Jörn Lausen

HNF4alpha reduces proliferation of kidney cells and affects genes deregulated in renal cell carcinoma

Hepatocyte nuclear factor 4α (HNF4α) is a tissue-specific transcription factor known to regulate a large number of genes in hepatocytes and pancreatic β cells. Although HNF4α is highly expressed in some sections of the kidney, little is known about its role in this organ and about HNF4α-regulated genes in the kidney cells. The abundance and activity of HNF4α are frequently reduced in renal cell carcinoma (RCC) indicating some tumor suppressing function of HNF4α in renal cells. To determine the potential role of HNF4α in RCC, we used Flp recombinase-mediated gene integration to generate human embryonic kidney cells (HEK293) that conditionally express wild-type or mutated HNF4α. Expression of wild-type HNF4α but not of the mutants led to reduction of proliferation and alterations of cell morphology. These effects were reversible and induced at physiological concentrations of HNF4α. Using gene expression profiling by microarrays, we determined genes regulated by HNF4α. Interestingly, many of the genes regulated by HNF4α have been shown to be deregulated in RCC microarray studies. These genes (ACY1, WT1, SELENBP1, COBL, EFHD1, AGXT2L1, ALDH5A1, THEM2, ABCB1, FLJ14146, CSPG2, TRIM9 and HEY1) are good candidates for genes whose activity is changed upon the decrease of HNF4α in RCC.

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.

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.

Histone arginine methylation keeps RUNX1 target genes in an intermediate state

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.

A minimal ubiquitous chromatin opening element (UCOE) effectively prevents silencing of juxtaposed heterologous promoters by epigenetic remodeling in multipotent and pluripotent stem cells

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.

Tissue-specific transcription factor HNF4alpha inhibits cell proliferation and induces apoptosis in the pancreatic INS-1 beta-cell line.

Abstract Hepatocyte nuclear factor 4α (HNF4α) is a tissue-specific transcription factor expressed in many cell types, including pancreatic β-cells. Mutations in the HNF4α gene in humans give rise to maturity-onset diabetes of the young (MODY1) characterized by defective insulin secretion by β-cells. To elucidate the mechanism underlying this disease, we introduced the splice form HNF4α2 or HNF4α8 into the rat β-cell line INS-1. Upon tetracycline-induced expression, both HNF4α isoforms caused distinct changes in cell morphology and a massive loss of cell numbers that was correlated with reduced proliferation and induced apoptosis. This differential activity was reflected in oligonucleotide microarray analysis that identified more genes affected by HNF4α2 compared to HNF4α8, and suggests that both isoforms regulate largely the same set of genes, with HNF4α2 being a stronger transactivator. We verified the induction of selected transcripts by real-time RT-PCR, including KAI1 and AIF, both known to have apoptotic potential. By establishing cell lines with inducible expression of these target genes, we deduce that both factors are insufficient to induce apoptosis. We propose that the anti-proliferative and apoptotic properties of HNF4α may be an essential feature impaired in MODY1 and possibly also in type 2 diabetes.

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.

Molecular Targeting of Aberrant Transcription Factors in Leukemia: Strategies for RUNX1/ETO

Malignant cell transformation is caused by mutations in distinct key regulatory genes involved in cell growth, apoptosis, senescence and differentiation. Particularly in human leukemia, chromosomal translocations involving crucial hematopoietic transcription factors are frequently causally linked to the disease. Transcription factors commonly have a modular structure, comprising distinct domains for DNA- binding, dimerization and protein-protein interaction. Each domain is functionally important and in principle accessible for a molecular-based therapeutic intervention. Uncovering the molecular structure of critical domains will allow the rational development of therapeutic agents that inhibit particular functions of leukemogenic transcription factors. However, so far most approaches are in the experimental stage. Among others, the RUNX1/ETO fusion protein, commonly found within acute myeloid leukemia cells carrying the translocation t(8;21), is currently intensively studied at the functional and structural level as well as in animal models. This combined effort has allowed the development of specific targeting approaches addressing different functional domains of the fusion protein. With a special focus on RUNX1/ETO we will discuss recent strategies to directly interfere with aberrant transcription factors to block their leukemogenic function.

Suppression of RUNX1/ETO oncogenic activity by a small molecule inhibitor of tetramerization

RUNX1/ETO, the product of the t(8;21) chromosomal translocation, is required for the onset and maintenance of one of the most common forms of acute myeloid leukemia (AML). RUNX1/ETO has a modular structure and, besides the DNA-binding domain (Runt), contains four evolutionary conserved functional domains named nervy homology regions 1-4 (NHR1 to NHR4). The NHR domains serve as docking sites for a variety of different proteins and, in addition, the NHR2 domain mediates tetramerization through hydrophobic and ionic/polar interactions. Tetramerization is essential for RUNX1/ETO oncogenic activity. Destabilization of the RUNX1/ETO high molecular weight complex abrogates RUNX1/ETO oncogenic activity. Using structure-based virtual screening, we identified several small molecule inhibitors mimicking the tetramerization hot spot within

Compatibility of RUNX1/ETO fusion protein modules driving CD34+ human progenitor cell expansion

Chromosomal translocations represent frequent events in leukemia. In t(8;21)+ acute myeloid leukemia, RUNX1 is fused to nearly the entire ETO protein, which contains four conserved nervy homology regions, NHR1-4. Furthermore RUNX1/ETO interacts with ETO-homologous proteins via NHR2, thereby multiplying NHR domain contacts. As shown recently, RUNX1/ETO retains oncogenic activity upon either deletion of the NHR3 + 4 N-CoR/SMRT interaction domain or substitution of the NHR2 tetramer domain. Thus, we aimed to clarify the specificities of the NHR domains. A C-terminally NHR3 + 4 truncated RUNX1/ETO containing a heterologous, structurally highly related non-NHR2 tetramer interface translocated into the nucleus and bound to RUNX1 consensus motifs. However, it failed to interact with ETO-homologues, repress RUNX1 targets, and transform progenitors. Surprisingly, transforming capacity was fully restored by C-terminal fusion with ETO’s NHR4 zinc-finger or the repressor domain 3 of N-CoR, while other repression domains failed. With an inducible protein assembly system, we further demonstrated that NHR4 domain activity is critically required early in the establishment of progenitor cultures expressing the NHR2 exchanged truncated RUNX1/ETO. Together, we can show that NHR2 and NHR4 domains can be replaced by heterologous protein domains conferring tetramerization and repressor functions, thus showing that the NHR2 and NHR4 domain structures do not have irreplaceable functions concerning RUNX1/ETO activity for the establishment of human CD34+ cell expansion. We could resemble the function of RUNX1/ETO through modular recomposition with protein domains from RUNX1, ETO, BCR and N-CoR without any NHR2 and NHR4 sequences. As most transcriptional repressor proteins do not comprise tetramerization domains, our results provide a possible explanation as to the reason that RUNX1 is recurrently found translocated to ETO family members, which all contain tetramer together with transcriptional repressor moieties.