Interplay between transcription regulators RUNX1 and FUBP1 activates an enhancer of the oncogene c-KIT and amplifies cell proliferation.

Runt-related transcription factor 1 (RUNX1) is a well-known master regulator of hematopoietic lineages but its mechanisms of action are still not fully understood. Here, we found that RUNX1 localizes on active chromatin together with Far Upstream Binding Protein 1 (FUBP1) in human B-cell precursor lymphoblasts, and that both factors interact in the same transcriptional regulatory complex. RUNX1 and FUBP1 chromatin localization identified c-KIT as a common target gene. We characterized two regulatory regions, at +700 bp and +30 kb within the first intron of c-KIT, bound by both RUNX1 and FUBP1, and that present active histone marks. Based on these regions, we proposed a novel FUBP1 FUSE-like DNA-binding sequence on the +30 kb enhancer. We demonstrated that FUBP1 and RUNX1 cooperate for the regulation of the expression of the oncogene c-KIT. Notably, upregulation of c-KIT expression by FUBP1 and RUNX1 promotes cell proliferation and renders cells more resistant to the c-KIT inhibitor imatinib mesylate, a common therapeutic drug. These results reveal a new mechanism of action of RUNX1 that implicates FUBP1, as a facilitator, to trigger transcriptional regulation of c-KIT and to regulate cell proliferation. Deregulation of this regulatory mechanism may explain some oncogenic function of RUNX1 and FUBP1

Functional and clinical consequences of clonal hematopoiesis of indeterminate potential on cardiovascular diseases and cancer

Studien aus den Jahren 2014 und 2015, die Sequenzierungsmethoden der neuesten Generation („next generation sequencing“, NGS) verwendet haben, brachten eine neue Entität hervor, klonale Hämatopoese von unbestimmtem Potenzial („clonal hematopoiesis of indeterminate potential“, CHIP). Sie wird definiert durch das Auftreten von somatischen Mutationen in Genen, die typischerweise in hämatologischen Krankheiten mutiert sind, bei CHIP aber in hämatologischen gesunden Menschen vorkommen. Diese Menschen zeigen ein nur leicht erhöhtes Risiko für spätere Leukämien, aber überraschenderweise eine signifikant verkürzte Lebenserwartung, die hauptsächlich durch ein erhöhtes Risiko für kardiovaskuläre Krankheiten erklärt wird. CHIP ist altersabhängig, sodass 10-20% der Altersgruppe über 70 Jahren CHIP aufweisen. Für CHIP wurde der Schwellenwert mutierter Allele („variant allele frequency“, VAF) auf 2 % definiert. Wegen der Korrelation mit kardiovaskulären Erkrankungen, wurde im Rahmen dieser Dissertation das Knochenmark einer Patientenkohorte (CHF-Kohorte) sequenziert, die an chronischer Herzinsuffizienz litten. Dazu wurde eine sehr genaue Amplikon-basierte NGS-Methode etabliert. Eine Analyse der Blutzellen im Knochenmark der CHF-Kohorte mit TET2-Mutationen hat eine signifikant erhöhte Anzahl von Leukozyten, sowie Stamm- und Vorläuferzellen gezeigt. Die vorliegende Studie konnte außerdem zeigen, dass sich ein signifikant verschlechterten Krankheitsverlauf und eine erhöhte Sterblichkeit ergab, wenn die Patienten eine Mutation in einem der am häufigsten mutierten Genen trugen, DNMT3A oder TET2. Mit der etablierten Sequenziermethode waren auch Mutationen unter einer VAF von 2 % detektierbar. Damit konnte eine Dosis-Abhängigkeit zwischen der Klongröße und klinischem Ausgang der Krankheit bei CHF-Patienten mit CHIP gezeigt werden. Das Risiko für eine kardiovaskuläre Erkrankung und Krebs steigt mit zunehmendem Alter und die Diagnose einer der Krankheiten bedeutet auch ein erhöhtes Risiko, die andere zu entwickeln. Zwischen Krebstherapien und der Entwicklung von kardiovaskulären Veränderungen, sowie sekundären, therapieinduzierten Leukämien, besteht ein oft beobachteter Zusammenhang. Therapieinduzierte Leukämien sind eine relativ häufige Folge von Chemotherapie und folgender autologer Stammzelltransplantation (ASZT). Darum war ein zweiter Schwerpunkt dieser Arbeit eine Patientenkohorte, die mit einer ASZT behandelt wurde. Die meisten Mutationen waren in den transplantierten Zellen zu finden, die dann zahlenmäßig nach der Therapie anstiegen, was für das Vorkommen der Mutationen in den 2 Stamm- und Vorläuferzellen spricht. Eine weitere Schlussfolgerung ist, dass Mutationen einen selektiven Vorteil bei der Wiederherstellung der Blutzellen nach der Therapie haben könnten.Clonal hematopoiesis of indeterminate potential (CHIP) is caused by recurrent somatic mutations leading to clonal blood cell expansion. However, direct evidence of the fitness of CHIP-mutated human hematopoietic stem cells (HSCs) in blood reconstitution is lacking. Because myeloablative treatment and transplantation enforce stress on HSCs, we followed 81 patients with solid tumors or lymphoid diseases undergoing autologous stem cell transplantation (ASCT) for the development of CHIP. We found a high incidence of CHIP (22%) after ASCT with a high mean variant allele frequency (VAF) of 10.7%. Most mutations were already present in the graft, albeit at lower VAFs, demonstrating a selective reconstitution advantage of mutated HSCs after ASCT. Thus, CHIP-mutated stem and progenitor cells largely gain on clone size upon ASCT-related blood reconstitution, leading to an increased future risk of CHIP-associated complications. CHIP increase with age and is also associated with atherosclerosis and inflammation. Age and inflammation are the major risk factors for heart failure, yet the association of CHIP with chronic ischemic heart failure (CHF) in humans is unknown. Therefore, we analyzed bone marrow-derived mononuclear cells from 200 patients with CHF by NGS to detect the presence of CHIP and associated such with long-term prognosis in patients with CHF. Forty-seven mutations with a VAF of at least 2% were found in 18.5% of 200 patients with CHF. The mutations most commonly occurred in the genes DNMT3A and TET2. During a median follow-up of 4.4 years, a significantly worse clinical outcome for patients with either DNMT3A or TET2 mutations compared with non-CHIP carriers was notable. Importantly, there was a significant dose-response association between VAF and clinical outcome. Our data suggest that somatic mutations in hematopoietic cells, may be significantly associated with the progression and poor prognosis of CHF

The four positively selected candidate genes are expressed during host infection.

<p><i>In planta</i> expression data for the four positively selected candidate genes <i>Zt80707</i>, <i>Zt89160</i>, <i>Zt103264</i> and <i>Zt110804</i> of <i>Z</i>. <i>tritici</i>, relative to expression during axenic growth. Values are normalized to the expression of the gene encoding GAPDH, a constitutively expressed housekeeping control. Error bars indicate the standard error of the mean (SEM) of three independent biological replicates per sample (** <i>p</i><0.01; <i>p</i>-values were calculated using the Mann-Whitney U-test).</p

Confocal laser scanning microscopy image analyses of pycnidia of wild and mutant <i>Z</i>. <i>tritici</i>.

<p>WGA-FITC / Propidium iodide double-stained pycnidia on infected wheat leaves 28 dpi of wild-type (A), mutant <i>Z</i>. <i>tritici</i> isolates; IPO323Δ<i>Zt80707</i> (B) and IPO323Δ<i>Zt103264</i> (C). Fungal cell walls (green) and fungal and plant nuclei (red) are visible in the infected plant tissue (scale bar: 50 μm). D) Average size of pycnidia measured 14 and 28 dpi. For each strain and time point, 50 pycnidia were measured. ** <i>p</i><0.01; <i>p</i>-values were calculated using a Mann-Whitney U test. Whiskers indicate standard deviations.</p

Rapidly Evolving Genes Are Key Players in Host Specialization and Virulence of the Fungal Wheat Pathogen <i>Zymoseptoria tritici</i> (<i>Mycosphaerella graminicola</i>)

<div><p>The speciation of pathogens can be driven by divergent host specialization. Specialization to a new host is possible via the acquisition of advantageous mutations fixed by positive selection. Comparative genome analyses of closely related species allows for the identification of such key substitutions via inference of genome-wide signatures of positive selection. We previously used a comparative genomics framework to identify genes that have evolved under positive selection during speciation of the prominent wheat pathogen <i>Zymoseptoria tritici</i> (synonym <i>Mycosphaerella graminicola</i>). In this study, we conducted functional analyses of four genes exhibiting strong signatures of positive selection in <i>Z</i>. <i>tritici</i>. We deleted the four genes in <i>Z</i>. <i>tritici</i> and confirm a virulence-related role of three of the four genes <i>ΔZt80707</i>, <i>ΔZt89160</i> and <i>ΔZt103264</i>. The two mutants <i>ΔZt80707</i> and <i>ΔZt103264</i> show a significant reduction in virulence during infection of wheat; the <i>ΔZt89160</i> mutant causes a hypervirulent phenotype in wheat. Mutant phenotypes of <i>ΔZt80707</i>, <i>ΔZt89160</i> and <i>ΔZt103264</i> can be restored by insertion of the wild-type genes. However, the insertion of the <i>Zt80707</i> and <i>Zt89160</i> orthologs from <i>Z</i>. <i>pseudotritici</i> and <i>Z</i>. <i>ardabiliae</i> do not restore wild-type levels of virulence, suggesting that positively selected substitutions in <i>Z</i>. <i>tritici</i> may relate to divergent host specialization. Interestingly, the gene <i>Zt80707</i> encodes also a secretion signal that targets the protein for cell secretion. This secretion signal is however only transcribed in <i>Z</i>. <i>tritici</i>, suggesting that <i>Z</i>. <i>tritici</i>-specific substitutions relate to a new function of the protein in the extracellular space of the wheat-<i>Z</i>. <i>tritici</i> interaction. Together, the results presented here highlight that <i>Zt80707</i>, <i>Zt103264</i> and <i>Zt89160</i> represent key genes involved in virulence and host-specific disease development of <i>Z</i>. <i>tritici</i>. Our findings illustrate that evolutionary predictions provide a powerful tool for the identification of novel traits crucial for host adaptation and pathogen evolution.</p></div

<i>In planta</i> phenotypic assay demonstrates impact of gene replacement in <i>Z</i>. <i>tritici</i>.

<p>To assess the functional relevance of non-synonymous changes in <i>Zt80707</i>, <i>Zt89160</i> and <i>Zt103264</i>, the orthologous genes from <i>Z</i>. <i>pseudotritici</i> and <i>Z</i>. <i>ardabiliae</i> were inserted at the respective native loci in the deletion mutants IPO323Δ<i>Zt80707</i>, IPO323Δ<i>Zt89160</i> and IPO323Δ<i>Zt103264</i>. For the gene <i>Zt80707</i>, the <i>Zp80707</i> ortholog was inserted with (IPO323Δ<i>Zt80707</i>::<i>sp</i>+<i>Zp80707)</i> and without (IPO323Δ<i>Zt80707</i>::<i>Zp80707</i>) the signal peptide of <i>Zt80707</i>. Quantitative differences in pycnidia levels were measured according to six categories: 0 (without any visible symptoms), 1 (1–20%), 2 (21–40%), 3 (41–60%), 4 (61–80%) and 5 (81–100%). Significant differences in pycnidia levels (** <i>p</i><0.01) were documented using a Mann-Whitney-U-Test for the <i>Zt80707</i> and <i>Zt89160</i> mutants. Only the replacement strain IPO323Δ<i>Zt103264</i>::<i>Za103264</i> could restore the wild-type phenotype of the <i>Z</i>. <i>tritici</i> isolate IPO323.</p

<i>Zt80707</i> encodes a signal peptide targeting the protein for cell secretion in <i>Z</i>. <i>tritici</i>.

<p>Western blot analyses of the proteins Zt111221-GFP (secreted LysM effector, positive control (+)), Zt80707-GFP (<i>Z</i>. <i>tritici</i>), Zp80707-GFP (<i>Z</i>. <i>pseudotritici</i>) and Zt77228-GFP (non-secreted protein, lysis control (-)) detected in the pellet fraction (P) and the cell culture supernatant (SN) with an anti-GFP antibody. The presence of the GFP tagged gene products in the supernatant demonstrates the secretion of the Zt80707 protein and the functioning of the predicted signal peptide of Zt80707. As Loading Control (LC) a Coomassie stained SDS gel is included to show comparable amounts of protein loaded in the lanes of the different samples.</p

<i>In planta</i> phenotypic assay demonstrates impact of gene deletion in <i>Z</i>. <i>tritici</i>.

<p>Mutant phenotypes of <i>Z</i>. <i>tritici</i> are characterized by measures of leaf area covered with pycnidia on the susceptible wheat variety Obelisk. To compare quantitative differences in pycnidia levels at 28 dpi, six categories ranging from 0 (without any visible symptoms), 1 (1–20%), 2 (21–40%), 3 (41–60%), 4 (61–80%) and 5 (81–100%) are used. Significant deviations in pycnidia levels between the wild-type strain and mutants are observed for the IPO323Δ<i>Zt80707</i>, IPO323Δ<i>Zt89160</i> and IPO323Δ<i>Zt103264</i> mutants (** p<0.01), but not the IPO323Δ<i>Zt110804</i> mutant. Wild-type pycnidia levels for IPO323Δ<i>Zt80707</i>, IPO323Δ<i>Zt89160</i> and IPO323Δ<i>Zt103264</i> are restored by insertion of the wild-type gene at the respective native loci in the mutants. P-values were calculated using the Mann-Whitney-U-Test.</p

Mutant IPO323Δ<i>Zt80707</i> and IPO323Δ<i>Zt103264</i> produce fewer pycnidiospores per pycnidium.

<p>Macroscopic images of infected wheat leaves 28 dpi with wild-type IPO323 (A) and the two mutants IPO323Δ<i>Zt80707</i> (B) and IPO323Δ<i>Zt103264</i> (C). Pycnidiospores are oozing from the pycnidia of IPO323 (A, white arrow), but not from the pycnidia of the two mutants. D) A quantitative comparison of pycnidiospores produced by the wild-type and the three mutant strains IPO323Δ<i>Zt80707</i>, IPO323Δ<i>Zt89160</i> and IPO323Δ<i>Zt103264</i> measured as counts of spores isolated from a known number of pycnidia. ** <i>p</i><0.01; <i>p</i>-values were calculated using a Mann-Whitney U test. Whiskers indicate standard deviations.</p

Candidate genes and genomic coordinates in <i>Z</i>. <i>tritici</i>.

<p>Candidate genes and genomic coordinates in <i>Z</i>. <i>tritici</i>.</p