism , DNA Helicases/physiology , Hematopoietic Stem Cells/pathology , Leukemia, Myeloid, Acute/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Transcription Factors/metabolism , Transcription Factors/physiology , Animals , Blotting, Southern , Chromatography, Liquid , DNA Helicases/genetics , Disease Models, Animal , Flow Cytometry , Hematopoietic Stem Cells/metabolism , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tandem Mass Spectrometry , Transcription Factors/geneticsABSTRACT
ATP-dependent SWI/SNF-like BAF chromatin remodeling complexes are emerging as key regulators of embryonic and adult stem cell function. Particularly intriguing are the findings that specialized assemblies of BAF complexes are required for establishing and maintaining pluripotent and multipotent states in cells. However, little is known on the importance of these complexes in normal and leukemic hemopoiesis. Here we provide the first evidence that the actin-related protein BAF53a, a subunit of BAF complexes preferentially expressed in long-term repopulating stem cells, is essential for adult hemopoiesis. Conditional deletion of BAF53a resulted in multilineage BM failure, aplastic anemia, and rapid lethality. These severe hemopoietic defects originate from a proliferative impairment of BM HSCs and progenitors and decreased progenitor survival. Using hemopoietic chimeras, we show that the impaired function of BAF53a-deficient HSCs is cell-autonomous and independent of the BM microenvironment. Altogether, our studies highlight an unsuspected role for BAF chromatin remodeling complexes in the maintenance of HSC and progenitor cell properties.
Subject(s)
Actins/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Hematopoietic Stem Cells/metabolism , Hematopoietic System/metabolism , Transcriptome , Actins/metabolism , Anemia, Aplastic/genetics , Anemia, Aplastic/metabolism , Animals , Apoptosis/genetics , Apoptosis/physiology , Bone Marrow Cells/metabolism , Bone Marrow Cells/physiology , Cell Cycle Proteins/genetics , Cell Proliferation , Cell Survival/genetics , Cell Survival/physiology , Cells, Cultured , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Female , Fetus , Flow Cytometry , Gene Expression Regulation, Developmental , Hematopoietic Stem Cells/physiology , Hematopoietic System/growth & development , Liver , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Pregnancy , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Plasma cholesterol from low- and high-density lipoproteins (LDL and HDL) are cleared from the circulation by specific receptors that either totally degrade lipoproteins as the LDL receptor or selectively take up their cholesteryl esters (CE) like the scavenger receptor class B type I (SR-BI). The aim of the present study was to define the effect of apoC-I on the uptake of LDL and HDL(3) by HepG2 cells. In experiments conducted with exogenously added purified apoC-I, no significant effect was observed on lipoprotein-protein association and degradation; however, LDL- and HDL(3)-CE selective uptake was significantly reduced in a dose-dependent manner. This study also shows that apoC-I has the ability to associate with HepG2 cells and with LDL and HDL(3). Moreover, pre-incubation of HepG2 cells with apoC-I reduces HDL(3)-CE selective uptake and pre-incubation of LDL and HDL(3) with apoC-I decreases their CE selective uptake by HepG2 cells. Thus, apoC-I can accomplish its inhibitory effect on SR-BI activity by either binding to SR-BI or lipoproteins. We conclude that by reducing hepatic lipoprotein-CE selective uptake, apoC-I has an atherogenic character.
