MiR144/451 Expression Is Repressed by RUNX1 During Megakaryopoiesis and Disturbed by RUNX1/ETO.

A network of lineage-specific transcription factors and microRNAs tightly regulates differentiation of hematopoietic stem cells along the distinct lineages. Deregulation of this regulatory network contributes to impaired lineage fidelity and leukemogenesis. We found that the hematopoietic master regulator RUNX1 controls the expression of certain microRNAs, of importance during erythroid/megakaryocytic differentiation. In particular, we show that the erythorid miR144/451 cluster is epigenetically repressed by RUNX1 during megakaryopoiesis. Furthermore, the leukemogenic RUNX1/ETO fusion protein transcriptionally represses the miR144/451 pre-microRNA. Thus RUNX1/ETO contributes to increased expression of miR451 target genes and interferes with normal gene expression during differentiation. Furthermore, we observed that inhibition of RUNX1/ETO in Kasumi1 cells and in RUNX1/ETO positive primary acute myeloid leukemia patient samples leads to up-regulation of miR144/451. RUNX1 thus emerges as a key regulator of a microRNA network, driving differentiation at the megakaryocytic/erythroid branching point. The network is disturbed by the leukemogenic RUNX1/ETO fusion product.

Essential control of early B-cell development by Mef2 transcription factors.

The sequential activation of distinct developmental gene networks governs the ultimate identity of a cell, but the mechanisms involved in initiating downstream programs are incompletely understood. The pre-B-cell receptor (pre-BCR) is an important checkpoint of B-cell development and is essential for a pre-B cell to traverse into an immature B cell. Here, we show that activation of myocyte enhancer factor 2 (Mef2) transcription factors (TFs) by the pre-BCR is necessary for initiating the subsequent genetic network. We demonstrate that B-cell development is blocked at the pre-B-cell stage in mice deficient for Mef2c and Mef2d TFs and that pre-BCR signaling enhances the transcriptional activity of Mef2c/d through phosphorylation by the Erk5 mitogen-activating kinase. This activation is instrumental in inducing Krüppel-like factor 2 and several immediate early genes of the AP1 and Egr family. Finally, we show that Mef2 proteins cooperate with the products of their target genes (Irf4 and Egr2) to induce secondary waves of transcriptional regulation. Our findings uncover a novel role for Mef2c/d in coordinating the transcriptional network that promotes early B-cell development.

Protein arginine methyltransferase 6 controls erythroid gene expression and differentiation of human CD34+ progenitor cells.

Hematopoietic differentiation is driven by transcription factors, which orchestrate a finely tuned transcriptional network. At bipotential branching points lineage decisions are made, where key transcription factors initiate cell type-specific gene expression programs. These programs are stabilized by the epigenetic activity of recruited chromatin-modifying cofactors. An example is the association of the transcription factor RUNX1 with protein arginine methyltransferase 6 (PRMT6) at the megakaryocytic/erythroid bifurcation. However, little is known about the specific influence of PRMT6 on this important branching point. Here, we show that PRMT6 inhibits erythroid gene expression during megakaryopoiesis of primary human CD34+ progenitor cells. PRMT6 is recruited to erythroid genes, such as glycophorin A Consequently, a repressive histone modification pattern with high H3R2me2a and low H3K4me3 is established. Importantly, inhibition of PRMT6 by shRNA or small molecule inhibitors leads to upregulation of erythroid genes and promotes erythropoiesis. Our data reveal that PRMT6 plays a role in the control of erythroid/megakaryocytic differentiation and open up the possibility that manipulation of PRMT6 activity could facilitate enhanced erythropoiesis for therapeutic use.

RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation.

The activity of antagonizing transcription factors represents a mechanistic paradigm of bidirectional lineage-fate control during hematopoiesis. At the megakaryocytic/erythroid bifurcation, the cross-antagonism 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(+) 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.

Flagella mediate endophytic competence rather than act as MAMPS in rice-Azoarcus sp. strain BH72 interactions.

Azoarcus sp. strain BH72 is an endophytic betaproteobacterium able to colonize rice roots without induction of visible disease symptoms. BH72 possesses one polar flagellum. The genome harbors three copies of putative fliC genes, generally encoding the major structural protein flagellin. It is not clear whether, in endophytic interactions, flagella mediate endophytic competence or act as MAMPs (microbe-associated molecular patterns) inducing plant defense responses. Therefore, possible functions of the three FliC proteins were investigated. Only fliC3 was found to be highly expressed in pure culture and in association with rice roots and to be required for bacterial motility, suggesting that it encodes the major flagellin. Endophytic colonization of rice roots was significantly reduced in the in-frame deletion mutant, while the establishment of microcolonies on the root surface was not affected. Moreover, an elicitation of defense responses related to FliC3 was not observed. In conclusion, our data support the hypothesis that FliC3 does not play a major role as a MAMP but is required for endophytic colonization in the Azoarcus-rice interaction, most likely for spreading inside the plant.

Dimer-tetramer transition controls RUNX1/ETO leukemogenic activity.

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 dimer-tetramer 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 self-renewal 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 dimer-tetramer interface, suitable for a molecular intervention in t(8;21) leukemias.

Two Functionally Deviating Type 6 Secretion Systems Occur in the Nitrogen-Fixing Endophyte Azoarcus olearius BH72.

Type VI protein secretion systems (T6SSs) have been identified in many plant-associated bacteria. However, despite the fact that effector proteins may modulate host responses or interbacterial competition, only a few have been functionally dissected in detail. We dissected the T6SS in Azoarcus olearius strain BH72, a nitrogen-fixing model endophyte of grasses. The genome harbors two gene clusters encoding putative T6SSs, tss-1 and tss-2, of which only T6SS-2 shared genetic organization and functional homology with the H1-T6SS of Pseudomonas aeruginosa. While tss-2 genes were constitutively expressed, tss-1 genes were strongly up-regulated under conditions of nitrogen fixation. A comparative analysis of the wild type and mutants lacking either functional tss-1 or tss-2 allowed to differentiate the functions of both secretion systems. Abundance of Hcp in the culture supernatant as an indication for T6SS activity revealed that only T6SS-2 was active, either under aerobic or nitrogen-fixing conditions. Our data show that T6SS-2 but not T6SS-1 is post-translationally regulated by phosphorylation mediated by TagE/TagG (PpkA/PppA), and by the phosphorylation-independent inhibitory protein TagF, similar to published work in Pseudomonas. Therefore, T6SS-1 appears to be post-translationally regulated by yet unknown mechanisms. Thus, both T6SS systems appear to perform different functions in Azoarcus, one of them specifically adapted to the nitrogen-fixing lifestyle.

PADI4 acts as a coactivator of Tal1 by counteracting repressive histone arginine methylation.

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.