ABSTRACT
The AML1-ETO fusion protein, which is present in 10-15% of cases of acute myeloid leukemia, is known to repress myeloid differentiation genes through DNA binding and recruitment of chromatin-modifying proteins and transcription factors in target genes. ChIP-chip analysis of human hematopoietic stem/progenitor cells transduced with the AML1-ETO fusion gene enabled us to identify 1168 AML1-ETO target genes, 103 of which were co-occupied by histone deacetylase 1 (HDAC1) and had lost the hyperacetylation mark at histone H4, and 264 showed a K9 trimethylation at histone H3. Enrichment of genes involved in hematopoietic differentiation and in specific signaling pathways was observed in the presence of these epigenetic modifications associated with an 'inactive' chromatin status. Furthermore, AML1-ETO target genes had a significant correlation between the chromatin marks studied and transcriptional silencing. Interestingly, AML1 binding sites were absent on a large number of selected AML1-ETO promoters and an Sp1 binding site was found in over 50% of them. Reversible silencing induced by the fusion protein in the presence of AML1 and/or Sp1 transcription factor binding site was confirmed. Therefore, this study provides a global analysis of AML1-ETO functional chromatin modifications and identifies the important role of Sp1 in the DNA binding pattern of AML1-ETO, suggesting a role for Sp1-targeted therapy in this leukemia subtype.
Subject(s)
Chromatin/genetics , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Hematopoietic Stem Cells/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Sp1 Transcription Factor/metabolism , Acetylation , Binding Sites , Cells, Cultured , Chromatin Immunoprecipitation , Core Binding Factor Alpha 2 Subunit/antagonists & inhibitors , Epigenesis, Genetic , Genomics , Hematopoietic Stem Cells/cytology , Histone Deacetylase 1/genetics , Histone Deacetylase 1/metabolism , Histones/metabolism , Humans , Oncogene Proteins, Fusion/antagonists & inhibitors , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RUNX1 Translocation Partner 1 Protein , Real-Time Polymerase Chain Reaction , Sp1 Transcription Factor/antagonists & inhibitors , Sp1 Transcription Factor/genetics , Umbilical Cord/cytology , Umbilical Cord/metabolismABSTRACT
The Kruppel-like factor (KLF) proteins are multitasked transcriptional regulators with an expanding tumor suppressor function. KLF2 is one of the prominent members of the family because of its diminished expression in malignancies and its growth-inhibitory, pro-apoptotic and anti-angiogenic roles. In this study, we show that epigenetic silencing of KLF2 occurs in cancer cells through direct transcriptional repression mediated by the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2). Binding of EZH2 to the 5'-end of KLF2 is also associated with a gain of trimethylated lysine 27 histone H3 and a depletion of phosphorylated serine 2 of RNA polymerase. Upon depletion of EZH2 by RNA interference, short hairpin RNA or use of the small molecule 3-Deazaneplanocin A, the expression of KLF2 was restored. The transfection of KLF2 in cells with EZH2-associated silencing showed a significant anti-tumoral effect, both in culture and in xenografted nude mice. In this last setting, KLF2 transfection was also associated with decreased dissemination and lower mortality rate. In EZH2-depleted cells, which characteristically have lower tumorigenicity, the induction of KLF2 depletion 'rescued' partially the oncogenic phenotype, suggesting that KLF2 repression has an important role in EZH2 oncogenesis. Most importantly, the translation of the described results to human primary samples demonstrated that patients with prostate or breast tumors with low levels of KLF2 and high expression of EZH2 had a shorter overall survival.
Subject(s)
DNA-Binding Proteins/metabolism , Kruppel-Like Transcription Factors/metabolism , Neoplasms/metabolism , Transcription Factors/metabolism , Adenosine/analogs & derivatives , Adenosine/pharmacology , Animals , Breast Neoplasms/metabolism , Enhancer of Zeste Homolog 2 Protein , Female , Humans , Kruppel-Like Transcription Factors/genetics , Male , Mice , Mice, Nude , Neoplasm Transplantation , Neoplasms/genetics , Polycomb Repressive Complex 2 , Prostatic Neoplasms/metabolism , RNA Interference , TransfectionABSTRACT
Understanding the mechanisms that link changes in DNA methylation with histone modifications is particularly relevant in the case of tumor suppressor genes that undergo transcriptional silencing in cancer cells in association with promoter CpG island hypermethylation. In this study, we show that two histone lysine methylation marks associated with active transcription, dimethylation of H3K79 (H3K79me2) and trimethylation of H3K4 (H3K4me3), are present in all the unmethylated promoters analysed, and both of them are lost when these promoters become hypermethylated. Most importantly, pharmacological and genetic interventions that cause DNA demethylation and partial recovery of gene transcription, result in the restoration of H3K4me3, but not of H3K79me2. We also show that DOT1L, the major H3K79 histone methyltransferase, is no longer recruited to the promoters that are demethylated after 5-aza-deoxycytidine treatment or genetic deletion of DNA methyltransferases. Knock-down and transfection experiments for DOT1L show that this enzyme has a direct role in maintaining the euchromatic and active status of these genes when unmethylated. These findings suggest that DNA demethylating interventions alone are not able to restore a complete euchromatic status and a full transcriptional reactivation of the epigenetically silenced tumor suppressor genes, and reinforce the necessity of targeting multiple elements of the epigenetics machinery for a successful treatment of malignancies.
