The RUNX1/AML1 gene is the most frequent target for chromosomal translocation, and often identified as a site for reciprocal rearrangement of chromosomes 8 and 21 in patients with acute myelogenous leukemia. Virtually all chromosome translocations in leukemia show no consistent homologous sequences at the breakpoint regions. However, specific chromatin elements (DNase I and topoisomerase II cleavage) have been found at the breakpoints of some genes suggesting that structural motifs are determinant for the double strand DNA-breaks. We analyzed the chromatin organization at intron 5 of the RUNX1 gene where all the sequenced breakpoints involved in t(8;21) have been mapped. Using chromatin immunoprecipitation assays we show that chromatin organization at intron 5 of the RUNX1 gene is different in HL-60 and HeLa cells. Two distinct features mark the intron 5 in cells expressing RUNX1: a complete lack or significantly reduced levels of Histone H1 and enrichment of hyperacetylated histone H3. Strikingly, induction of DNA damage resulted in formation of t(8;21) in HL-60 but not in HeLa cells. Taken together, our results suggest that H1 depletion and/or histone H3 hyperacetylation may have a linkage with an increase susceptibility of specific chromosomal regions to undergo translocations.

One of the most frequent chromosomal translocation found in patients with acute myeloid leukemia (AML) is the t(8;21). This translocation involves the RUNX1 and ETO genes. The breakpoints regions for t(8;21) are located at intron 5 and intron 1 of the RUNX1 and ETO gene respectively. To date, no homologous sequences have been found in these regions to explain their recombination. The breakpoint regions of RUNX1 gene are characterized by the presence of DNasaI hypersensitive sites and topoisomerase II cleavage sites, but no information exists about complementary regions of ETO gene. Here, we report analysis of chromatin structure of ETO breakpoint regions. Chromatin immunoprecipitation (ChIP) were performed with antibodies specific to acetylated histone H3, H4, and total histone H1. Nucleosomal distribution at the ETO locus was evaluated by determining total levels of histone H3. Our data show that in myeloid cells, the breakpoint regions at the ETO gene are enriched in hyperacetylated histone H3 compared to a control region of similar size where no translocations have been described. Moreover, acetylated H4 associates with both the whole ETO breakpoint regions as well as the control intron. Interestingly, we observed no H1 association either at the breakpoint regions or the control region of the ETO gene. Our data indicate that a common chromatin structure enriched in acetylated histones is present in breakpoint regions involved in formation of (8;21) leukemic translocation.

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Human RUNX1 gene is the most frequent target for chromosomal translocations associated with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). The highest prevalence in acute myeloid leukemia (12‐15% of all cases) is noted with (8;21) translocation. Interestingly, all the breakpoints mapped to date in t(8;21) are clustered in intron 5 of the RUNX1 gene and intron 1 of the partner ETO gene. Even though, no homologous sequences have been found at the t(8:21) recombination regions; DNaseI hypersensitive sites (DHS) and topoisomerase II cleavage sites have been mapped to these areas of the genes. Presence of DHS sites is commonly associated with regulatory elements such as promoters, enhancers and silencers. In this study we used a combination of comparative genomics and chromatin analysis to identify transcriptionally active regions within intron 5 of the RUNX1 gene. Our genomic analysis identified nine potential enhancer regions that are evolutionarily conserved. Analysis of histone acetylation status of these regions in myeloid cells demonstrate a high association of 9,14 diacetylated histone H3; a hallmark of enhancer modules . Our results suggest that some of the identified conserved non coding sequences in the RUNX1 gene locus may act as regulatory sequences in hematopoietic cells.Grants: ACT 44; NIH R03 TW007170