The aim of the study was to investigate the expression NDST transcripts in a wide variety of tissues using RNA-sequencing experimental data from five published studies, using two common in silico tools: the Tophat-Cufflink pipeline and the HTSeq-DEXSeq pipeline. We show that to detect NDST alternative transcripts, paired-end sequencing should be used with replicates of samples or conditions together with 100 base read length to allow for reliable detection of the low expressed transcripts in the NDST family.  As a demonstration project, we also characterized HS synthesized by the adrenal carcinoma (ACC) cell line H295R and determined expression of NDSTs in the cells and in ACC tumor samples. We could show that roughly 65% of newly synthesized proteoglycans isolated after metabolic 35S-sulfate labeling of the cells are made up of heparan sulfate (HS) with an average chain length of 45 kDa. The HS chains show a high frequency of N-sulfation and a high total degree of sulfation. Interestingly, disaccharide analysis demonstrated a three-time higher amount of stored chondroitin sulfate (CS) compared to HS in the ACC cell line.

HDAC6 est une histone déacétylase hautement conservée, principalement cytoplasmique. Contrairement à d'autres désacétylases, HDAC6 a une spécificité de substrat unique pour les protéines non - histones . Outre les domaines de désacétylation, HDAC6 contient également un domaine de liaison à l'ubiquitine , qui relie HDAC6 de la voie ubiquitine / protéasome .L’atrophie du muscle squelettique est une condition sévère de perte progressive de masse musculaire au cours de certaines maladies telles le cancer, le diabète, le SIDA ou également immobilizations prolongées. Le contrôle de la masse musculaire est sous la dépendance d’un équilibre entre les processus anaboliques et cataboliques. L’atrophie se caractérise par une augmentation substantielle de la dégradation des protéines par le système ubiquitine-protéasome, causée par l'expression d'une série de gènes spécifiques, les atrogenes . Un des atrogenes induits plus spectaculaire est le muscle spécifique de l'ubiquitine ligase E3 MAFbx/Atrogin-1, qui prend soin de la dégradation de MyoD et de eIF3 -f. La dégradation de ces deux protéines inhibe l'expression de gènes et la traduction myotrophiques empêchant le remplacement de protéines dégradées.Récemment, nous avons identifié l’Histone Deacetylase 6 (HDAC6) comme un nouvel atrogène. L’expression de HDAC6 augmente au cours de l’atrophie musculaire, à la fois chez la souris et l’homme, à travers un mécanisme FOXO3 -dépendante. La déplétion de cet enzyme in vivo (electroporation de l’shRNA contre HDAC6 dans des muscle squelettiques de souris ou analyse de souris invalidées pour ce gène) protège contre l’atrophie. De plus, l’inhibition de HDAC6 après déclenchement de l’atrophie peut aussi atténuer le phénotype. Lors de la caractérisation du mécanisme d’action de HDAC6, nous avons montré que HDAC6 intéragit avec MAFbx et que elle est nécessaire pour l’ubiquitination de MyoD par MAFbx. Nos résultats montrent que la surexpression d’un mutant MyoD resistant à la degradation par MAFbx protège contre l’atrophie provoqué par la denervation.. De plus, certaines données préliminaires indiquent une implication de HDAC6 dans la dégradation de eIF3-f et dans le processus de autophagy dans le tissu musculaire , révélant une double rôle de HDAC6 dans le muscle squelettique .Ces preuves suggèrent que HDAC6 représente potentiellement une cible utile pour des traitements curatifs. / HDAC6 is a highly conserved histone deacetylase, mostly cytoplasmic. Unlike other deacetylases, HDAC6 has unique substrate specificity for non-histone proteins. Besides the deacetylation domains, HDAC6 also contains an ubiquitin-binding domain, which links HDAC6 to the ubiquitin/proteasome pathway. Skeletal muscle atrophy is a severe condition of muscle mass loss occurring during aging or in many clinical disorders as cancer, diabetes and AIDS. The maintenance of muscle mass is subtly controlled by an equilibrium between catabolic and anabolic processes. Muscle atrophy results as a partial suppression of protein synthesis and a substantial increase of protein breakdown by the ubiquitin-proteasome system, caused by the expression of a series of specific genes, the atrogenes. One of the atrogenes induced more dramatically is the muscle specific E3 ubiquitin ligase MAFbx/Atrogin-1, which takes care of the degradation of MyoD and of eIF3-f. Degradation of those two proteins inhibits expression of myotrophic genes and translation preventing the replacement of degraded proteins.We identified HDAC6 as a new atrogene. HDAC6 expression is up regulated during muscle atrophy in mouse and human through a mechanism FoxO3-dependent. In vivo depletion of this enzyme by shRNA electroporation or homologous recombination gives protection against atrophy and its inhibition during atrophy can partially reverse the muscle wasting phenotype. HDAC6 can interact with MAFbx and is required for MAFbx-mediated degradation of MyoD. According to our results, forced expression of a MyoD mutant resistant to HDAC6 and MAFbx dependent degradation prevents muscle wasting induced by denervation. Furthermore, some preliminary data show an involvement of HDAC6 in the degradation of eIF3-f and in the autophagy process in muscle tissue, revealing a double role of HDAC6 in skeletal muscle.These evidences suggest that HDAC6 potentially represents a valuable target for curative treatments.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Faustová Supervisor: Prof. Doutor Jorge Miguel de Ascenção Oliveira PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: The impact of SIRT1 inhibition on zebrafish morphology and behavior After discovery of connection between yeast Silent Information Regulator 2 (Sir2) and its ability to alter lifespan, Sir2 and its seven mammalian orthologs became very attractive therapeutic target. These so called sirtuins are members of a histone deacetylase family. They possess unique catalytic activity having nicotinamide adenine dinucleotide as a cofactor and their function can be influenced by environmental factors. The aim of this diploma thesis was to extend knowledge of Sirtuin 1 (SIRT1), which is from all mammalian sirtuins considered to have the closest relation to yeast Sir2. At first we tested the impact of SIRT1 inhibition on early developmental stages of zebrafish (Danio rerio) embryos and larvae, finding out that SIRT1 is important for normal development and SIRT1 inhibition or malfunction result in cardiovascular defects, delayed development, and death. Additionally, we tried to learn more about SIRT1 and its connection with Parkinson's disease by combining nontoxic doses...

The enhanced expression of histone deacetylases (HDACs) in a variety of malignancies drew attention to investigate a new category of anti-cancer drugs that are based on the inhibition of those enzymes. Valproic acid (VPA) is a well-known antiepileptic drug that exhibits antitumor activities through inhibition of HDACs class I and IIa. Cancer stem cells (CSCs) have been recognized to drive the tumor growth and progression hence; attention has been given to target this small subpopulation of CSCs rather than the whole bulk tumor cells. CD133 is considered to be a CSC marker in several tumors and its transcription is strongly influenced by epigenetic changes that will be altered upon administration of histone deacetylase inhibitors (HDACi) in cancer treatment. Therefore, we evaluated the epigenetic and cytotoxic effects of treatment with 1 mM VPA in combination with other chemotherapeutics and its influence on the expression of CD133 in human neuroblastoma (NB) cell lines. Our results revealed that addition of VPA to DNA-damaging chemotherapeutics induced a synergistic anti-tumor effect that was associated with caspase-3 dependent induction of apoptosis in UKF-NB-4 cells. This synergism was related to the increase of the acetylation status of histones H3 and H4 and was only produced either by...

The NAD-dependent deacetylase SIRT1 regulates several factors involved in stress response and cell survival but its function in cancer is largely unknown. Research suggests that SIRT1 influences several transcription factors and molecules that are important components of pathways often deregulated in cancer. Our experiments have shown that SIRT1 knock down by short hairpin RNA accelerates tumor xenograft formation by HCT116 colon cancer cells, whereas SIRT1 overexpression inhibits tumor formation. We have also found that, pharmacological inhibition of SIRT1 stimulates cell proliferation under conditions of growth factor deprivation suggesting a tumor suppressive function of SIRT1. Paradoxically, SIRT1 inhibition sensitizes the same cells to apoptosis by chemotherapeutic drugs. Immunohistochemical staining of a colon tumor microarray revealed high SIRT1 expression levels in normal colon mucosa and benign adenomas. SIRT1 overexpression was observed in nearly 25% of stage I/II/III colorectal adenocarcinomas but rarely found in advanced stage IV tumors. Furthermore, about 30% of carcinomas showed lower than normal SIRT1 expression. These results suggest a pleiotropic effect of SIRT1 in cancer, i.e., anti-proliferative as well as anti-apoptotic. Further experiments along these lines and examination of a larger patient cohort could provide a rationale for the use of SIRT1 activators and inhibitors in the prevention and treatment of cancer.

Neuroblastoma is the most common extracranial solid tumor that occurs during infancy. Despite the great progress has been made in contemporary clinic medicine some forms of neuroblastoma disease are still found very difficult to treat . This work focuses on the effects of histone deacetylase inhibitors (HDAC) in the neuroblastoma cell lines. It is known that HDAC inhibitors may contribute to recurrence of the tumor cells by affecting the chromatin structure and thus increase the expression of critical tumor suppressor genes. These genes activate apoptotic pathways that may even be independent of caspases. We observed the efficiency of used HDAC inhibitors as under standard conditions an in hypoxia (1 % O2). Inadequate amount of oxygen supply is one of the characteristic features of tumors and it also may contribute to chemoresistance. With the hypoxia-induced chemoresistance of tumor cells, the influence of HIF-1α is expected. Some HDAC inhibitors reduce the amount of HIF-1α in hypoxia and thus HIF transcription factor activity. Thus, the first part of this study is concerned with the acquisition of suitable experimental arrangement for the monitoring of induction of cellular death in human neuroblastoma cell lines SK-N-AS and UKF-NB-3. Secondly, this paper provides the evaluation of the influence...

Acetyl-CoA is a metabolite at the crossroads of central metabolism and the substrate of histone acetyltransferases regulating gene expression. In many tissues fasting or lifespan extending calorie restriction (CR) decreases glucose-derived metabolic flux through ATP-citrate lyase (ACLY) to reduce cytoplasmic acetyl-CoA levels to decrease activity of the p300 histone acetyltransferase (HAT) stimulating pro-longevity autophagy. Because of this, compounds that decrease cytoplasmic acetyl-CoA have been described as CR mimetics. But few authors have highlighted the potential longevity promoting roles of nuclear acetyl-CoA. For example, increasing nuclear acetyl-CoA levels increases histone acetylation and administration of class I histone deacetylase (HDAC) inhibitors increases longevity through increased histone acetylation. Therefore, increased nuclear acetyl-CoA likely plays an important role in promoting longevity. Although cytoplasmic acetyl-CoA synthetase 2 (ACSS2) promotes aging by decreasing autophagy in some peripheral tissues, increased glial AMPK activity or neuronal differentiation can stimulate ACSS2 nuclear translocation and chromatin association. ACSS2 nuclear translocation can result in increased activity of CREB binding protein (CBP), p300/CBP-associated factor (PCAF), and other HATs to increase histone acetylation on the promoter of neuroprotective genes including transcription factor EB (TFEB) target genes resulting in increased lysosomal biogenesis and autophagy. Much of what is known regarding acetyl-CoA metabolism and aging has come from pioneering studies with yeast, fruit flies, and nematodes. These studies have identified evolutionary conserved roles for histone acetylation in promoting longevity. Future studies should focus on the role of nuclear acetyl-CoA and histone acetylation in the control of hypothalamic inflammation, an important driver of organismal aging.

Multiple regulatory mechanisms work to ensure that eukaryotic transcription maintains mRNA pools and subsequent protein synthesis. When errors in transcription occur, deleterious effects on cellular fitness can develop. RNA degradation as well as histone modifications, specifically at promoter proximal nucleosomes, play a critical role in maintaining transcription, but, exact mechanisms are not fully understood. In this dissertation, I investigate the role of RNA degradation and chromatin dynamics in transcription regulation as well as further understand, through biochemical analysis, a critical histone deacetylase. Using various genome-wide methodologies in Saccharomyces cerevisiae, we find a functional interaction between the nuclear RNA exosome and histone variant H2A.Z that maintains mRNA levels. There is a reduction in RNA polymerase II nascent transcription following RNA exosome subunit Rrp6 depletion that is further globally accentuated with H2A.Z deposition loss. To understand the mechanism leading to this global reduction, we identify the mRNA of Sirtuin histone deacetylase Hst3 as a target of the RNA exosome, revealing a means to link degradation to the transcription machinery. These findings show that even slight changes in deacetylase or acetylase activity can have significant effects on transcription. Additionally, we reveal a global impact of H2A.Z on transcription. We further investigate the functional and structural significance of human surtuin histone deacetylase SIRT6 (yeast homolog Hst3). Using histone deacetylase assays, we confirm the significance of specific residues of SIRT6 in nucleosome binding and deacetylase activity. Additionally, we show SIRT6 has reduced deacetylase activity in vitro on acetylated lysine 56 as compared to acetylated lysine 9 on histone H3. Finally, we confirm structural findings that the histone tail of H2A impacts SIRT6 H3K9Ac deacetylation activity. Together, these findings indicate a critical importance of histone deacetylase activity in maintaining transcription, a novel role of H2A.Z in global transcription regulation that furthers our understanding of SIRT6 structure and function.

Despite the remarkable success of adoptive T cell therapy in the treatment of melanoma and hematological malignancies, therapeutic capacity in a broad range of solid tumours is impaired due to immunosuppressive events that render tumour-specific T cells unable to persist and kill transformed cells. To address some of the limitations of ACT in solid tumours, our laboratory has developed a therapeutic modality utilizing oncolytic virus, which expresses a tumour-associated antigen, known as an oncolytic viral vaccine (OVV), in combination with tumour specific central memory T cells. With this therapeutic approach (ACT), we can achieve robust in vivo expansion of transferred cells resulting in the complete and durable tumour regression in multiple solid murine tumour models. However, we demonstrate that the curative potential is lost when the tumour stage and burden increase as expanded transferred cells differentiate to a dysfunctional state resulting in the progressive decline in the tumour-specific CD8+ T cell response. Thus, we believe that restoring the T cell response in late-stage tumours will lead to enhanced curative potential of ACT in late-stage tumours. We have previously shown that HDACi, MS-275, can enhance the therapeutic capacity of a T cell-based therapy in an aggressive brain tumour model. In addition, concomitant delivery of MS-275 with ACT ensures durable cures through immunomodulatory mechanisms. Strikingly, concomitant delivery of MS-275, a class 1 histone deacetylase inhibitor (HDACi), with ACT in late-stage tumours completely restores the transferred T cell response to similar levels observed in early-stage tumours resulting in the complete regression of advance-stage tumours. Furthermore, MS-275 enhanced the proliferative capacity and tumour-specific cytotoxic function of transferred cells, independently of tumour stage, type and mouse strain. Interestingly, we did not observe a complete reversal of T cell dysfunction, but rather observed that MS-275 conferred unique properties to T cells as the expression of some markers typically associated with T cell dysfunction was enhanced in addition to persistence and proliferation capacity. Moreover, concomitant delivery of MS-275 also restored the therapeutic capacity of endogenously primed tumour-specific CD8+ T cells expanded by an OVV in late-stage tumours, demonstrating the potential for general use for MS-275 in T cell-based therapies. Our data suggests the use of HDACi may potentiate T cell-based immunotherapies to overcome tumour-mediated T cell dysfunction in advanced stage solid tumours. / Thesis / Master of Science in Medical Sciences (MSMS)

Vitamin D plays an important role in preserving healthy bones, and has additional roles in the body, including modulation of cell growth, differentiation, neuromuscular and immune function, and anti-inflammatory function. The vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily and regulates transcription of vitamin D-dependent target genes, such as those for key proteins involved in calcium and phosphorus absorption and bone development. Histone acetylation weakens the association of histones with DNA, and increases the accessibility of transcriptional regulatory proteins to chromatin templates, thereby increasing transcriptional activity of gene expression. Histone deacetylases remove the acetyl groups and condense chromatin structure, thereby preventing transcription. TSA is a potent histone deacetylase inhibitor and can significantly enhance gene expression. Bioactive food component, sulforaphane (SFN) is found in cruciferous vegetables and is known to be a histone deacetylase inhibitor, leading to transcriptional activation of gene expression. The objective of this study is to demonstrate that the bioactive food components modulate vitamin D action in adipocytes. To investigate the effects of TSA and SFN on vitamin D response, 3T3L1 mouse preadipocytes were treated with the combination of various concentrations of 1,25(OH)2 vitamin D, TSA, and SFN. Upon harvesting cells, the amounts of 24-hydroxylase mRNA, marker of vitamin D response, were measured by semiquantitative reverse transcriptase-PCR analysis. The results showed that the cells treated with 1μM TSA increased 1,25(OH)2 vitamin D-induced CYP24 mRNA level nearly 3.5-fold (p < 0.05) at 1nM 1,25(OH)2 vitamin D and nearly 2.5-fold (p < 0.05) in 10 nM 1,25(OH)2 vitamin D, and the cells treated with 5μM SFN increased 1,25(OH)2 vitamin D-induced CYP24 mRNA level nearly 1.4-fold at 1nM 1,25(OH)2 vitamin D and nearly 1.2-fold at 10 nM 1,25(OH)2 vitamin D.