A distinct epigenetic signature at targets of a leukemia protein

BMC Genomics A distinct epigenetic signature at targets of a leukemia protein Stefano Rossetti 2 Andr T Hoogeveen 1 Ping Liang 2 Cornel Stanciu 2 Peter van der Spek 0 Nicoletta Sacchi 2 0 Department of Bioinformatics, Erasmus MC , Dr Molewaterplein 50, 3015GE Rotterdam , The Netherlands 1 Department of Clinical Genetics, Erasmus MC , Dr Molewaterplein 50, 3015GE Rotterdam , The Netherlands 2 Department of Cancer Genetics, Roswell Park Cancer Institute , Elm and Carlton Streets, Buffalo, NY 14263 , USA Background: Human myelogenous leukemia characterized by either the non random t(8; 21)(q22; q22) or t(16; 21)(q24; q22) chromosome translocations differ for both their biological and clinical features. Some of these features could be consequent to differential epigenetic transcriptional deregulation at AML1 targets imposed by AML1-MTG8 and AML1-MTG16, the fusion proteins deriving from the two translocations. Preliminary findings showing that these fusion proteins lead to transcriptional downregulation of AML1 targets, marked by repressive chromatin changes, would support this hypothesis. Here we show that combining conventional global gene expression arrays with the power of bioinformatic genomic survey of AML1-consensus sequences is an effective strategy to identify AML1 targets whose transcription is epigenetically downregulated by the leukemia-associated AML1-MTG16 protein. Results: We interrogated mouse gene expression microarrays with probes generated either from 32D cells infected with a retroviral vector carrying AML1-MTG16 and unable of granulocyte differentiation and proliferation in response to the granulocyte colony stimulating factor (G-CSF), or from 32D cells infected with the cognate empty vector. From the analysis of differential gene expression alone (using as criteria a p value < 0.01 and an absolute fold change > 3), we were unable to conclude which of the 37 genes downregulated by AML1-MTG16 were, or not, direct AML1 targets. However, when we applied a bioinformatic approach to search for AML1-consensus sequences in the 10 Kb around the gene transcription start sites, we closed on 17 potential direct AML1 targets. By focusing on the most significantly downregulated genes, we found that both the AML1-consensus and the transcription start site chromatin regions were significantly marked by aberrant repressive histone tail changes. Further, the promoter of one of these genes, containing a CpG island, was aberrantly methylated. Conclusion: This study shows that a leukemia-associated fusion protein can impose a distinct epigenetic repressive signature at specific sites in the genome. These findings strengthen the conclusion that leukemia-specific oncoproteins can induce non-random epigenetic changes. - Background Nuclear hormone receptors and transcription factors can regulate the transcription of their target genes by inducing chromatin changes. Paradigmatic are the retinoic acid receptor alpha (RAR) and the transcription factor core binding factor (CBF), which regulate in this way the transcription of target genes involved in hematopoietic processes [1,2]. Differently from RAR, which epigenetically activates its targets by recruiting coactivator protein complexes with histone acetyl transferase (HAT) activity only when bound to retinoic acid, CBF can directly recruit HAT-containing complexes to activate its targets [3-6]. One of the two CBF subunits, CBF or AML1, can bind target genes endowed with the AML1-consensus sequence TG(T/C)GGT via its N-terminal DNA-binding domain [7]. AML1, encoding a master hematopoietic transcription factor, is frequently affected by different chromosome translocations in leukemic cells [8]. Moreover, AML1 haploinsufficiency was found to be associated with familial platelet disorder, a condition predisposing to acute myeloid leukemia [9]. Two leukemia-associated chromosome translocations, the t(8;21)(q22;q22) and the t(16;21)(q24;q22), result in the fusion between the N-terminal region of AML1 and the Cterminal regions of two almost identical chromatin corepressors, MTG8 and MTG16, leading to the formation of AML1-MTG8 and AML1-MTG16, respectively [10-13]. Upon fusion with either MTG8 or MTG16, AML1 is converted from a transcriptional activator into a transcriptional repressor of AML1-targets. Specific MTG domains in the wild type, as well as in the MTG fusion proteins, can interact, directly or via other corepressors such as NCoR and Sin3A, with histone deacetylases (HDACs), thus creating a repressive chromatin state at AML1 target sites (reviewed in [14,15]). Repression at these sites is further enhanced by the formation of oligomers between the fusion proteins and wild-type MTG proteins [16-18]. Myeloid cell differentiation systems, such as the 32D mouse myeloid cell line, ectopically expressing either AML1-MTG8 or AML1-MTG16, were used as models to simulate some of the effects of these fusion proteins in myelogenesis and leukemogenesis. Both fusion proteins, when exogenously expressed in the 32D background, were shown to affect granulocytic differentiation and produce distinct effects on cell proliferation [19-21]. In a preliminary study, we found that AML1-MTG16, when exogenously expressed in 32D cells, can induce aberrant myeloid phenotypes in association with repressive modifications at the chromatin of the Colony stimulating factor 1 receptor (Csf1r), an AML1-target gene encoding the macrophage colony stimulating factor receptor [19]. Based on this finding, we hypothesize that the comparative epigenetic analysis of the changes induced by different AML1-MTG fusion proteins in an identical cell context (e.g. the 32D context) might provide a lead to elucidating the differences observed in leukemic cells carrying either one of the two proteins [8]. The objective of this study was to demonstrate whether AML1-MTG16 induces epigenetic changes at AML1-target genes in the 32D myeloid cell genome. Only by coupling global gene expression array analysis with a bioinformatic genomic survey for the AML1-consensus sequence, we were able to close onto AML1-targets downregulated by AML1-MTG16. AML1MTG16-induced transcriptional downregulation was marked by the acquisition of a distinct repressive chromatin signature. Results Global gene expression array analysis of AML1-MTG16expressing cells To study the molecular and biological consequences of AML1-MTG16 expression in a myeloid differentiation cell model, we previously developed, by infecting 32D mouse myeloblasts with retroviral particles carrying either the pLNCX2 vector containing the AML1-MTG16 cDNA or the cognate empty vector, stable independent clones expressing AML1-MTG16 (hereafter called A16 clones) and stable independent control clones (hereafter called "mock" clones), respectively (Figure 1A). Upon treatment with granulocyte colony stimulating factor (G-CSF), A16 clones do not undergo granulocytic differentiation and proliferate significantly less than mock (...truncated)