title: Identifying targets for the restoration and reactivation of BRM.
abstract: Brahma (BRM) is a novel anticancer gene, which is frequently inactivated in a variety of tumor types. Unlike many anticancer genes, BRM is not mutated, but rather epigenetically silenced. In addition, histone deacetylase complex (HDAC) inhibitors are known to reverse BRM silencing, but they also inactivate it via acetylation of its C-terminus. High-throughput screening has uncovered many compounds that are effective at pharmacologically restoring BRM and thereby inhibit cancer cell growth. As we do not know which specific proteins, if any, regulate BRM, we sought to identify the proteins, which underlie the epigenetic suppression of BRM. By selectively knocking down each HDAC, we found that HDAC3 and HDAC9 regulate BRM expression, whereas HDAC2 controls its acetylation. Similarly, we ectopically overexpressed 21 different histone acetyltransferases and found that KAT6A, KAT6B and KAT7 induce BRM expression, whereas KAT2B and KAT8 induce its acetylation. We also investigated the role of two transcription factors (TFs) linked to either BRM (GATA3) or HDAC9 (MEF2D) expression. Knockdown of either GATA3 and/or MEF2D downregulated HDAC9 and induced BRM. As targets for molecular biotherapy are typically uniquely, or simply differentially expressed in cancer cells, we also determined if any of these proteins are dysregulated. However, by sequencing, no mutations were found in any of these BRM-regulating HDACs, HATs or TFs. We selectively knocked down GATA3, MEF2D, HDAC3 and HDAC9, and found that each gene-specific knockdown induced growth inhibition. We observed that both GATA3 and HDAC9 were greatly overexpressed only in BRM-negative cell lines indicating that HDAC9 may be a good target for therapy. We also found that the mitogen-activated protein (MAP) kinase pathway regulates both BRM acetylation and BRM silencing as MAP kinase pathway inhibitors both induced BRM as well as caused BRM deacetylation. Together, these data identify a cadre of key proteins, which underlie the epigenetic regulation of BRM.
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, University of Florida, Gainesville, FL, USA.
authors.forename: B
authors.initials: B
authors.lastname: Kahali
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, University of Florida, Gainesville, FL, USA.
authors.forename: S J B
authors.initials: SJ
authors.lastname: Gramling
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, University of Florida, Gainesville, FL, USA.
authors.forename: S B
authors.initials: SB
authors.lastname: Marquez
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, University of Florida, Gainesville, FL, USA.
authors.forename: K
authors.initials: K
authors.lastname: Thompson
authors.affiliation: Department of Pathology, University of Florida, Gainesville, FL, USA.
authors.forename: L
authors.initials: L
authors.lastname: Lu
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, University of Florida, Gainesville, FL, USA.
authors.forename: D
authors.initials: D
authors.lastname: Reisman
mesh_terms: D000107:Acetylation; D002460:Cell Line; D049109:Cell Proliferation; D015536:Down-Regulation; D050990:GATA3 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D051548:Histone Acetyltransferases / Q000378:metabolism; D056464:Histone Deacetylase 2 / Q000235:genetics / Q000378:metabolism*; D006655:Histone Deacetylases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System; D064326:MEF2 Transcription Factors / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D050880:p300-CBP Transcription Factors / Q000378:metabolism; D000099224:p300-CBP-Associated Factor; D000099186:Histone Deacetylase 3
chemical_list: D050990:GATA3 Transcription Factor; D051548:Histone Acetyltransferases; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases; D064326:MEF2 Transcription Factors; D020928:Mitogen-Activated Protein Kinases; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; D000099186:Histone Deacetylase 3; C494749:GATA3 protein, human; C578043:MEF2D protein, human; C084011:SMARCA2 protein, human; C108712:KAT6A protein, human; C487137:KAT6B protein, human; C496553:KAT7 protein, human; C496552:KAT8 protein, human; C533423:HDAC2 protein, human; C486544:HDAC9 protein, human