title: Binding of a Smad4/Ets-1 complex to a novel intragenic regulatory element in exon12 of FPGS underlies decreased gene expression and antifolate resistance in leukemia.
abstract: Polyglutamylation of antifolates catalyzed by folylpoly-γ-glutamate synthetase (FPGS) is essential for their intracellular retention and cytotoxic activity. Hence, loss of FPGS expression and/or function results in lack of antifolate polyglutamylation and drug resistance. Members of the TGF-β/Smad signaling pathway are negative regulators of hematopoiesis and deregulation of this pathway is considered a major contributor to leukemogenesis. Here we show that FPGS gene expression is inversely correlated with the binding of a Smad4/Ets-1 complex to exon12 of FPGS in both acute lymphoblastic leukemia cells and acute myeloid leukemia blast specimens. We demonstrate that antifolate resistant leukemia cells harbor a heterozygous point mutation in exon12 of FPGS which disrupts FPGS activity by abolishing ATP binding, and alters the binding pattern of transcription factors to the genomic region of exon12. This in turn results in the near complete silencing of the wild type allele leading to a 97% loss of FPGS activity. We show that exon12 is a novel intragenic transcriptional regulator, endowed with the ability to drive transcription in vitro, and is occupied by transcription factors and chromatin remodeling agents (e.g. Smad4/Ets-1, HP-1 and Brg1) in vivo. These findings bear important implications for the rational overcoming of antifolate resistance in leukemia.
authors.affiliation: The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Shachar
authors.initials: S
authors.lastname: Raz
authors.affiliation: The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Stark
authors.affiliation: The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
mesh_terms: D000328:Adult; D000368:Aged; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D019175:DNA Methylation / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D020868:Gene Silencing; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics; D008297:Male; D008875:Middle Aged; D009687:Nuclear Proteins / Q000378:metabolism; D010453:Peptide Synthases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D050784:Proto-Oncogene Protein c-ets-1 / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D051901:Smad4 Protein / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors; D055815:Young Adult
chemical_list: D002868:Chromosomal Proteins, Non-Histone; C494041:ETS1 protein, human; D005493:Folic Acid Antagonists; D009687:Nuclear Proteins; D050784:Proto-Oncogene Protein c-ets-1; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D000090266:Chromobox Protein Homolog 5; C084108:SMARCA4 protein, human; D004265:DNA Helicases; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase