Epigenetic role of Jumonji C (JmjC)-domain-containing proteins in angiogenesis

The ability of endothelial cells to properly adapt to changes in a dynamic blood perfused environment is essential to maintain the physiological function of the vascular system and of the organs. Epigenetic control of gene expression is believed to be the mechanism controlling cell-fate determination and cell-phenotype maintenance. In the thesis, two JmjC demethylases were screened for their function in endothelial biology. Both of them were proved to play a central role in angiogenesis. The histone 3 lysine 4 demethylase JARID1B was identified as the most highly expressed demethylase in endothelial cell. Knockdown of JARID1B in human umbilical vein endothelial cells (HUVEC) attenuated cell migration, angiogenic sprouting and tube formation. Jarid1b null mice exhibited attenuated retinal angiogenesis and reduced endothelial sprout outgrowth from aortic segments. Microarray data identified that the antiangiogenic transcription factor HOXA5 was suppressed by JARID1B. Consistently, chromatin immunoprecipitation experiment revealed that JARID1B occupies and reduces the histone 3 lysine 4 methylation levels at the HOXA5 promoter, demonstrating a direct function of JARID1B in endothelial HOXA5 gene regulation. Hence, as a highly expressed JmjC protein in endothelial cells, JARID1B fundamentally maintains endothelial angiogenic phenotypes perhaps through suppression of HOXA5. As second enzyme it was identified that the histone plant homeodomain finger protein 8 (PHF8) plays a role in endothelial angiogenic sprouting as well as tube formation and cell migration. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. To characterize the underlying mechanism, E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4, but not E2F1, expression in endothelial cells. Likewise, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Thus, it is proposed that PHF8 maintains endothelial function by controlling E2F4 expression. On the other hand, microarray and subsequent qPCR validation revealed that the expressions of small nuclear RNAs (snRNAs) were regulated by PHF8. Co-immunoprecipitation experiment demonstrated that PHF8 interacts with spliceosome related proteins SNRP70 and SRPK1 as well as snRNA. Indeed, PHF8 contributed to splicing: GLS and VEGF-A displayed alternative splicing in PHF8 depleted cells. In addition, c-FOS introns were showed to be retained after knockdown of PHF8 in endothelial cells. These results demonstrated that, by controlling angiogenic mRNA splicing, PHF8 could affect endothelial properties. Collectively, the results uncover the important roles of JARID1B and PHF8 in endothelial cells in the control of angiogenesis. Changing histone modifiers appears as an attractive concept for pro- and antiangiogenic therapy. The present work adds JARID1B and PHF8 as novel potential targets to this emerging field.Die besondere Fähigkeit von Endothelzellen sich den dynamischen Veränderungen der Umgebung des Blutkreislaufes anzupassen ist essentiell, um die physiologische Funktion des vaskulären Systems und der Organe aufrecht zu erhalten. Die epigenetische Kontrolle von differenzieller Genexpression wird als Mechanismus in der Festlegung des Zellschicksals und der Erhaltung des Zellphänotyps angesehen. In dieser Arbeit wurden die zwei Histondemethylasen JARID1B und PHF8 in einem Screening ausgewählt, um ihre Funktion in der Endothelzellbiologie zu untersuchen. Für beide Demethylasen wurde gezeigt, dass sie eine essentielle Rolle bei der Regulation der Angiogenese spielen. Zunächst wurden die Transkriptionslevel der Jumonji-domain-containing (JmjC) Demethylasen in Endothelzellen mit einem Affymetrix-Microarray und mittels RT-qPCR-Validierung ermittelt. Die Histon 3 Lysin 4 Demethylase JARID1B wurde als die in Endothelzellen am höchsten exprimierte JmjC-Demethylase identifiziert. Funktionsverlust-Studien mittels RNAi von JARID1B in Human umbilical vein endothelial cells (HUVECs) verminderten die endotheliale angiogene Kapazität bei der Bildung von Sphäroid-Auswüchsen und bei der Bildung von kapillar-ähnlichen Strukturen. Um diese Befunde im physiologischen Kontext zu bestätigen, wurden tamoxifen-induzierbare globale Knockout-Mäuse generiert, in denen die Depletion des Exons 6 zu einer Mutation mit einer Frame-Verschiebung führt. Die Aktivierung der Cre-Rekombinase durch Tamoxifen führte zu einem signifikanten Verlust von JARID1B auf Proteinebene in verschiedenen Geweben. Es wurde mittels qRT-PCR bestätigt, dass die Aktivierung der Cre-Rekombinase die Expression von JARID1B um etwa 80% in der Lunge reduzierte. Zehn Wochen alte Mäuse mit einem induzierten Knockout von JARID1B waren vital und zeigten keinen offensichtlichen Phänotyp im Erscheinungsbild. Der Aussprossung von Endothelzellen, detektiert anhand der Anzahl der CD31-positiven Zellen, war vermindert in explantierten Aortenringen: Beides, Spross-Anzahl und absolute Spross-Länge waren signifikant vermindert in Aortenringen von JARID1B Knockout-Mäusen. JARID1B beeinflusste die Physiologie der Angiogenese, da sowohl die Rate der retinalen Vaskularisierung als auch die Dichte des entwickelten vaskulären Plexus in Mäusen mit postnatalem Knockdown vermindert war. Zellen aus dem Knochenmark und Zellen, die bei der neuronalen Entwicklung beteiligt sind, könnten am Angiogeneseprozess potenziell involviert sein. Um diesen Einfluss auszuschließen, wurden die Versuche mit Tamoxifen induzierbaren Endothel-spezifischen JARID1B Knockout-Mäusen (Cdh5-Cre-Jarid1bF/F) wiederholt. In diesen Tieren war JARID1B spezifisch in Endothelzellen vermindert und der Phänotyp des verminderten retinalen vaskulären Wachstums und der verminderten vaskulären Dichte des Plexus wurde reproduziert. ... In dieser Arbeit wurde die Bedeutung der Histondemethylasen JARID1B und PHF8 in essentiellen Funktionen in Endothelzellen aufgezeigt. Das Verständnis der bisher wenig untersuchten epigenetischen Regulation in Endothelzellen wurde erweitert und neue Funktionen von JmjC-Histondemethylasen aufgedeckt. Es konnte gezeigt werden, dass die epigenetische Kontrolle ein zentraler Mechanismus in der Vaskulären Biologie darstellt und seine Störung die Endothelzellfunktion beeinträchtigt und damit als potentieller pharmakologischer Angriffspunkt fungieren kann

The histone demethylase PHF8 is essential for endothelial cell migration

Epigenetic marks critically control gene expression and thus the cellular activity state. The functions of many epigenetic modifiers in the vascular system have not yet been studied. We screened for histone modifiers in endothelial cells and observed a fairly high expression of the histone plant homeodomain finger protein 8 (PHF8). Given its high expression, we hypothesize that this histone demethylase is important for endothelial cell function. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. As a functional readout endothelial migration and tube formation was studied. PHF8 siRNA attenuated the capacity for migration and developing of capillary-like structures. Given the impact of PHF8 on cell cycle genes, endothelial E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4 but not E2F1 expression in endothelial cells. Consistently, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Conclusion: PHF8 by controlling E2F4 expression maintains endothelial function

PHF8 maintains endothelial migration in an E2F4-dependent manner.

<p>A: Flow cytometry analyses of HUVEC proliferation with the CFSE dye after RNAi transfection against PHF8 with and without overexpression of PHF8 or E2F or E2F1 or DDK (FLAG tag) as control (Ctl) n = 5. Tube formation (B) Boyden chamber (C) and scratch wound assay (D&E&F&G) each with statistics of HUVECs transfected with control siRNA (siScr) or two different PHF8 siRNAs (siPHF8-1, siPHF8-2) or E2F4 siRNAs (siE2F4-1, siE2F4-2) with and without electroporation of plasmids coding for E2F4 and control (DDK (FLAG tag). n = 3. *p<0.05.</p

PHF8 is required for endothelial proliferation and survival.

<p>A: Proliferation analysis determined by cell counting of HUVECs transduced with control shRNA shScrambled (shScr) or shRNA against PHF8 (shPHF8), n = 5. B&C: Flow cytometry analysis of HUVEC proliferation with the CFSE dye (B) or cell cycle analysis (C) with propidium iodide after 72 h RNAi transfection, n = 5. D: Apoptosis/survival assay for late apoptosis (Q3: annexin V and propidium iodide positive cells), early apoptosis (Q2: only annexin V positive) and annexin V/propidium iodide negative cells (Q1: normal). Tumor necrosis factor alpha (TNFα, 20 ng/ml, 3h) and cycloheximide (CHX, 25 μg/ml, 3h) served as positive control. HUVECs were transduced with control shRNA (shGFP) or shPHF8, n = 3, *p<0.05.</p

PHF8 is expressed in endothelial cells.

<p>A: Expression profile of the KDM7 family members PHF8 and JHDM1D as determined by qRT-PCR normalized to β-Actin, n = 3. B: Representative western blot of PHF8 and RNA-polymerase II (Pol II) from the nucleus of human umbilical vein endothelial cell (HUVEC), human microvascular endothelial cell line (HMEC), human aortic smooth muscle cells (HAoSMC), fibroblast and HEK293. C&D: Western blot from the cytosol and nuclear fraction (C) or only nuclear fraction (D) of HUVECs stained with anti-PHF8 (C: abcam #ab36068, D: bethyl #A301-772A) and transfected with siRNA against PHF8 or scrambled as control M = Protein Ladder.</p

PHF8 demethylates H3K9me1/2 and H4K20me1 in endothelial cells.

<p>A: Representative western blot and densitometry for histones and their modifications as indicated from HUVECs with overexpression (A) or knockdown (B) of PHF8 by RNAi. Anti-PHF8 from bethyl was used. Scr = scrambled RNAi, Ctl = pcDNA3-GFP. Numbers above the blots indicate the results of the relative densitometry. n = 3, *p<0.05.</p