Chromatin modifications induced by the AML1-ETO fusion protein reversibly silence its genomic targets through AML1 and Sp1 binding motifs

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.Leukemia ( Keywords: AML1-ETO; myeloid leukemia ; histone deacetylation; H3K9me3; Sp1 INTRODUCTIONThe presence of the AML1-ETO fusion protein indicates acute myeloid leukemia (AML) with t(8;21)(q22;q22), which accounts for B10 --15% of all AML cases.1,2 This product is the consequence of a reciprocal translocation that fuses the DNA binding domain of the AML1 transcription factor essential for transcriptional activation of genes involved in normal hematopoiesis in frame with nearly the entire ETO protein, a transcriptional repressor molecule expressed in a variety of tissues.

Epigenetic alterations in acute myeloid leukemias

“…In a recent study, chromatin immunoprecipitation chip analysis of human hematopoietic stem/progenitor cells transduced with the AML1-ETO fusion gene resulted in the identification of 1168 AML1-ETO target genes, 103 of which were co-occupied by HDAC1 and had lost the hyperacetylation marks on histone H4 at Lys5, Lys8, Lys12, Lys16 and 264 of which showed induction of trimethylation at Lys9 of histone H3 (H3K9me3). In the presence of these epigenetic modifications, enrichment of genes involved in hematopoietic differentiation and in specific signaling pathways was observed; this was associated with an 'inactive' chromatin status, so AML1-ETO binding correlates with changes in the histone modification pattern and increased association of HDACs [47]. On the other hand, AML1-ETO can interact, via its Nervy homology region (which is involved in oligomerization of ETO), with the histone acetyltransferase p300, which acetylates lysines of histones in its target genes, thus activating gene transcription [48].…”

Abrogation of RUNX1 gene expression in de novo myelodysplastic syndrome with t(4;21)(q21;q22)

“…To evaluate this hypothesis and the putative leukemic role of the RUNX1 abrogation in the context of our patient, we decided to explore the level of expression of some other genes that had previously been found to be down-regulated by the presence of the RUNX1/ETO fusion. 6 In detail, we used quantitative real-time RT-PCR to study the expression of 4 genes (YES1, MLLT3, CTCF and MAPK1) and we observed a significant downregulation in all of them, both in our patient and in Kasumi-1 ( Figure 1D). Taken together, we observed that these 5 genes, which are undoubtedly involved in cell differentiation, are significantly down-regulated, indicating that the abrogation of RUNX1 observed in our patient results in the loss of its capacity to transactivate RUNX1 target genes, mimicking what has been observed in the presence of the RUNX1/ETO fusion protein.…”

“…The calibrated normalized relative quantity, taking into account targetand run-specific amplification efficiencies, was calculated using endogenous GAPDH expression with the Qbase software application. The same approach was used to explore the level of expression of some other genes that were previously found down-regulated by the presence of the RUNX1/ETO fusion (YES1, MLLT3, CTCF and MAPK1), 6 both in our patient and in Kasumi-1 (a cell line carrying the RUNX1/ETO fusion gene).…”

MAPK8‐mediated stabilization of SP1 is essential for RUNX1‐RUNX1T1 – driven leukaemia

“…We previously reported relatively low SP1 mRNA levels in human haematopoetic stem progenitor cells expressing RUNX1-RUNX1T1 (referred to hereafter as HSPC-RR) and the SKNO1 cell line compared with HSPC (selected CD34 + cells with an empty vector transduction) (Maiques-Diaz et al, 2012). However, abundant SP1 protein levels were observed in RUNX1-RUNX1T1 -expressing cells compared with normal HSPC (Fig 1A).…”

“…Several studies have reinforced the importance of the interaction between RUNX1-RUNX1T1 and other transcription factors on its DNA-binding profile, and we previously identified the Sp1 transcription factor (SP1) binding site in more than 50% of DNA-bound RUNX1-RUNX1T1 protein (Maiques-Diaz et al, 2012). SP1 induces the expression of essential haematopoietic genes (i.e.…”