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.

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.

Tal1 regulates osteoclast differentiation through suppression of the master regulator of cell fusion DC-STAMP.

The balance between bone-forming osteoblasts and bone-resorbing osteoclasts is crucial to bone homeostasis, an equilibrium that is disturbed in many bone diseases. The transcription factor Tal1 is involved in the establishment of hematopoietic stem cells in the embryo and is a master regulator of hematopoietic gene expression in the adult. Here, we show that Tal1 is expressed in osteoclasts and that loss of Tal1 in osteoclast progenitors leads to altered expression of >1200 genes. We found that DC-STAMP, a key regulator of osteoclast cell fusion, is a direct target gene of Tal1 and show that Tal1 represses DC-STAMP expression by counteracting the activating function of the transcription factors PU.1 and MITF. The identification of Tal1 as a factor involved in cell fusion contributes to the understanding of osteoclast-associated diseases, including osteoporosis.

Targets of the Tal1 transcription factor in erythrocytes: E2 ubiquitin conjugase regulation by Tal1.

The Tal1 transcription factor is essential for the development of the hematopoietic system and plays a role during definitive erythropoiesis in the adult. Despite the importance of Tal1 in erythropoiesis, only a small number of erythroid differentiation target genes are known. A chromatin precipitation and cloning approach was established to uncover novel Tal1 target genes in erythropoiesis. The BirA tag/BirA ligase biotinylation system in combination with streptavidin chromatin precipitation (Strep-CP) was used to co-precipitate genomic DNA bound to Tal1. Tal1 was found to bind in the vicinity of 31 genes including the E2-ubiquitin conjugase UBE2H gene. Binding of Tal1 to UBE2H was confirmed by chromatin immunoprecipitation. UBE2H expression is increased during erythroid differentiation of hCD34(+) cells. Tal1 expression activated UBE2H expression, whereas Tal1 knock-down reduced UBE2H expression and ubiquitin transfer activity. This study identifies parts of the ubiquitinylation machinery as a cellular target downstream of the transcription factor Tal1 and provides novel insights into Tal1-regulated erythropoiesis.

PRMT6 activates cyclin D1 expression in conjunction with the transcription factor LEF1.

The establishment of cell type specific gene expression by transcription factors and their epigenetic cofactors is central for cell fate decisions. Protein arginine methyltransferase 6 (PRMT6) is an epigenetic regulator of gene expression mainly through methylating arginines at histone H3. This way it influences cellular differentiation and proliferation. PRMT6 lacks DNA-binding capability but is recruited by transcription factors to regulate gene expression. However, currently only a limited number of transcription factors have been identified, which facilitate recruitment of PRMT6 to key cell cycle related target genes. Here, we show that LEF1 contributes to the recruitment of PRMT6 to the central cell cycle regulator CCND1 (Cyclin D1). We identified LEF1 as an interaction partner of PRMT6. Knockdown of LEF1 or PRMT6 reduces CCND1 expression. This is in line with our observation that knockdown of PRMT6 increases the number of cells in G1 phase of the cell cycle and decreases proliferation. These results improve the understanding of PRMT6 activity in cell cycle regulation. We expect that these insights will foster the rational development and usage of specific PRMT6 inhibitors for cancer therapy.

The transcription factor TAL1 and miR-17-92 create a regulatory loop in hematopoiesis.

A network of gene regulatory factors such as transcription factors and microRNAs establish and maintain gene expression patterns during hematopoiesis. In this network, transcription factors regulate each other and are involved in regulatory loops with microRNAs. The microRNA cluster miR-17-92 is located within the MIR17HG gene and encodes six mature microRNAs. It is important for hematopoietic differentiation and plays a central role in malignant disease. However, the transcription factors downstream of miR-17-92 are largely elusive and the transcriptional regulation of miR-17-92 is not fully understood. Here we show that miR-17-92 forms a regulatory loop with the transcription factor TAL1. The miR-17-92 cluster inhibits expression of TAL1 and indirectly leads to decreased stability of the TAL1 transcriptional complex. We found that TAL1 and its heterodimerization partner E47 regulate miR-17-92 transcriptionally. Furthermore, miR-17-92 negatively influences erythroid differentiation, a process that depends on gene activation by the TAL1 complex. Our data give example of how transcription factor activity is fine-tuned during normal hematopoiesis. We postulate that disturbance of the regulatory loop between TAL1 and the miR-17-92 cluster could be an important step in cancer development and progression.

FUSE binding protein 1 (FUBP1) expression is upregulated by T-cell acute lymphocytic leukemia protein 1 (TAL1) and required for efficient erythroid differentiation.

During erythropoiesis, haematopoietic stem cells (HSCs) differentiate in successive steps of commitment and specification to mature erythrocytes. This differentiation process is controlled by transcription factors that establish stage- and cell type-specific gene expression. In this study, we demonstrate that FUSE binding protein 1 (FUBP1), a transcriptional regulator important for HSC self-renewal and survival, is regulated by T-cell acute lymphocytic leukaemia 1 (TAL1) in erythroid progenitor cells. TAL1 directly activates the FUBP1 promoter, leading to increased FUBP1 expression during erythroid differentiation. The binding of TAL1 to the FUBP1 promoter is highly dependent on an intact GATA sequence in a combined E-box/GATA motif. We found that FUBP1 expression is required for efficient erythropoiesis, as FUBP1-deficient progenitor cells were limited in their potential of erythroid differentiation. Thus, the finding of an interconnection between GATA1/TAL1 and FUBP1 reveals a molecular mechanism that is part of the switch from progenitor- to erythrocyte-specific gene expression. In summary, we identified a TAL1/FUBP1 transcriptional relationship, whose physiological function in haematopoiesis is connected to proper erythropoiesis.

Fine structure and putative feeding mechanism of the archigregarine Selenidium orientale (Apicomplexa: Gregarinomorpha).

Archigregarines are considered one of the most plesiomorphic groups of Apicomplexa. Until recently, however, this viewpoint was based mainly on the results of the detailed investigation of a single species, Selenidium hollandei. The present study of the fine structure of trophozoites of another archigregarine species Selenidium orientale Bogolepova, 1953. (Apicomplexa, Archigregarinida, as proposed by Grassé and Schrével), with special reference to the forebody structure of the attached individuals, allows more confident discussion of the plesiomorphic status of the archigregarines. Specimens of S. orientale were collected from the midgut of the Pacific sipunculid Themiste pyroides Chamberlain, 1920. The ultrastructure of the trophozoites generally corresponds to that of other studied species of Selenidium. Differences in the forebody structure between S. orientale and S. hollandei do not conflict with Schrével's hypothesis on the feeding function of the apical complex in archigregarine trophozoites, although they suggest that, in S. orientale at least, the cytostome is not a persistent structure, but re-opens for each sucking event, and Selenidium trophozoites feed by intermittent sucking of host cytoplasm. Microtubules in the axial zone of the mucron neck may mediate the transport of food vacuoles.

Hematopoietic transcription factors and differential cofactor binding regulate PRKACB isoform expression.

Hematopoietic differentiation is controlled by key transcription factors, which regulate stem cell functions and differentiation. TAL1 is a central transcription factor for hematopoietic stem cell development in the embryo and for gene regulation during erythroid/megakaryocytic differentiation. Knowledge of the target genes controlled by a given transcription factor is important to understand its contribution to normal development and disease. To uncover direct target genes of TAL1 we used high affinity streptavidin/biotin-based chromatin precipitation (Strep-CP) followed by Strep-CP on ChIP analysis using ChIP promoter arrays. We identified 451 TAL1 target genes in K562 cells. Furthermore, we analysed the regulation of one of these genes, the catalytic subunit beta of protein kinase A (PRKACB), during megakaryopoiesis of K562 and primary human CD34+ stem cell/progenitor cells. We found that TAL1 together with hematopoietic transcription factors RUNX1 and GATA1 binds to the promoter of the isoform 3 of PRKACB (Cß3). During megakaryocytic differentiation a coactivator complex on the Cß3 promoter, which includes WDR5 and p300, is replaced with a corepressor complex. In this manner, activating chromatin modifications are removed and expression of the PRKACB-Cß3 isoform during megakaryocytic differentiation is reduced. Our data uncover a role of the TAL1 complex in controlling differential isoform expression of PRKACB. These results reveal a novel function of TAL1, RUNX1 and GATA1 in the transcriptional control of protein kinase A activity, with implications for cellular signalling control during differentiation and disease.

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.

Molecular phylogeny and surface morphology of Colpodella edax (Alveolata): insights into the phagotrophic ancestry of apicomplexans.

The molecular phylogeny of colpodellids provides a framework for inferences about the earliest stages in apicomplexan evolution and the characteristics of the last common ancestor of apicomplexans and dinoflagellates. We extended this research by presenting phylogenetic analyses of small subunit rRNA gene sequences from Colpodella edax and three unidentified eukaryotes published from molecular phylogenetic surveys of anoxic environments. Phylogenetic analyses consistently showed C. edax and the environmental sequences nested within a colpodellid clade, which formed the sister group to (eu)apicomplexans. We also presented surface details of C. edax using scanning electron microscopy in order to supplement previous ultrastructural investigations of this species using transmission electron microscopy and to provide morphological context for interpreting environmental sequences. The microscopical data confirmed a sparse distribution of micropores, an amphiesma consisting of small polygonal alveoli, flagellar hairs on the anterior flagellum, and a rostrum molded by the underlying (open-sided) conoid. Three flagella were present in some individuals, a peculiar feature also found in the microgametes of some apicomplexans.

The phylogeny of colpodellids (Alveolata) using small subunit rRNA gene sequences suggests they are the free-living sister group to apicomplexans.

In an attempt to reconstruct early alveolate evolution, we have examined the phylogenetic position of colpodellids by analyzing small subunit rDNA sequences from Colpodella pontica Myl'nikov 2000 and Colpodella sp. (American Type Culture Collection 50594). All phylogenetic analyses grouped the colpodellid sequences together with strong support and placed them strongly within the Alveolata. Most analyses showed colpodellids as the sister group to an apicomplexan clade, albeit with weak support. Sequences from two perkinsids, Perkinsus and Parvilucifera, clustered together and consistently branched as the sister group to dinoflagellates as shown previously. These data demonstrate that colpodellids and perkinsids are plesiomorphically similar in morphology and help provide a phylogenetic framework for inferring the combination of character states present in the last common ancestor of dinoflagellates and apicomplexans. We can infer that this ancestor was probably a myzocytotic predator with two heterodynamic flagella, micropores, trichocysts, rhoptries, micronemes, a polar ring, and a coiled open-sided conoid. This ancestor also very likely contained a plastid, but it is presently not certain whether it was photosynthetic, and it is not clear whether extant perkinsids or colpodellids have retained the organelle.