Chromatin modifications induced by PML-RARα repress critical targets in leukemogenesis as analyzed by ChIP-Chip

Abstract

IntroductionBalanced translocations that lead to expression of aberrant fusion proteins are a hallmark of acute leukemias. 1 Many of these fusion proteins function as aberrant transcription factors that are an initiating event in leukemogenesis. 2 Some translocations, eg those involving the MLL gene can lead to phenotypically diverse forms of leukemia, whereas the t(15;17) uniformly leads to acute promyelocytic leukemia. The reasons why some translocations are leukemia-type specific, whereas others are not, is unknown.Histone modifications such as methylation of histone H3 at lysine 4 and 9 and acetylation of histone H3 are closely linked to the transcriptional activation status. In t(15;17), the chimeric PML-RAR␣ fusion protein has been shown to recruit corepressors such as DAXX, histone deacetylase (HDAC) activity, DNA methyltransferase activity, and the SUV39H1 histone methyltransferase to RAR␤2, the most extensively studied target gene. [3][4][5][6][7] PML-RAR␣ also interacts with histone deacetylase 1. 8 PML-RAR␣ homodimerization has been shown to relax the relatively stringent RAR␣ DNA binding specificity. 9,10 This gain of function is supposed to lead to many additional genomic binding sites that are not well defined. As a consequence, virtually all direct genomic targets of PML-RAR␣ are currently unknown. In addition, on a global level, the mechanistic alterations occurring at PML-RAR␣ target genes remain to be clarified.On a phenotypic level, the PML-RAR␣ fusion protein blocks differentiation and apoptosis and enhances self-renewal. 11,12 In mouse models, PML-RAR␣ induces a disease similar to acute promyelocytic leukemia (APL). 13,14 Microarray analyses elucidated several leukemogenic mechanisms and pathways. [15][16][17][18][19] For example, PML-RAR␣ induces activation of the Wnt signaling pathway. 17 Also, PML-RAR␣ alters the apoptotic response and expression of differentiation genes. 15 However, these studies do not distinguish between direct and indirect effects on gene expression. This knowledge is crucial to understand the mechanistic implications of PML-RAR␣ and to elucidate the reasons for the unique phenotype that is associated with its activities.The possibilities to understand transcription factor functions has recently been significantly improved by genome-wide approaches that identify target genes in vivo using Chromatin immunoprecipitation (ChIP)-Chip approaches. 20,21 In addition, the ability to use this method to map epigenetic modifications such as histone acetylation at promoters known to bind the transcription factor allows for the identification of the functional consequences of transcription factor binding to its genomic targets. 22,23 Using ChIP-chip analyses, we identified 372 direct PML-RAR␣ genomic targets and show that PML-RAR␣ induces heterochromatin formation on virtually all of its identified target genes. Several of the identified genes are known tumor suppressors and for one of the novel genes (S100P), we show a potential role in the PML-RAR␣-associated block in differenti...

EZH2 is a mediator of EWS/FLI1 driven tumor growth and metastasis blocking endothelial and neuro-ectodermal differentiation

“…ChIP was carried out as described previously with slight modifications (38). In brief, 3 ϫ 10 7 A673 cells were formaldehyde (1% final) fixed for 10 min.…”

“…ChIP was carried out with 3 g of antibody either anti-FLI1 (C-19), or anti-IgG (Santa Cruz) added to 0.5 mg of precleared chromatin. Quantitative real-time PCR by using Sybr Green (Applied Biosystems) with quantitation performed by the ⌬⌬Ct-method (38). FLI1 data at individual genomic loci were normalized to the IgG control to compensate for differences in PCR efficiency, and standardized to 2 nonregulated genomic loci outside of the EZH2 locus.…”

Loss of imprinting at the 14q32 domain is associated with microRNA overexpression in acute promyelocytic leukemia

“…Accordingly, PML-RARa has been shown to induce APL in transgenic mice. 3 However, deregulation of the retinoic acid pathway is insufficient to initiate APL, 4 and several studies have shown additional genetic and epigenetic processes that accompany the expression of the PML-RARa protein, 5,6 also involving master transcription regulators 7 and modulators of chromatin structure. 8,9 MicroRNAs (miRNAs) are single-stranded small noncoding RNAs (sncRNAs) that negatively regulate the expression of target genes.…”

Acute Promyelocytic Leukemia: A Model Disease for Targeted Cancer Therapy

“…In addition to increased repressor activity, the PML/RARA fusion also appears to possess a considerable expanded repertoire of target genes compared to the n o r m a l R A R A p r o t e i n . T h i s n o t i o n i s supported by in vitro binding studies showing that PML/RARA has a broader and more relaxed DNA binding specificity compared to RARA (Hauksdottir & Privalsky, 2001;Kamashev et al, 2004), and by a genome wide screen revealing a wide range of PML/RARA target genes (Hoemme et al, 2008). The altered DNA binding and transcription repression properties of PML/RARA are partially due to the ability of this chimeric protein to form homodimeres through protein-protein interactions mediated by the TRIM motif of PML (Jansen et al, 1995;Perez et al, 1993).…”