ABSTRACT
The mitochondrial deacetylase SIRT3 regulates several important metabolic processes. SIRT3 is transcriptionally upregulated in multiple tissues during nutrient stresses such as dietary restriction and fasting, but the molecular mechanism of this induction is unclear. We conducted a bioinformatic study to identify transcription factor(s) involved in SIRT3 induction. Our analysis identified an enrichment of binding sites for nuclear respiratory factor 2 (NRF-2), a transcription factor known to play a role in the expression of mitochondrial genes, in the DNA sequences of SIRT3 and genes with closely correlated expression patterns. In vitro, knockdown or overexpression of NRF-2 modulated SIRT3 levels, and the NRF-2α subunit directly bound to the SIRT3 promoter. Our results suggest that NRF-2 is a regulator of SIRT3 expression and may shed light on how SIRT3 is upregulated during nutrient stress.
Subject(s)
GA-Binding Protein Transcription Factor/metabolism , Sirtuin 3/genetics , Animals , Binding Sites , Cells, Cultured , Computational Biology , Enzyme Induction/genetics , GA-Binding Protein Transcription Factor/deficiency , HEK293 Cells , Humans , Promoter Regions, Genetic/genetics , Sirtuin 3/biosynthesisABSTRACT
Mitochondria are membrane-bound cytoplasmic organelles that serve as the major source of ATP production in eukaryotic cells. GABP (also known as nuclear respiratory factor 2) is a nuclear E26 transformation-specific transcription factor (ETS) that binds and activates mitochondrial genes that are required for electron transport and oxidative phosphorylation. We conditionally deleted Gabpa, the DNA-binding component of this transcription factor complex, from mouse embryonic fibroblasts (MEFs) to examine the role of Gabp in mitochondrial biogenesis, function, and gene expression. Gabpα loss modestly reduced mitochondrial mass, ATP production, oxygen consumption, and mitochondrial protein synthesis but did not alter mitochondrial morphology, membrane potential, apoptosis, or the expression of several genes that were previously reported to be GABP targets. However, the expression of Tfb1m, a methyltransferase that modifies ribosomal rRNA and is required for mitochondrial protein translation, was markedly reduced in Gabpα-null MEFs. We conclude that Gabp regulates Tfb1m expression and plays an essential, nonredundant role in mitochondrial biogenesis.
Subject(s)
GA-Binding Protein Transcription Factor/metabolism , Mitochondria/metabolism , Adenosine Triphosphate/biosynthesis , Animals , Apoptosis , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/metabolism , GA-Binding Protein Transcription Factor/deficiency , GA-Binding Protein Transcription Factor/genetics , Gene Expression Regulation , Genes, Mitochondrial , Membrane Potential, Mitochondrial , Mice , Mice, Knockout , Mitochondria/genetics , Oxygen Consumption , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. Transformation of HSCs by the breakpoint cluster region-ABL tyrosine kinase (BCR-ABL) oncogene causes chronic myelogenous leukemia (CML). The E-twenty six (ets) transcription factor GA binding protein (GABP) is a tetrameric transcription factor complex that contains GABPα and GABPß proteins. Deletion in bone marrow of Gabpa, the gene that encodes the DNA-binding component, caused cell cycle arrest in HSCs and profound loss of hematopoietic progenitor cells. Loss of Gabpα prevented development of CML, although mice continued to generate BCR-ABL-expressing Gabpα-null cells for months that were serially transplantable and contributed to all lineages in secondary recipients. A bioinformatic screen identified the serine-threonine kinase protein kinase D2 (PRKD2) as a potential effector of GABP in HSCs. Prkd2 expression was markedly reduced in Gabpα-null HSCs and progenitor cells. Reduced expression of PRKD2 or pharmacologic inhibition decreased cell cycling, and PRKD2 rescued growth of Gabpα-null BCR-ABL-expressing cells. Thus, GABP is required for HSC cell cycle entry and CML development through its control of PRKD2. This offers a potential therapeutic target in leukemia.
Subject(s)
GA-Binding Protein Transcription Factor/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/etiology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Protein Kinases/metabolism , Animals , Antineoplastic Agents/pharmacology , Benzamides , Cell Cycle , GA-Binding Protein Transcription Factor/deficiency , GA-Binding Protein Transcription Factor/genetics , Gene Expression , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Mice , Mice, Transgenic , Piperazines/pharmacology , Protein Kinase D2 , Protein Kinase Inhibitors/pharmacology , Protein Kinases/genetics , Pyrimidines/pharmacologyABSTRACT
Hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) are both capable of self-renewal, with HSCs sustaining multiple blood lineage differentiation and LSCs indefinitely propagating leukemia. The GABP complex, consisting of DNA binding GABPα subunit and transactivation GABPß subunit, critically regulates HSC multipotency and self-renewal via controlling an essential gene regulatory module. Two GABPß isoforms, GABPß1L and GABPß2, contribute to assembly of GABPα(2)ß(2) tetramer. We demonstrate that GABPß1L/ß2 deficiency specifically impairs HSC quiescence and survival, with little impact on cell cycle or apoptosis in differentiated blood cells. The HSC-specific effect is mechanistically ascribed to perturbed integrity of the GABP-controlled gene regulatory module in HSCs. Targeting GABPß1L/ß2 also impairs LSC self-renewal in p210(BCR-ABL)-induced chronic myelogenous leukemia (CML) and exhibits synergistic effects with tyrosine kinase inhibitor imatinib therapy in inhibiting CML propagation. These findings identify the tetramer-forming GABPß isoforms as specific HSC regulators and potential therapeutic targets in treating LSC-based hematological malignancy.
Subject(s)
GA-Binding Protein Transcription Factor/metabolism , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Leukemia/pathology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Animals , Cell Differentiation , GA-Binding Protein Transcription Factor/deficiency , GA-Binding Protein Transcription Factor/genetics , Hematopoietic Stem Cells/cytology , Mice , Mice, Knockout , Protein Isoforms/deficiency , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
GABP is an ets transcription factor that regulates genes that are required for myeloid differentiation. The tetrameric GABP complex includes GABPα, which binds DNA via its ets domain, and GABPß, which contains the transcription activation domain. To examine the role of GABP in myeloid differentiation, we generated mice in which Gabpa can be conditionally deleted in hematopoietic tissues. Gabpa knockout mice rapidly lost myeloid cells, and residual myeloid cells were dysplastic and immunophenotypically abnormal. Bone marrow transplantation demonstrated that Gabpα null cells could not contribute to the myeloid compartment because of cell intrinsic defects. Disruption of Gabpa was associated with a marked reduction in myeloid progenitor cells, and Gabpα null myeloid cells express reduced levels of the transcriptional repressor, Gfi-1. Gabp bound and activated the Gfi1 promoter, and transduction of Gabpa knockout bone marrow with Gfi1 partially rescued defects in myeloid colony formation and myeloid differentiation. We conclude that Gabp is required for myeloid differentiation due, in part, to its regulation of the tran-scriptional repressor Gfi-1.
Subject(s)
DNA-Binding Proteins/physiology , GA-Binding Protein Transcription Factor/physiology , Myelopoiesis/physiology , Transcription Factors/physiology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , CD11b Antigen/metabolism , DNA-Binding Proteins/genetics , GA-Binding Protein Transcription Factor/deficiency , GA-Binding Protein Transcription Factor/genetics , Gene Knockout Techniques , Mice , Mice, Knockout , Myelopoiesis/genetics , Myelopoiesis/immunology , Phenotype , Precursor Cells, B-Lymphoid/cytology , Precursor Cells, B-Lymphoid/immunology , Precursor Cells, T-Lymphoid/cytology , Precursor Cells, T-Lymphoid/immunology , Transcription Factors/geneticsABSTRACT
Maintaining a steady pool of self-renewing hematopoietic stem cells (HSCs) is critical for sustained production of multiple blood lineages. Many transcription factors and molecules involved in chromatin and epigenetic modifications have been found to be critical for HSC self-renewal and differentiation; however, their interplay is less understood. The transcription factor GA binding protein (GABP), consisting of DNA-binding subunit GABPα and transactivating subunit GABPß, is essential for lymphopoiesis as shown in our previous studies. Here we demonstrate cell-intrinsic, absolute dependence on GABPα for maintenance and differentiation of hematopoietic stem/progenitor cells. Through genome-wide mapping of GABPα binding and transcriptomic analysis of GABPα-deficient HSCs, we identified Zfx and Etv6 transcription factors and prosurvival Bcl-2 family members including Bcl-2, Bcl-X(L), and Mcl-1 as direct GABP target genes, underlying its pivotal role in HSC survival. GABP also directly regulates Foxo3 and Pten and hence sustains HSC quiescence. Furthermore, GABP activates transcription of DNA methyltransferases and histone acetylases including p300, contributing to regulation of HSC self-renewal and differentiation. These systematic analyses revealed a GABP-controlled gene regulatory module that programs multiple aspects of HSC biology. Our studies thus constitute a critical first step in decoding how transcription factors are orchestrated to regulate maintenance and multipotency of HSCs.
Subject(s)
GA-Binding Protein Transcription Factor/physiology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/physiology , Animals , Binding Sites/genetics , Cell Differentiation/genetics , Cell Differentiation/physiology , Forkhead Box Protein O3 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , GA-Binding Protein Transcription Factor/deficiency , GA-Binding Protein Transcription Factor/genetics , Gene Expression Profiling , Gene Expression Regulation, Developmental , Genome-Wide Association Study , Hematopoiesis/genetics , Hematopoiesis/physiology , Humans , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-ets/genetics , Proto-Oncogene Proteins c-ets/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , ETS Translocation Variant 6 ProteinABSTRACT
The GA-binding protein (GABP) transcription factor has been shown in vitro to regulate the expression of the neuromuscular proteins utrophin, acetylcholine esterase, and acetylcholine receptor subunits delta and epsilon through the N-box promoter motif (5'-CCGGAA-3'), but its in vivo function remains unknown. A single point mutation within the N-box of the gene encoding the acetylcholine receptor epsilon subunit has been identified in several patients suffering from postsynaptic congenital myasthenic syndrome, implicating the GA-binding protein in neuromuscular function and disease. Since conventional gene targeting results in an embryonic-lethal phenotype, we used conditional targeting to investigate the role of GABPalpha in neuromuscular junction and skeletal muscle development. The diaphragm and soleus muscles from mutant mice display alterations in morphology and distribution of acetylcholine receptor clusters at the neuromuscular junction and neurotransmission properties consistent with reduced receptor function. Furthermore, we confirmed decreased expression of the acetylcholine receptor epsilon subunit and increased expression of the gamma subunit in skeletal muscle tissues. Therefore, the GABP transcription factor aids in the structural formation and function of neuromuscular junctions by regulating the expression of postsynaptic genes.