Histone deacetylase inhibitors

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The assessment of histone deacetylase inhibitors to correct human macrophages in the context of neuroinflammation / submitted by Bettina Zierfuss

The assessment of histone deacetylase inhibitors to correct human macrophages in the context of neuroinflammation / submitted by Bettina Zierfuss

Endoplasmic reticulum Fatty acid-binding protein 4 Fluorescence-activated cell sorting Fatty acid synthase Fetal calf serum Fc gamma receptor Food and Drug Administration Glycerol 3-phos[r]

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OPUS 4 | Effect of chromatin modeling by histone deacetylase inhibitors (HDIs) on hematopoietic stem cell (HSC) fate

OPUS 4 | Effect of chromatin modeling by histone deacetylase inhibitors (HDIs) on hematopoietic stem cell (HSC) fate

Ergänzend zu den Analysen zur Proliferation und Selbsterneuerung der Maus-HSCs wurde auf zellbiologischer Ebene der Zellzyklus-Status und die Expression des Cyclin-abhängigen Kinase-Inhibitors (cyclin dependent kinase inhibitor, CDKI) p21 cip-1/waf-1 in VPA-behandelten HSCs analysiert, um den Einfluss auf die Zellzyklusregulation in der hämatopoetischen Kaskade beurteilen zu können. Von Histon-Deacetylase-Inhibitoren ist bekannt, dass sie einen Zellzyklusarrest durch die Aktivierung von p21 cip-1/waf-1 induzieren können, worauf es zur Differenzierung und/oder Apoptose sowohl von leukämischen Blasten, als auch von Tumorzellen kommt. Überdies gibt es Hinweise darauf, dass p21 cip-1/waf-1 möglicherweise Stadien-spezifische Funktionen in der frühen Hämatopoese hat. In der vorliegenden Arbeit wurde gezeigt, dass Valproinsäure, im Gegensatz zu t-RA, die Zellzyklusprogression muriner HSCs von der G1- in die S-Phase beschleunigt, was sich in einer Erhöhung des Anteils von S- Phasen-Zellen bei gleichzeitiger Verringerung des Prozentsatzes von Zellen in der G1-Phase äußerte.
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Expression of calcium pumps is differentially regulated by histone deacetylase inhibitors and estrogen receptor alpha in breast cancer cells

Expression of calcium pumps is differentially regulated by histone deacetylase inhibitors and estrogen receptor alpha in breast cancer cells

AP-1: activator protein 1; ATCC: American Type Culture Collection; ChIP- seq: chromatin immunoprecipitation sequencing; DMEM: Dulbecco ’s Modified Eagle Medium; DMSO: dimethyl sulfoxide; E2: 17 β-estradiol; ERE: estrogen response element; ER- α: estrogen receptor alpha; FBS: fetal bovine serum; FDA: US Food and Drug Administration; GEO: Gene Expression Omnibus; GPER/GRP30: G protein-coupled estrogen receptor 1; HBSS: Hank ’s balanced salt solution; HDAC: histone deacetylase; HEPES: 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid; HER2: human epidermal growth factor receptor 2; HSD17B: hydroxysteroid 17 β-dehydrogenase; MEGM: Mammary Epithelial Cell Growth Medium; PMA: phorbol 12-myristate 13-acetate; PMCA ( ATP2B): plasma membrane Ca 2+
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Gutenberg Open Science: Histone deacetylase inhibitors reverse resistance to methylating agents : mechanisms in malignant melanoma and glioblastoma cells

Gutenberg Open Science: Histone deacetylase inhibitors reverse resistance to methylating agents : mechanisms in malignant melanoma and glioblastoma cells

downregulation underline that both effects are likely attributed to the HDAC inhibitory function and that HDAC1, HDAC2 and HDAC3 are the relevant targets, as these are impeded by MS-275. Early studies of HDAC inhibition in melanoma cells reported a reduction of KU proteins and DNA-PKcs, parts of NHEJ DSB repair pathway, to confer radiosensitivity (Munshi et al., 2005; Munshi et al., 2006). These findings were not reproduced in this work, since VPA did not impact on KU80 (Figure 17). On the other hand, research in prostate, colon and bone cancer cells confirmed the effect of HDAC inhibition on RAD51 expression, which coincided with an enhanced sensitivity to irradiation (Chinnaiyan et al., 2005; Adimoolam et al., 2007; Kachhap et al., 2010; Blattmann et al., 2010). This study confirms the aforementioned studies, as VPA pretreatment sensitized melanoma cells to irradiation. However, the effect of HDAC inhibition on alkylating agent-induced apoptosis was more pronounced than on IR-induced apoptosis in the cell lines tested (Figure 13). This supports the role of RAD51 and HR in HDAC inhibitor-mediated sensitization. DSBs induced by IR are primarily repaired via NHEJ, whereas TMZ or FM induce DSBs replication-dependently or block replication and therefore require HR for recovery. This makes alkylating agents a better choice in combination with HDAC inhibition. Further, if HDAC inhibitor-mediated impairment of HR also applies to other solid tumors a combination of HDAC inhibition with agents that replication-dependently introduce DNA-strand breaks might be a reasonable therapy approach. These agents are for instance topoisomerase inhibitors that are used for colon or lung cancer therapy.
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Histone deacetylase inhibitor valproic acid in pancreas differentiation

Histone deacetylase inhibitor valproic acid in pancreas differentiation

Use of histone deacetylase inhibitors as small molecules is a promising approach to increase the differentiation efficiency of various cell types. In the present study, efficiency of the Histone deacetylase inhibitor Valproic acid (VPA) to induce endocrine differentiation in human exocrine pancreatic ductal adenocarcinoma cell line (Panc-1) was investigated. Panc-1 cells were cultured and treated with different concentrations of VPA and using quantitative real-time polymerase chain reaction regulation of pancreatic developmental genes were studied. The real-time PCR studies revealed an enhanced expression of pancreatic developmental genes Pdx1, Sox17, Ngn3, Pax6, Isl1, whereas very low regulation was observed in Foxa2 expression. Regulation of Ngn3 and Pdx1 were further looked at protein level by Western blots. Glucagon expression was found in cells treated with VPA, which was confirmed at protein level by Western blot, immunocytochemistry and measured glucagon content in the lysates by enzyme-linked immunoassay. Results from Western blots demonstrate enhanced acetylation of histones H3 and H4, which marks in the most cases active chromatin, indicating that the action of VPA on pancreatic differentiation occurred through the prevention of deacetylation of histones H3 and H4.
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The histone deacetylase inhibitor SAHA acts in synergism with fenretinide and doxorubicin to control growth of rhabdoid tumor cells

The histone deacetylase inhibitor SAHA acts in synergism with fenretinide and doxorubicin to control growth of rhabdoid tumor cells

tumor cells and sensitize cancer cells to the cytotoxic effects of conventional cytostatic drugs [4-6]. These characteristics have led to the use of several HDACi in a number of single agent or combinatorial clinical trials (more than 100 currently listed) (e.g. in lung, breast bladder cancer, glioblastoma, leukemias and lymphomas) [7,8]. Recently the importance of deregulation of epigenetic mechanisms in the development of embryonal tumors such as medulloblastoma, CNS PNET and AT/RT has been demonstrated. Epigenetically active compounds including histone deacetylase inhibitors (HDACi) and demethylating agents (e.g. azacitidine) have been identified as attractive tools for the treatment of embryonal tumors, including rhabdoid tumors [9-11].
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Investigation of the effects of the histone deacetylase inhibitor SAHA on the medulloblastoma cell line DAOY

Investigation of the effects of the histone deacetylase inhibitor SAHA on the medulloblastoma cell line DAOY

Packer, 1991). Therefore, there is an urgent need to develop new and innovative therapies than can improve overall survival rate and reduce detrimental long term effects. 1.1.4. DAOY cell line DAOY is one of the most widely used model cell lines for investigation of medulloblastoma. This cell line was first established from a desmoplastic medulloblastoma tumor in the posterior fossa of a 4 years old Caucasian boy (Jacobsen et al., 1985) and grew as attached polygonal cells in the culture medium with a population doubling time of 30.5 h (Li et al., 2004). Like other medulloblastoma cell lines, the DAOY cell line has been shown to be relatively sensitive to a variety of chemotherapeutic drugs such as doxorubin, etoposide, and cisplastin or anticancer drugs, for example VPA, D85, SAHA, MS275, TSA and NaB (Jaboin et al., 2002; Li et al., 2005; Furchert et al., 2007). Moreover, treatment with histone deacetylase inhibitors like SAHA, TSA and NaB enhances the susceptibility of DAOY cells to other therapeutic regimens including ionizing radiation (IR) and tumor necrosis factor related apoptosis inducing ligand (TRAIL) (Sonnemann et al., 2006).
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Lysine Ethylation by Histone Lysine Methyltransferases

Lysine Ethylation by Histone Lysine Methyltransferases

Enzymatic assays by means of MALDI-TOF MS: The reactions were performed in a total volume of 25 mL in an Eppendorf vial by using a thermomixer. A typical enzymatic assay included histone peptides (40 mm), cosubstrate AdoMet (200 mm with SETD8 and SETD7; 500 mm with GLP and G9a), AdoEth (1 mm) or AdoSeEth (1 mm), and KMT enzyme (2 mm) in a reaction buffer of 50 mm Tris·HCl at pH 8.0. The reactions were incubated at 37 8C and ali- quots were removed from the reaction vial at different time points (1, 3, and 5 h) to measure the conversion of histone peptide sub- strates into alkylated products. The reaction was stopped by mixing the reaction mixture (3 mL) with MeOH (3 mL). An aliquot (3 mL) of these samples was directly mixed onto the MALDI target plate with a-cyano-4-hydroxycinnamic acid (CHCA) matrix (3 mL, 5 mg mL 1
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Histone methylation and neuroprotection in cerebral ischemia

Histone methylation and neuroprotection in cerebral ischemia

For mRNA expression analysis, quantitative real time Polymerase Chain Reaction (qRT-PCR) was performed using LightCycler 2.0 (Roche). Briefly, whole cell mRNA was harvested in Trizol (1ml per 3 Mio cells) and stored at -20°C. Following chloroform extraction, RNA containing pellets were solved in diethylpyrocarbonate treated water. After DNase digest, PCR-inhibitors were removed with the help of NucleoSpin RNA clean-up KIT (Macherey Nagel). RNA concentration was measured using Nanodrop and probes were uniformly diluted. RNA was reverse transcribed to complementary DNA (cDNA) with M-MLV reverse transcriptase and random hexamers. For qRT-PCR the LightCycler-FastStartDNA-Master SYBR-Green-I Kit (Roche) was used. Amplification and detection relied on LightCyclerRelative Quantification Software (Roche Molecular Biochemicals). All PCRs were performed in duplicate. The relative expression of each gene of interest (GOI) was calculated compared to a reference gene (ref) by the delta Cp (crossing point) method with efficiency correction using the equation E (GOI) –Cp(GOI) / E (ref) –Cp (ref) . Mean values of
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Charakterisierung der Interaktion der humanen Histon-Deacetylase 3 mit der MAP Kinase 11

Charakterisierung der Interaktion der humanen Histon-Deacetylase 3 mit der MAP Kinase 11

In den letzten Jahren rückte die Regulation der „Genexpression“ in den Mittelpunkt vieler Forschungsarbeiten. Dabei wird untersucht, wie eine Zelle exakt bestimmen kann, welches Gen exprimiert und bei welchem Gen die Expression unterdrückt wird. Eine große Rolle bei der Aufklärung der Steuerung der Genexpression spielte die Entdeckung von Enzymen, die Einfluss auf das Acetylierungsgleichgewicht einer Zelle nehmen: Histon-Acetyltransferasen (HATs) führen zu einer Hyperacetylierung bestimmter Proteine, während Histon-Deacetylasen (HDACs) eine Hypoacetylierung hervorrufen. Diese (De-)Acetylierung findet an Lysinresten basischer Histone statt, die zusammen mit der DNA die Bausteine des Chromatins bilden. Dass Lysinreste an Histonen acetyliert werden können, wurde schon im Jahre 1964 von Allfrey und Kollegen herausgefunden. Die molekularen Hintergründe waren allerdings über Jahrzehnte unklar, denn humane Histon-Deacetylasen konnten erst vor einigen Jahren isoliert und charakterisiert werden. HDACs werden aufgrund ihrer Homologie zu Hefeproteinen in drei Klassen eingeteilt, wobei die erste Klasse von den HDACs 1, 2, 3 und 8 repräsentiert wird, welche zum Hefeprotein RPD3 (reduced potassium dependency) homolog sind. Die zweite Klasse, die aus den HDACs 4, 5, 6, 7, 9 und 10 gebildet wird, ist HDA1 (histone deacetylase 1) in der Sequenz ähnlich. Die dritte HDAC-Klasse wurde erst kürzlich entdeckt; sie ist homolog zum Hefeprotein SIR2 (silencing information regulator) und besteht aus den humanen Proteinen SIRT1-7. Der Einfluss von HDACs und HATs bei der Regulation der Genexpression ist heute unbestritten: Generell wird es Transkriptionsfaktoren und anderen regulatorischen Proteinen durch eine Hyperacetylierung ermöglicht, an die DNA zu binden und eine Genexpression zu induzieren, während eine Hypoacetylierung meist den gegenteiligen Effekt hervorruft. Die Bedeutung von HDACs in malignen Erkrankungen wurde ebenfalls in den letzten Jahren untersucht. Inzwischen sind eine Reihe von HDAC-Hemmstoffen entwickelt worden, die neben den üblichen Therapien im Kampf gegen Krebserkrankungen eingesetzt werden können. Die Forschungen hierzu sind allerdings noch am Anfang, und in der nächsten Zeit werden noch weitere Untersuchungen nötig sein.
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Regulation of the NAD + -dependent deacetylase SIRT2 by CDK5

Regulation of the NAD + -dependent deacetylase SIRT2 by CDK5

1.2.1 SIRT1 Among the seven human sirtuins SIRT1 shares the highest sequence homology with yeast Sir2 (Voelter-Mahlknecht and Mahlknecht, 2006). In addition SIRT1, similar to its ancestor Sir2, is localized in the nucleus and involved in chromatin remodeling as it deacetylates several lysine residues of histone tails, including acetylated lysines 9 of histones H3 (H3K9ac), H3K14ac, H4K16ac, and H1K26ac (Vaquero et al., 2004). Moreover SIRT1 targets also non-histone proteins and its activity can be regulated by its ability to shuttle between nuclear and cytoplasmic compartments (Tanno et al., 2007; Hisahara et al., 2008). The increasing number of known SIRT1 substrates includes the transcription factor and tumor suppressor p53 as well as several other transcriptional regulators and cofactors, among them NF-κB, members of the forkhead family (FOXOs), PPAR (peroxisome proliferator-activated receptors) and p300 (Rahman and Islam, 2011). Molecular studies revealed that SIRT1 is involved in the regulation of diverse cellular processes ranging from lipid and glucose metabolism, cancer, aging to stress response. Of particular relevance for many of these processes is the AMP-activated protein kinase (AMPK)-SIRT1 signaling axis. AMPK is activated in response to increasing amounts of AMP and thus functions as an energy sensor that responds to cellular metabolic stress, including calorie restriction. Thereupon it increases the intracellular NAD + /NADH ratio in a nicotinamide (NAM) phosphoribosyltransferase (NAMPT)-dependent way, promoting the activity of SIRT1 (Fulco et al., 2008). Maybe not surprising then is the finding that Sirt1 knockout mice display a severe phenotype. These mice are viable but sterile and have a high prenatal or early postnatal death rate. Surviving animals are smaller than their wildtype littermates and exhibit developmental defects, e.g. delayed eyelid opening. Furthermore they are reported to suffer from several dysfunctions of the heart, lung and pancreas (Cheng et al., 2003; McBurney et al., 2003).
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Strukturelle Untersuchung Histon-Deacetylase-ähnlicher Enzyme aus Pseudomonas aeruginosa

Strukturelle Untersuchung Histon-Deacetylase-ähnlicher Enzyme aus Pseudomonas aeruginosa

Eine enzymatische Analyse der Enzyme bestätigte die Vorhersage, dass es sich bei den Enzymen PA1409 und PA0321 um APAHs handelt. Im Gegensatz dazu zeigte das Enzym PA3774 keinen signifikanten Umsatz von acetylierten Polyaminen. Die hohen Umsatzraten von artifiziellen HDAC- Substraten lassen darauf schließen, dass acetylierte Proteine die eigentlichen Substrate dieses Enzyms sind. Im weiteren Verlauf gelang es die Struktur des Enzyms PA3774 aufzuklären. Neben der nativen Struktur konnten zusätzlich Enzym-Ligand-Komplexe in hoher Auflösung determiniert werden, die detaillierte Einblicke in das Bindungsverhalten der Liganden geben. Wie sich herausstellte, zeigt PA3774 eine hohe strukturelle Ähnlichkeit mit der Histon-Deacetylase-ähnlichen Amidohydrolase (FB188-HDAH) aus Bordetella/Alcaligenes und sollte treffender als HDAH anstatt als APAH klassifiziert werden. Zum besseren Verständnis und Vergleich dieser beiden Proteine wurde die Kristallisation der FB188-HDAH reproduziert und es konnten verschiedene neue Enzym-Ligand-Komplexe erhalten werden. Beide Enzyme zeigen eine analoge Tetramerisierung, deren spezifische Assemblierung den Bindungskanal entscheidend beeinflusst. Aufgrund dessen ist anzunehmen, dass diese Quartärstruktur eine essenzielle Funktion besitzt. Zwar gelang die Kristallisation von PA0321 und PA1409 im Rahmen dieser Arbeit nicht, jedoch ist die Struktur der eng verwandten APAH aus M. ramosa bereits bekannt. Ein Vergleich der HDAH und APAH Enzyme zeigt gravierende Unterschiede hinsichtlich ihrer Quartärstruktur, was eine plausible Erklärung für ihre unterschiedlichen Substratselektivitäten liefert. Zusätzlich wurden an zwei Nebenprojekten gearbeitet. Im Ersten wurden vielversprechende photoschaltbare Inhibitoren auf bakterielle sowie menschliche HDAC-Enzyme untersucht. Hierbei zeigten die Inhibitoren in ihrer metastabilen cis-Konfiguration eine um bis zu 10-fach erhöhte Wirkung Inhibition im Vergleich zur thermostabileren trans-Konfiguration. Solche photoschaltbaren Inhibitoren eröffnen einen Weg hin zu zielgerichteten nebenwirkungsärmeren Chemotherapien.
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Synthese hochfunktionalisierter Indolin-2-one als Histon-Deacetylase-Inhibitoren

Synthese hochfunktionalisierter Indolin-2-one als Histon-Deacetylase-Inhibitoren

Die geplante reduktive Aminierung (siehe Kap. 73 verlief leider erfolglos. Es konnte jedoch in relativ guten Ausbeuten der Enolether 73 zum Alkylether 76 reduziert werden. [r]

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Characterization of histone lysine demethylation

Characterization of histone lysine demethylation

inhibitors in vivo. 100 µM of inhibitor JMJ-1, JMJ-4 and JMJ-6 were added to the culture medium individually and incubated at 26 o C for 48 hours. Equivalent amounts of solvent at final concentration of 1 % DMSO (v/v) and no treatment conditions were used as control groups. Cell pellets were harvested and followed by nuclei isolation. Acid extraction has been performed with nuclei fraction. Extracted histone proteins were separated by SDS- PAGE gel followed by western blotting detection or LC-MS/MS measurement. A schematic illustration of the workflow with histone proteins were indicated in Figure 3.6 a. Histone proteins in SDS-PAGE gel were stained by coomassie G250 as shown in Figure 3.6 b. Variation of dKDM4a substrate H3K36me3 was detected by western blotting using anti-H3K36me3 specific antibody. Comparing to L2-4 cells with no treatment, 1 % DMSO (v/v) treatment led to decrease of H3K36me3. Cells treated with 100 µM final concentration of inhibitor JMJ-1, JMJ-4 and JMJ-6 individually shown increased level of H3K36me3 based on antibody detection by western blotting. Total histone protein H3 was used as loading control (see Figure 3.6 c). Chemiluminescence detection was applied using ECL Prime Western Blotting Detection Reagent from Amersham.
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Epigenetic reprogramming involving histone H3 variants, histone modifications and DNA methylation in mouse zygotes

Epigenetic reprogramming involving histone H3 variants, histone modifications and DNA methylation in mouse zygotes

Histone H1 is defined as a linker histone, which consists of three different regions, that is, a short C-terminal tail of around 35 amino acids, a central globular domain with roughly 80 amino acids long and a rather long N-terminal tail of approximately 100 amino acids (Hartman et al., 1977). It is located between the adjacent nucleosomes, binds to the nucleosome where the DNA enters or exits and protects the linker DNA via its evolutionarily conserved globular domain, and facilitates to stabilize the formation of higher-order chromatin structures by its two highly variable flanking terminal tails (Allan et al., 1980; Harshman et al., 2013; Zhou et al., 2013). In mice, there are at least 9 members of histone H1 family have been identified in mice so far, including five somatic subtypes H1a, H1b, H1c, H1d and H1e, one testis-specific linker histone H1t, one differentiation-specific linker histone H1 0 as well as another two isoforms of oocyte-specific linker histone H1FOO (formerly H1OO), namely H1FOO α and β. The variety of H1 isoforms is summarized in Fig. 1.1 (Lennox and Cohen, 1983; Lennox and Cohen, 1984; Seyedin and Kistler, 1979; Tanaka et al., 2005). Apart from H1 0 and H1FOO, the others follow a replication dependent way (Marzluff et al., 2002). Notably, oocyte-specific linker histone H1FOO appears in both pronuclei during 1-cell stage, decreases in the nuclei at 2-cell stage and becomes undetectable after 4-cell stage (Gao et al., 2004), with H1FOO α isoform being much more abundant than the β one in the zygotes (Tanaka et al., 2005). The somatic variants are absent throughout oogenesis up to 1-cell stage and become detectable from 2-cell-stage on (Gao et al., 2004). However, an opposite observation has been reported that the depositions of somatic variants into the chromatin occur as soon as the formation of the pronuclei after fertilization (Adenot et al., 2000). The discrepancy could be resulted from the specificities of the antibodies. As for the differentiation-specific linker histone H1 0 , it has been proved that H1 0 knockout mice have a normal development, suggesting that it is indispensable for mouse embryogenesis (Sirotkin et al., 1995).
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Crosstalk between DNA methylation and histone modifications

Crosstalk between DNA methylation and histone modifications

Figure 3. Structure and H3 N-terminal tail binding of the tandem Tudor domain. (A) Schematic drawing of the multi-domain architecture of Np95 (top) and alignment of tandem Tudor domains from vertebrate Np95 homologs (bottom). Arrows show the end and start positions of the crystallized tandem Tudor domain shown in (B). Residues forming the aromatic cage shown in (C) are indicated by arrowheads. Absolutely conserved residues of the tandem Tudor domain are black shaded, while positions showing conservative substitutions are boxed with residues in bold face. Secondary-structure elements were generated with EsPript (37) using the crystal structure of human UHRF1 (PDB 3db3 and 3db4) and are shown above the amino acid sequence: a-helices (g), b-strands, strict alpha turns (TT) and strict beta turns (TTT). Accession numbers: Homo sapiens Q96T88.1; Pan troglodytes XP_001139916.1; Bos Taurus AAI51672.1; Mus musculus Q8VDF2.2; Rattus norvergicus Q7TPK1.2; Dario rerio NP_998242.1; Xenopus laevis ABY28114.1, Gallus gallus XP_418269.2. (B) Side view of the tandem Tudor domain as a cartoon model (left) and as surface representation (right) in complex with a histone H3 N-terminal tail peptide trimethylated at lysine 9 (green stick model; only Arg8-Lys9-Ser10 of the H3 peptide are resolved). The image was generated with PyMOL (38). (C) An aromatic cage is formed by Phe152, Tyr188 and Tyr191 and accommodates the trimethylated lysine 9 of H3 (H3K9me3). (D) Histone H3 N-terminal tail binding specificity of GFP–Np95, GFP–Tudor and GFP–Tudor (Y188A Y191A) in vitro. Shown are fluorescence intensity ratios of bound probe/bound GFP fusion. GFP was used as negative control. Shown are means ± SEM from four to ten independent experiments and two-sample t-tests were performed that do or do not assume equal variances, respectively. Statistical significance compared to the binding ratio of H3K9me3 is indicated: *P < 0.05, **P < 0.001.
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The effect of depletion of histone demethylase Jarid1A on cell proliferation, histone modifications, radiation response and gene expression

The effect of depletion of histone demethylase Jarid1A on cell proliferation, histone modifications, radiation response and gene expression

is, however, also possible that enhanced activity of a histone acetyltransferase is responsible for the observed hyperacetylation at H3K9, H3K56 and H4K16. To gain more insights in the interplay between Jarid1A and histone deacetylation further experiments will be required. Histone hyperacetylation is generally assumed to result in decondensation of chromatin facilitating accessibility to transcription factors or repair proteins (Görisch et al. 2005). Especially acetylation at H4K16 was reported to interfere with DNA fiber compaction, leading to a rather open chromatin structure by reducing the inter-nucleosome interaction (Zhang et al. 2016). To study chromatin condensation I used the MNase assay, whereby the DNA is cleaved in the linker regions between the nucleosomes by micrococcal nuclease (Telford and Stewart 1989). The sensitivity of the DNA to the MNase is dependent on the grade of decondensation and can be investigated by partial MNase digestion (Liu et al. 2013). In my hands chromatin of control cells and of Jarid1A depleted cells showed a similar sensitivity to the MNase treatment. As the MNase assay is a rather insensitive method requiring broad changes in chromatin compaction, it is possible that the grade of histone hyperacetylation upon Jarid1A depletion is not sufficient to make the decondensation visible (Goodarzi et al. 2011). Other groups also reported that histone hyperacetylation does not translate into altered MNase digestion pattern (Perry and Chalkley 1981; Gilbert et al. 2007). Although reduced formation of compact chromatin fibres by acetylation of H4K16 was clearly demonstrated by certain groups (Shogren-Knaak et al. 2006; Dorigo et al. 2003, Zhang et al. 2016) , it may not visibly influence chromatin compaction at all size scales of analyzed structures (Taylor et al. 2013). Even if no distinct alteration in the chromatin structure is visible, the impact of the observed hyperacetylation on cellular processes upon Jarid1A depletion may be wide-ranging.
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Characterization of H3K56me3, a novel histone core modification

Characterization of H3K56me3, a novel histone core modification

29 Profiling of H3K122ac-containing nucleosomes showed they are enriched for H3K56ac as well as other hallmarks of active transcription, but not H3K36me3 (117), which is typically found at the 3’end of actively transcribed genes and is associated with elongation (181). These findings substantiate genome-wide profiling data of H3K122ac, which showed that its distribution is limited to the flanking regions of the TSS (Figure 6), similar to H3K27ac and H2A.Zac. Likewise, H3K64ac is also enriched around the TSS (Figure 6) of active genes and is anti-correlated with repressive marks (140). H3K56ac has also been shown to overlap with H2A.Z at vertebrate promoters. Interestingly, recent findings show that H3K56Q-containing nucleosomes enhance the replacement of H2A.Z with H2A (144) indicating that H3K56ac may prepare H2A.Z nucleosomes for exchange. H3K122ac levels are proportional to the amount of mRNA expression (117), suggesting a role in transcriptional activation. The correlation to transcriptional activation is interesting, given the promotion of nucleosome disassembly when H3K122 is acetylated (108). One could speculate that H3K56ac allows limited access to a small region of nucleosome-obscured DNA, requiring relatively little energy, given the weaker DNA-histone contacts at these sites, and maintaining it in a poised state. Indeed, experimental evidence implicates H3K56ac in nucleosome disassembly during transcription (184). When the conditions favour transcriptional activation, acetylation at H3K122 could act as a switch to reinforce the signal and facilitate the more energy-demanding nucleosome disassembly or nucleosome sliding required for promoter clearance. The latter point is substantiated, by the confirmation of a direct function for H3K122ac in transcriptional activation, by the group of R. Schneider (117). Using an in vitro transcription assay, they showed that unlike the tail modification H3K18ac, H3K122ac alone could stimulate transcription from a chromatin template. Furthermore, histone eviction experiments demonstrated that nucleosomes displaying H3K122ac were more susceptible to displacement, reiterating the likely mechanism by which it functions. Given the negative effects H3K64ac has on nucleosome stability and its correlation with transcriptional activation (140), it will be interesting, in future studies, to assess any cross-talk that may occur between these modifications. The role of H3K56ac in the proposed hypothesis, as a mechanism of opening up chromatin but not activating transcription, is supported experimentally. In both yeast and humans, H3K56ac is found at some repressed genes and regions of DNA repair and therefore does not necessarily correlate with mRNA expression levels (37,99).
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Histone lysine methylation in the context of nuclear architecture

Histone lysine methylation in the context of nuclear architecture

__________________________________________________________ constitutive heterochromatin via these interaction (Bannister et al., 2001; Lachner et al., 2001). In mouse a high overlap of H3K9me3 with chromocenters (visualized by DAPI- counterstaining) was observed linking it to (peri-) centromeric heterochromatin (Peters et al., 2003; Rice et al., 2003). ChIP analysis of the bulk of repetitive sequences in mouse confirmed a strong and stable enrichment of H3K9me3 in the major and minor tandem satellite repeats in all investigated cell types (Martens et al., 2005) whereas enrichment for different subsets of interspersed repetitive elements with H3K9me3 was found to be rather cell cycle dependent. The observation in this thesis that H3K9me3 exists either in smaller and more dispersed clusters or in big intense clusters posed the question if there is a dynamic movement of constitutive heterochromatin dependent on cell cycle stage. Changes in pattern formation after exit of the cell cycle and also during the cell cycle were already ovserved in the past (Cremer et al., 2004). These results are to align with the claim that dynamic properties of heterochromatin are required for its nuclear function (Baxter et al., 2004; Manuelidis, 1990). Recently the absence of several histone methylation sites in resting B and T lymphocytes was described which were remarkably increased after mitotic stimulation (Baxter et al., 2004). The discrepancy of this observation with data obtained in this work is probably due to the fact that lymphocytes represent a very special case and exit of the cell cycle as investigated for tumor cells may differ fundamentally from mechanisms in senescent cells or during terminal differentiation.
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Vergleich des Proteasom-Inhibitors Bortezomib und des Immunoproteasom-Inhibitors ONX-0914 in ihrer Wirkung auf kortikale Primärkulturen der Ratte

Vergleich des Proteasom-Inhibitors Bortezomib und des Immunoproteasom-Inhibitors ONX-0914 in ihrer Wirkung auf kortikale Primärkulturen der Ratte

Die Ergebnisse dieser Arbeit zeigen eindeutige Unterschiede in der Wirkung des Proteasom-Inhibitors BZ und des Immunoproteasom-Inhibitors ONX. Man könnte daher annehmen, dass die beobachteten Effekte auf die unterschiedliche Wirkweise von BZ und ONX zurückzuführen sind. Wahrscheinlich aber haben beide Pharmaka viele off- target-Effekte und bewirken nicht nur eine spezifische )nhibition der - bzw. der LMP7-Untereinheit. Ob Neurone überhaupt eine basale oder induzierbare Expression des Immunoproteasoms aufweisen wird diskutiert. In einem Schlaganfall-Modell scheinen sie die Hauptquelle für LMP7 nach zerebraler Ischämie zu sein (Lü und Wang 2012). Eine andere Arbeit widerlegt dies (Chen et al. 2015). Ein hier beobachteter passiv neuroprotektiver Effekt von ONX ist daher nicht unbedingt nur auf seine Wirkweise, sondern auf eine verminderte bzw. fehlende Expression des Immunoproteasoms in Neuronen zurückzuführen. Da hier Glia-Zellen, die in der Diskussion sind das Immunoproteasom sicher zu exprimieren, nicht untersucht wurden, konnte keine Auswirkung von BZ und ONX auf diese Zelltypen ermittelt werden. Möglicherweise sind Mikroglia sogar geeignetere Zellen, um die direkte Wirkung der Immunoproteasom- Inhibition zu ermitteln. Eine ONX-vermittelte Reduktion der durch Mikroglia ausgelösten Inflammation nach ZNS-Traumata ist bereits erwiesen (Moritz et al. 2017). Hier sollten weitere Untersuchungen erfolgen.
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