ERG and FLI1 binding sites demarcate targets for aberrant epigenetic regulation by AML1-ETO in acute myeloid leukemia

Abstract

Introduction E-twenty-six (ETS) specific transcription factors are a family of Ͼ 20 helix-loop-helix domain transcription factors that have been implicated in a myriad of cellular processes, including hematopoiesis. 1 The hallmark ETS factor protein involved in hematopoietic development is SPI1 (Spleen focus forming virus Proviral Integration site 1; PU.1), which activates gene expression during myeloid and B-lymphoid cell development. Other ETS factors include the 2 closely related transcriptional activator proteins ERG (Ets Related Gene) and FLI1 (Friend Leukemia virus Integration site 1), which both play crucial roles in hematopoietic development 2,3 and multiple forms of cancer. 4,5 Recently, SPI1 was identified as a binding partner of the PML-RAR-␣ oncofusion protein complex in an inducible overexpression model. 6 The PML-RAR-␣ oncofusion protein is the result of a translocation t(15;17)(q22;q21) involving the promyelocytic leukemia (PML) gene on chromosome 15 and the retinoic acid receptor-␣ (RAR-␣) on chromosome 17. 7,8 Another translocation, t(8;21)(q22;q22), is present in ϳ 10% of all de novo acute myeloid leukemia (AML) cases, and results in the expression of the AML1-ETO (RUNX1-RUNX1T1) oncofusion protein. Expression of the AML1-ETO oncofusion protein in hematopoietic cells results in a stage-specific arrest of maturation and increased cell survival, predisposing cells to develop leukemia. 9 At the molecular level RUNX1 (Runt-related transcription factor 1; AML1, CBFA2) represents a DNA-binding transcriptional activator factor required for hematopoiesis, 10,11 while ETO (eight-twenty-one; MTG8, RUNX1T1) acts as a corepressor molecule. 12 The t(8;21) translocation replaces the transactivation domain of RUNX1/AML1 with the almost complete ETO protein, thereby converting an essential transcriptional activator into a strong repressor. 13,14 Here we extend genome-wide AML1-ETO studies 15,16 and reveal that a subset of AML1-ETO binding sites are bound by CBF-␤ (core binding factor-␤), whereas nearly all are bound by HEB (HeLa E-box-binding factor), RUNX1/AML1 as well as by the ETS factors ERG and FLI1. Subsequent analysis in t(8;21) cells revealed cell type specific ETS factor binding and preferential AML1-ETO binding to the cell type specific ETS factor binding sites, suggesting that these proteins facilitate oncofusion protein binding. In addition, we uncovered that binding of the ETS factors correlates with the "active" histone acetylation mark. Together, our results suggest that ETS factors demarcate hematopoietic regulatory sites that provide a target for (aberrant) epigenetic regulation by oncofusion proteins. Methods ChIPChromatin was harvested as described. 17 ChIPs were performed using specific antibodies to ETO, HEB, ERG, FLI1 (Santa Cruz Biotechnology), H3K9K14ac, AML1-ETO, ETO, CBF-␤, RNAPII (Diagenode), RUNX1, and FLI1 (Abcam), and H4panAc (Millipore) and analyzed by quantitative PCR or ChIP-seq. Primers for quantitative PCR are described in supplemental Methods (available on the Blood Web site...

CBFB–MYH11/RUNX1 together with a compendium of hematopoietic regulators, chromatin modifiers and basal transcription factors occupies self-renewal genes in inv(16) acute myeloid leukemia

“…Nevertheless, this analysis showed increased tag density for both CBFb as well as MYH11 at all of the high-confidence CBFb-MYH11-binding sites (Figure 1f), thereby confirming the binding results obtained in the cell line. Comparing the genomic location of CBFb-MYH11 with PML-RARa and AML1-ETO, two other AML-associated oncofusion proteins, 25,26 revealed a preferential localization of CBFb-MYH11 to promoter regions as compared with AML1-ETO and PML-RARa, which both target mostly non-promoter regions (Figure 1g). To further evaluate this finding, we partitioned our newly discovered binding sites in three categories.…”

“…Our motif analysis further revealed the presence of the E-box and GATA motif at CBFb-MYH11-binding sites. To investigate whether TAL1, E-box and GATA factors are present at CBFb-MYH11 bound regions, we extended our ChIP-seq analysis and included specific antibodies to TAL1, GATA2, the GATA factor highest expressed in ME-1 cells, HEB, a protein previously implicated in CBF leukemogenesis, 26,28,29 and also included several ETS factors, such as ELF1, FLI1, ERG and PU.1/SPI1 in our analysis. This revealed the increased occupancy of all factors at the CBFb-MYH11/RUNX1 occupied promoters of the C8ORF55 and SOCS1 genes (Figure 3b) whereas, in contrast, not all factors were enriched at the CBFb/RUNX1 occupied TAL1 or RUNX1 occupied PSMC3 gene.…”

“…In the case of AML, our analysis of PML-RARa and AML1-ETO were among the first to report on the genome-wide actions of oncofusion proteins. 25,26,44 Here, we analyzed the genome-widebinding pattern of CBFb-MYH11 and its interplay with other regulators of hematopoiesis in cell lines and patient primary blasts.…”

Structural and dynamic studies of the transcription factor ERG reveal DNA binding is allosterically autoinhibited

“…Several DNA-binding proteins, including the androgen receptor and the transcription factor RUNX1 (also known as AML1), have recently been shown to interact with ERG (6,30). Even PU.1, which is one of only three Ets family proteins not having this conserved tyrosine (substituted with an asparagine that could also form hydrogen bonds), has been shown to interact with Bcl6 (31).…”

Flow-induced protein kinase A–CREB pathway acts via BMP signaling to promote HSC emergence

“…We then conducted a bioinformatics-based screen using microarray data on CREB overexpression and CREB chromatin immunoprecipitation-sequencing (ChIP-Seq) data using data available at Encyclopedia of DNA Elements (ENCODE) and elsewhere to identify regulators of CREB function in hematopoietic cells (Esparza et al, 2008;Jolma et al, 2010;Pencovich et al, 2011;Raney et al, 2011;Trompouki et al, 2011;Martens et al, 2012). Using insight gained from bioinformatics, we discover that the bone morphogenetic protein (BMP) signaling pathway acts downstream of PKA-CREB signaling in regulating AGM hematopoiesis.…”