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...

Epigenetic silencing of transgene expression represents a major obstacle for the efficient genetic modification of multipotent and pluripotent stem cells. We and others have demonstrated that a 1.5 kb methylation-free CpG island from the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) effectively prevents transgene silencing and variegation in cell lines, multipotent and pluripotent stem cells, and their differentiated progeny. However, the bidirectional promoter activity of this element may disturb expression of neighboring genes. Furthermore, the epigenetic basis underlying the anti-silencing effect of the UCOE on juxtaposed promoters has been only partially explored. In this study we removed the HNRPA2B1 moiety from the A2UCOE and demonstrate efficient anti-silencing properties also for a minimal 0.7 kb element containing merely the CBX3 promoter. This DNA element largely prevents silencing of viral and tissue-specific promoters in multipotent and pluripotent stem cells. The protective activity of CBX3 was associated with reduced promoter CpG-methylation, decreased levels of repressive and increased levels of active histone marks. Moreover, the anti-silencing effect of CBX3 was locally restricted and when linked to tissue-specific promoters did not activate transcription in off target cells. Thus, CBX3 is a highly attractive element for sustained, tissue-specific and copy-number dependent transgene expression in vitro and in vivo.

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.

Glioblastoma is the most common primary brain tumor with a very poor prognosis, calling for novel treatment strategies. Here, we provide first evidence that histone deacetylase inhibitors (HDACI) prime glioblastoma cells for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -induced apoptosis at least in part by c-myc-mediated downregulation of cellular FLICE-inhibitory protein (cFLIP). Pretreatment with distinct HDACI (MS275, suberoylanilide hydroxamic acid, valproic acid) significantly enhances TRAIL-induced apoptosis in several glioblastoma cell lines. Monitoring a panel of apoptosisregulatory proteins revealed that MS275 reduces the expression of cFLIP L and cFLIP S . This leads to decreased recruitment of cFLIP L and cFLIP S and increased activation of caspase-8 to the TRAIL death-inducing signaling complex, resulting in enhanced cleavage of caspase-8, -9 and -3 and caspase-dependent apoptosis. Also, MS275 promotes TRAIL-triggered processing of Bid, activation of Bax, loss of mitochondrial membrane potential and release of cytochrome c. MS275-mediated downregulation of cFLIP occurs at the mRNA level independent of proteasome-or caspase-mediated degradation, and is preceded by upregulation of nuclear levels of c-myc, a transcriptional repressor of cFLIP. Notably, MS275 causes increased binding of c-myc to the cFLIP promoter and reduces cFLIP promoter activity. Indeed, knockdown of c-myc partially rescues cFLIP L from MS275-inferred downregulation and significantly decreases TRAIL-and MS275-induced apoptosis. Also, overexpression of cFLIP L or cFLIP S significantly reduces MS275-and TRAIL-induced apoptosis. Importantly, MS275 sensitizes primary cultured glioblastoma cells towards TRAIL and cooperates with TRAIL to reduce long-term clonogenic survival of glioblastoma cells and to suppress glioblastoma growth in vivo underscoring the clinical relevance of this approach. Thus, these findings demonstrate that HDACI represent a promising strategy to prime glioblastoma for TRAIL-induced apoptosis by targeting cFLIP.