Key Points• RUNX1 inhibits erythroid differentiation by downregulation of the erythroid gene expression program.• RUNX1 can act as an activator and repressor during megakaryocytic differentiation and counteracts the activity of TAL1.The activity of antagonizing transcription factors represents a mechanistic paradigm of bidirectional lineage-fate control during hematopoiesis. At the megakaryocytic/erythroid bifurcation, the crossantagonism of krueppel-like factor 1 (KLF1) and friend leukemia integration 1 (FLI1) has such a decisive role. However, how this antagonism is resolved during lineage specification is poorly understood. We found that runt-related transcription factor 1 (RUNX1) inhibits erythroid differentiation of murine megakaryocytic/erythroid progenitors and primary human CD34 1 progenitor cells. We show that RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation by epigenetic repression of the erythroid master regulator KLF1. RUNX1 binding to the KLF1 locus is increased during megakaryocytic differentiation and counterbalances the activating role of T-cell acute lymphocytic leukemia 1 (TAL1). We found that corepressor recruitment by RUNX1 contributes to a block of the KLF1-dependent erythroid gene expression program.Our data indicate that the repressive function of RUNX1 influences the balance between erythroid and megakaryocytic differentiation by shifting the balance between KLF1 and FLI1 in the direction of FLI1. Taken together, we show that RUNX1 is a key player within a network of transcription factors that represses the erythroid gene expression program. (Blood. 2015;125(23):3570-3579) IntroductionThe hematopoietic system is in a constant process of cell proliferation, differentiation, and cell death. Progenitor cells produced by hematopoietic stem cells undergo a hierarchical progression in which the selfrenewal capability is lost and a specific lineage determination is adopted. [1][2][3] In this process, genes important for stem cell functions are downregulated and the expression of genes important for differentiation and cell type-specific functions is upregulated. Transcription factors initiate and maintain cell-specific expression by binding to regulatory sequences of target genes and by recruitment of generegulative complexes with DNA-and histone-modifying activity. These epigenetic modifications reorganize the chromatin locally and genome-wide to sustain a cell type-specific gene expression pattern. [4][5][6] Antagonizing transcription factors play an important role in the establishment of cell type-specific gene expression programs during hematopoietic differentiation. 7 At the megakaryocytic/erythroid bifurcation, the crossantagonism of the transcription factors krueppel-like factor 1 (KLF1) and friend leukemia integration 1 (FLI1) plays such a decisive role. 8,9 However, the mechanism of how this antagonism is resolved is poorly understood. During differentiation of common megakaryocyte/erythroid progenitor cells (MEPs) 10 toward the megakaryoc...

RUNX1/ETO, the fusion protein resulting from the chromosomal translocation t(8;21), is one of the most frequent translocation products in acute myeloid leukemia. Several in vitro and in vivo studies have shown that the homo-tetramerization domain of ETO, the nervy homology region 2 (NHR2), is essential for RUNX1/ETO oncogenic activity. We analyzed the energetic contribution of individual amino acids within the NHR2 to RUNX1/ETO dimertetramer transition and found a clustered area of 5 distinct amino acids with strong contribution to the stability of tetramers. Substitution of these amino acids abolishes tetramer formation without affecting dimer formation. Similar to RUNX1/ ETO monomers, dimers failed to bind efficiently to DNA and to alter expression of RUNX1-dependent genes. RUNX1/ETO dimers do not block myeloid differentiation, are unable to enhance the selfrenewal capacity of hematopoietic progenitors, and fail to induce leukemia in a murine transplantation model. Our data reveal the existence of an essential structural motif (hot spot) at the NHR2 dimertetramer interface, suitable for a molecular intervention in t(8;21) leukemias. IntroductionChromosomal translocations are frequent events during malignant cell transformation, particularly during leukemogenesis. 1 The translocation t(8;21), one of the most frequent chromosomal anomalies in acute myeloid leukemia (AML), involves the RUNX1 gene (also known as AML1, CBF␣2, or PEBP2␣B) on chromosome 21 and the ETO gene (also known as MTG8 or RUNX1T1) on chromosome 8. The ubiquitously expressed RUNX1 is a transcription factor and belongs to the key regulators of hematopoietic cell differentiation. 2 The fusion protein RUNX1/ETO contains the DNA-binding domain (Runt, RHD) of the RUNX1 transcription factor but lacks the C-terminal transactivation sequence that is replaced by almost the entire ETO protein. 2 forms of RUNX1/ETO coexist in AML-leukemia samples: the originally discovered full-length RUNX1/ETO and a splice variant called RUNX1/ETO9a, which lacks 178 amino acids at the C-terminus. Only RUNX1/ETO9a does not require cooperative events for inducing leukemia development in mice. 3,4 We and others have shown that RUNX1/ETO has a modular structure. Besides the Runt domain, RUNX1/ETO contains 4 functional domains, which are generally referred to as nervy homology region (NHR1 to NHR4). The NHR domains serve as docking interface for a variety of different proteins, including the E-protein HEB, 5,6 the apoptosis-related protein SON, 7 and nuclear corepressor proteins, such as N-CoR, SMRT, mSIN3A, and MTGR1, as well as histone deacetylases (HDACs). [8][9][10][11] In addition, the NHR2 domain mediates tetramer formation through hydrophobic and ionic/polar interactions. Two ␣-helices align in a head-to-tail fashion to form an antiparallel dimer. Two dimers subsequently are positioned on top of each other in a sandwich-like fashion. The total interaction area composing all contact points of the 4 ␣-helices is approximately 10 000 Å. 2 Substitution of 7 leucines withi...

The coordinated recruitment of epigenetic regulators of gene expression by transcription factors such as RUNX1 (AML1, acute myeloid leukemia 1) is crucial for hematopoietic differentiation. Here, we identify protein arginine methyltransferase 6 (PRMT6) as a central functional component of a RUNX1 corepressor complex containing Sin3a and HDAC1 in human hematopoietic progenitor cells. PRMT6 is recruited by RUNX1 and mediates asymmetric histone H3 arginine-2 dimethylation (H3R2me2a) at megakaryocytic genes in progenitor cells. H3R2me2a keeps RUNX1 target genes in an intermediate state with concomitant H3K27me3 and H3K4me2 but not H3K4me3. Upon megakaryocytic differentiation PRMT6 binding is lost, the H3R2me2a mark decreases and a coactivator complex containing WDR5/MLL and p300/pCAF is recruited. This leads to an increase of H3K4me3 and H3K9ac, which result in augmented gene expression. Our results provide novel mechanistic insight into how RUNX1 activity in hematopoietic progenitor cells maintains differentiation genes in a suppressed state but poised for rapid transcriptional activation.

The transcription factor Tal1 is a critical activator or repressor of gene expression in hematopoiesis and leukaemia. The mechanism by which Tal1 differentially influences transcription of distinct genes is not fully understood. Here we show that Tal1 interacts with the peptidylarginine deiminase IV (PADI4). We demonstrate that PADI4 can act as an epigenetic coactivator through influencing H3R2me2a. At the Tal1/PADI4 target gene IL6ST the repressive H3R2me2a mark triggered by PRMT6 is counteracted by PADI4, which augments the active H3K4me3 mark and thus increases IL6ST expression. In contrast, at the CTCF promoter PADI4 acts as a repressor. We propose that the influence of PADI4 on IL6ST transcription plays a role in the control of IL6ST expression during lineage differentiation of hematopoietic stem/progenitor cells. These results open the possibility to pharmacologically influence Tal1 in leukaemia.