Einfluss Diabetes-assoziierter Mutationen im Transkriptionsfaktor HNF4alpha

Der zellspezifische Transkriptionsfaktor HNF4alpha spielt eine zentrale Rolle in der Differenzierung von Hepatocyten und seine Expression geht in Nierenzellkarzinomen des Menschen verloren. In der Hepatomazelllinie HepG2 wird ein mutiertes HNF4alpha-Allel exprimiert, diese Mutation betrifft die DNA-Bindedomäne des Transkriptionsfaktors. Im Menschen führen Mutationen im HNF4alpha-Gen zu "maturity onset diabetes of the young 1" (MODY1), einer monogenen Form des nicht Insulin abhängigen Diabetes-mellitus. Ausserdem führt eine Mutation in der HNF4alpha-Bindestelle im Promotor des HNF1alpha-Gens zu MODY3. In dieser Arbeit wurde gezeigt, dass die D69A-Mutante aus der Hepatomazelllinie HepG2 auf einigen Promotoren stärker als der Wildtyp transaktiviert. Die D69A-Mutante verhält sich im Hinblick auf die Hemmung der Zellvermehrung in stabilen INS-1-Klonen wie der Wildtyp. Die geringe Funktionsänderung gegenüber dem Wildtyp macht eine ursächliche Beteiligung der Mutante bei der Tumorentstehung wenig wahrscheinlich. Die vorgestellten Daten zeigen, dass eine MODY3-assoziierte Mutation im HNF1alpha-Gen einen Verlust der HNF4alpha-Bindestelle des HNF1alpha-Promotors verursacht, durch diese Mutation wird im betroffenen MODY3-Patienten also wahrscheinlich die Expression des HNF1alpha-Gens gestört. Die Daten zeigen weiterhin, dass die Transaktivierung durch die MODY1-assoziierten Mutanten R127W, R154X, V255M, Q268X und E276Q abhängig von der Art der Mutation im Vergleich zum Wildtyp verringert ist. Eine Inaktivierung des Wildtyp Proteins durch die Mutanten ist kein allgemeiner Mechanismus bei der Entstehung von MODY1, denn es gibt nur bei der R154X-Mutante Hinweise auf einen schwachen dominant-negativen Effekt. Sowohl die verringerte Transaktivierung der MODY1-Mutanten als auch der festgestellte Funktionsverlust der HNF4alpha-Bindestelle unterstützt die These, dass sich MODY1-Mutationen über eine verminderte Expression des HNF1alpha-Gens auswirken. In dieser Arbeit wurden erstmals Daten präsentiert, die eine Hemmung der Zellvermehrung durch HNF4alpha belegen. Die Überexpression von HNF4alpha in der beta-Zelllinie INS-1 der Ratte führt zu einer geringeren Vermehrung der Zellen, diese Eigenschaft die Zellvermehrung zu hemmen ist in einigen MODY1-Mutanten verlorengegangen. Ausserdem ist die Zellvermehrung in induzierbaren HNF4alpha-INS-1-Klonen nach Induktion verlangsamt. Dies spiegelt sich in der progressiven Verringerung des Anteils HNF4alpha-exprimierender Zellen innerhalb eines Klones wider. Eine ähnliche Verminderung des Anteils HNF4alpha-exprimierender Zellen zeigt sich in induzierbaren HEK293-Klonen. Dieser Einfluss von HNF4alpha in den undifferenzierten HEK293-Zellen unterstützt die Annahme HNF4alpha könnte an der Entstehung von Nierentumoren beteiligt sein. In einem Modell zur Entstehung von MODY1 wird der fortschreitende Verlust der HNF4alpha-Aktivität in beta-Zellen des Pankreas als Ursache für die Krankheit vorgeschlagen. Nach diesem Modell geht die Expression des intakten Allels bei MODY1-Patienten in einigen beta-Zellen verloren. Diese HNF4alpha-defizienten beta-Zellen sind in der Insulinsekretion defekt und haben einen Wachstumsvorteil gegenüber den beta-Zellen, die noch HNF4alpha exprimieren. Durch den Wachstumsvorteil nimmt der Anteil der defekten beta-Zellen zu, dies trägt zur Progression der Krankheit bei. Die Eigenschaft von HNF4alpha die Vermehrung von Zellen zu hemmen könnte in MODY1- Patienten gestört sein, ein Verlust dieser Eigenschaft könnte zur Entstehung von Tumoren beitragen

The T-cell oncogene Tal2 is a Target of PU.1 and upregulated during osteoclastogenesis

Transcription factors play a crucial role in regulating differentiation processes during human life and are important in disease. The basic helix-loop-helix transcription factors Tal1 and Lyl1 play a major role in the regulation of gene expression in the hematopoietic system and are involved in human leukemia. Tal2, which belongs to the same family of transcription factors as Tal1 and Lyl1, is also involved in human leukaemia. However, little is known regarding the expression and regulation of Tal2 in hematopoietic cells. Here we show that Tal2 is expressed in hematopoietic cells of the myeloid lineage. Interestingly, we found that usage of the Tal2 promoter is different in human and mouse cells. Two promoters, hP1 and hP2 drive Tal2 expression in human erythroleukemia K562 cells, however in mouse RAW cells only the mP1 promoter is used. Furthermore, we found that Tal2 expression is upregulated during oesteoclastogenesis. We show that Tal2 is a direct target gene of the myeloid transcription factor PU.1, which is a key transcription factor for osteoclast gene expression. Strikingly, PU.1 binding to the P1 promoter is conserved between mouse and human, but PU.1 binding to P2 was only detected in human K562 cells. Additionally, we provide evidence that Tal2 influences the expression of the osteoclastic differentiation gene TRACP. These findings provide novel insight into the expression control of Tal2 in hematopoietic cells and reveal a function of Tal2 as a regulator of gene expression during osteoclast differentiation

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 (Cβ3). During megakaryocytic differentiation a coactivator complex on the Cβ3 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-Cβ3 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

Zika virus infection studies with CD34+ hematopoietic and megakaryocyte-erythroid progenitors, red blood cells and platelets

BACKGROUND: To date, several cases of transfusion-transmitted ZIKV infections have been confirmed. Multiple studies detected prolonged occurrence of ZIKV viral RNA in whole blood as compared to plasma samples indicating potential ZIKV interaction with hematopoietic cells. Also, infection of cells from the granulocyte/macrophage lineage has been demonstrated. Patients may develop severe thrombocytopenia, microcytic anemia, and a fatal course of disease occurred in a patient with sickle cell anemia suggesting additional interference of ZIKV with erythroid and megakaryocytic cells. Therefore, we analyzed whether ZIKV propagates in or compartmentalizes with hematopoietic progenitor, erythroid, and megakaryocytic cells. METHODS: ZIKV RNA replication, protein translation and infectious particle formation in hematopoietic cell lines as well as primary CD34+ HSPCs and ex vivo differentiated erythroid and megakaryocytic cells was monitored using qRT-PCR, FACS, immunofluorescence analysis and infectivity assays. Distribution of ZIKV RNA and infectious particles in spiked red blood cell (RBC) units or platelet concentrates (PCs) was evaluated. RESULTS: While subsets of K562 and KU812Ep6EPO cells supported ZIKV propagation, primary CD34+ HSPCs, MEP cells, RBCs, and platelets were non-permissive for ZIKV infection. In spiking studies, ZIKV RNA was detectable for 7 days in all fractions of RBC units and PCs, however, ZIKV infectious particles were not associated with erythrocytes or platelets. CONCLUSION: Viral particles from plasma or contaminating leukocytes, rather than purified CD34+ HSPCs or the cellular component of RBC units or PCs, present the greatest risk for transfusion-transmitted ZIKV infections

Protein arginine methyltransferase 6 controls brythroid Ggene 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

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

G9a-mediated Lysine Methylation Alters the Function of CCAAT/Enhancer-binding Protein-β*S⃞

The functional capacity of the transcriptional regulatory CCAAT/enhancer-binding protein-β (C/EBPβ) is governed by protein interactions and post-translational protein modifications. In a proteome-wide interaction screen, the histone-lysine N-methyltransferase, H3 lysine 9-specific 3 (G9a), was found to directly interact with the C/EBPβ transactivation domain (TAD). Binding between G9a and C/EBPβ was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation. Metabolic labeling showed that C/EBPβ is post-translationally modified by methylation in vivo. A conserved lysine residue in the C/EBPβ TAD served as a substrate for G9a-mediated methylation. G9a, but not a methyltransferase-defective G9a mutant, abrogated the transactivation potential of wild type C/EBPβ. A C/EBPβ TAD mutant that contained a lysine-to-alanine exchange was resistant to G9a-mediated inhibition. Moreover, the same mutation conferred super-activation of a chromatin-embedded, endogenous C/EBPβ target gene. Our data identify C/EBPβ as a direct substrate of G9a-mediated post-translational modification that alters the functional properties of C/EBPβ during gene regulation

Oncogenic Deregulation of Cell Adhesion Molecules in Leukemia

Numerous cell–cell and cell–matrix interactions within the bone marrow microenvironment enable the controlled lifelong self-renewal and progeny of hematopoietic stem and progenitor cells (HSPCs). On the cellular level, this highly mutual interaction is granted by cell adhesion molecules (CAMs) integrating differentiation, proliferation, and pro-survival signals from the surrounding microenvironment to the inner cell. However, cell–cell and cell–matrix interactions are also critically involved during malignant transformation of hematopoietic stem/progenitor cells. It has become increasingly apparent that leukemia-associated gene products, such as activated tyrosine kinases and fusion proteins resulting from chromosomal translocations, directly regulate the activation status of adhesion molecules, thereby directing the leukemic phenotype. These observations imply that interference with adhesion molecule function represents a promising treatment strategy to target pre-leukemic and leukemic lesions within the bone marrow niche. Focusing on myeloid leukemia, we provide a current overview of the mechanisms by which leukemogenic gene products hijack control of cellular adhesion to subsequently disturb normal hematopoiesis and promote leukemia development

Oncogenic Deregulation of Cell Adhesion Molecules in Leukemia

Numerous cell–cell and cell–matrix interactions within the bone marrow microenvironment enable the controlled lifelong self-renewal and progeny of hematopoietic stem and progenitor cells (HSPCs). On the cellular level, this highly mutual interaction is granted by cell adhesion molecules (CAMs) integrating differentiation, proliferation, and pro-survival signals from the surrounding microenvironment to the inner cell. However, cell–cell and cell–matrix interactions are also critically involved during malignant transformation of hematopoietic stem/progenitor cells. It has become increasingly apparent that leukemia-associated gene products, such as activated tyrosine kinases and fusion proteins resulting from chromosomal translocations, directly regulate the activation status of adhesion molecules, thereby directing the leukemic phenotype. These observations imply that interference with adhesion molecule function represents a promising treatment strategy to target pre-leukemic and leukemic lesions within the bone marrow niche. Focusing on myeloid leukemia, we provide a current overview of the mechanisms by which leukemogenic gene products hijack control of cellular adhesion to subsequently disturb normal hematopoiesis and promote leukemia development