Caractérisation de la fonction des complexes histone déacétylases Rpd3S et Set3C

La chromatine est essentielle au maintien de l’intégrité du génome, mais, ironiquement, constitue l’obstacle principal à la transcription des gènes. Plusieurs mécanismes ont été développés par la cellule pour pallier ce problème, dont l’acétylation des histones composant les nucléosomes. Cette acétylation, catalysée par des histones acétyl transférases (HATs), permet de réduire la force de l’interaction entre les nucléosomes et l’ADN, ce qui permet à la machinerie transcriptionnelle de faire son travail. Toutefois, on ne peut laisser la chromatine dans cet état permissif sans conséquence néfaste. Les histone déacétylases (HDACs) catalysent le clivage du groupement acétyle pour permettre à la chromatine de retrouver une conformation compacte. Cette thèse se penche sur la caractérisation de la fonction et du mécanisme de recrutement des complexes HDACs Rpd3S et Set3C. Le complexe Rpd3S est recruté aux régions transcrites par une interaction avec le domaine C-terminal hyperphosphorylé de Rpb1, une sous-unité de l’ARN polymérase II. Toutefois, le facteur d’élongation DSIF joue un rôle dans la régulation de cette association en limitant le recrutement de Rpd3S aux régions transcrites. L’activité HDAC de Rpd3S, quant à elle, dépend de la méthylation du résidu H3K36 par l’histone méthyltransférase Set2. La fonction du complexe Set3C n’est pas clairement définie. Ce complexe est recruté à la plupart de ses cibles par l’interaction entre le domaine PHD de Set3 et le résidu H3K4 di- ou triméthylé. Un mécanisme indépendant de cette méthylation, possiblement le même que pour Rpd3S, régit toutefois l’association de Set3C aux régions codantes des gènes les plus transcrits. La majorité de ces résultats ont été obtenus par la technique d’immunoprécipitation de la chromatine couplée aux biopuces (ChIP-chip). Le protocole technique et le design expérimental de ce type d’expérience fera aussi l’objet d’une discussion approfondie.

Targeting Histone deacetylases (HDAC) for the treatment of soft tissue sarcoma

Targeting Histone deacetylases (HDAC) for the treatment of genetically complex soft tissue sarcoma Histone deactylase inhibitors (HDACi) are a new class of anticancer therapeutics; however, little is known about HDACi or the individual contribution of HDAC isoform activity in soft tissue sarcoma (STS). We investigated the potential efficacy of HDACi as monotherapy and in combination with chemotherapy in a panel of genetically complex STS. We found that HDACi combined with chemotherapy significantly induced anti-STS effects in vitro and in vivo. We then focused our study of HDACi in malignant peripheral nerve sheath tumor (MPNST), a subtype of highly aggressive, therapeutically resistant, and commonly fatal malignancies that occur in patients with neurofibromatosis type-1 (NF1) or sporadically. The therapeutic efficacy of HDACi was investigated in a panel of NF1-associated and sporadic MPNST cell lines. Our results demonstrate the NF1-assocaited cohort to be highly sensitive to HDACi while sporadic cell lines exhibited resistance. HDACi-induced productive autophagy was found to be a mode of resistance and inhibiting HDACi-induced autophagy significantly induced pro-apoptotic effects of HDACi in vitro and in vivo. HDACs are not a single enzyme consisting of 11 currently known isoforms. HDACis used in these studies inhibit a variety of these isoforms, namely class I HDACs which include HDAC1, 2, 3, and 8. Recently, HDAC8-specific inhibitors (HDAC8i) have been created and tested in various cancer cell lines. Lastly, the potential therapeutic efficacy of HDAC8i was investigated in human (NF1-associated and sporadic) and NF1-associated murine-derived MPNST. HDAC8i abrogated cell growth in human and murine-derived MPNST cells. Similar to the pattern noticed with pan-HDACis NF1-associated cells, especially murine-derived, were more sensitive to HDAC8i compared to human sporadic MPNST cell lines. S-phase arrest was observed in human and murine MPNST cells, independent of p53 mutational and NF1 status. HDAC8i induced apoptosis is all cell lines tested, with a more pronounced effects in human and murine-derived NF1-associated cells. Most importantly, HDAC8i abrogated murine-derived MPNST xenograft growth in vivo. Taken together, these findings support the evaluation of pan-HDACi and isoform-specific inhibitors as a novel therapy to treat MPNST, including in combination with autophagy blocking combination regimens in particular for patients with sporadic MPNST.

Fourier transform infrared microspectroscopy identifies protein propionylation in histone deacetylase inhibitor treated glioma cells

Histone deacetylase inhibitors (HDIs) have attracted considerable attention as potential drug molecules in tumour biology. In order to optimise chemotherapy, it is important to understand the mechanisms of regulation of histone deacetylase (HDAC) enzymes and modifications brought by various HDIs. In the present study, we have employed Fourier transform infrared microspectroscopy (FT-IRMS) to evaluate modifications in cellular macromolecules subsequent to treatment with various HDIs. In addition to CH3 (methyl) stretching bands at 2872 and 2960 cm1, which arises due to acetylation, we also found major changes in bands at 2851 and 2922 cm1, which originates from stretching vibrations of CH2 (methylene) groups, in valproic acid treated cells. We further demonstrate that the changes in CH2 stretching are concentration-dependent and also induced by several other HDIs. Recently, HDIs have been shown to induce propionylation besides acetylation [1]. Since propionylation involves CH2 groups, we hypothesized that CH2 vibrational frequency changes seen in HDI treated cells could arise due to propionylation. As verification, pre-treatment of cells with propionyl CoA synthetase inhibitor resulted in loss of CH2 vibrational changes in histones, purified from valproic acid treated cells. This was further proved by western blot using propionyl-lysine specific antibody. Thus we demonstrate for the first time that propionylation could be monitored by studying CH2 stretching using IR spectroscopy and further provide a platform for monitoring HDI induced multiple changes in cells. (C) 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

On the function of the internal cavity of histone deacetylase protein 8: R37 is a crucial residue for catalysis

Biochemical studies reveal that a conserved arginine residue (R37) at the centre of the 14 angstrom internal cavity of histone deacetylase (HDAC) 8 is important for catalysis and acetate affinity. Computational studies indicate that R37 forms multiple hydrogen bonding interactions with the backbone carbonyl oxygen atoms of two conserved glycine residues, G303 and G305, resulting in a 'closed' form of the channel. One possible rationale for these data is that water or product (acetate) transit through the catalytically crucial internal channel of HDAC8 is regulated by a gating interaction between G139 and G303 tethered in position by the conserved R37. (C) 2011 Elsevier Ltd. All rights reserved.

Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis

FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-speci?c inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as ef?cient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as ‘FLIP inhibitors’ © 2012 Macmillan Publishers Limited.

Histone deacetylase 7 controls endothelial cell growth through modulation of beta-catenin

Rationale: Histone deacetylase (HDAC)7 is expressed in the early stages of embryonic development and may play a role in endothelial function.

Objective: This study aimed to investigate the role of HDAC7 in endothelial cell (EC) proliferation and growth and the underlying mechanism.

Methods and Results: Overexpression of HDAC7 by adenoviral gene transfer suppressed human umbilical vein endothelial cell (HUVEC) proliferation by preventing nuclear translocation of ß-catenin and downregulation of T-cell factor-1/Id2 (inhibitor of DNA binding 2) and cyclin D1, leading to G1 phase elongation. Further assays with the TOPFLASH reporter and quantitative RT-PCR for other ß-catenin target genes such as Axin2 confirmed that overexpression of HDAC7 decreased ß-catenin activity. Knockdown of HDAC7 by lentiviral short hairpin RNA transfer induced ß-catenin nuclear translocation but downregulated cyclin D1, cyclin E1 and E2F2, causing HUVEC hypertrophy. Immunoprecipitation assay and mass spectrometry analysis revealed that HDAC7 directly binds to ß-catenin and forms a complex with 14-3-3 e, ?, and ? proteins. Vascular endothelial growth factor treatment induced HDAC7 degradation via PLC?-IP3K (phospholipase C?–inositol-1,4,5-trisphosphate kinase) signal pathway and partially rescued HDAC7-mediated suppression of proliferation. Moreover, vascular endothelial growth factor stimulation suppressed the binding of HDAC7 with ß-catenin, disrupting the complex and releasing ß-catenin to translocate into the nucleus.

Conclusions: These findings demonstrate that HDAC7 interacts with ß-catenin keeping ECs in a low proliferation stage and provides a novel insight into the mechanism of HDAC7-mediated signal pathways leading to endothelial growth

Galectin-9 protein expression in endothelial cells is positively regulated by histone deacetylase 3

Galectin-9 expression in endothelial cells can be induced in response to inflammation. However, the mechanism of its expression remains unclear. In this study, we found that interferon-? (IFN-?) induced galectin-9 expression in human endothelial cells in a time-dependent manner, which coincided with the activation of histone deacetylase (HDAC). When endothelial cells were treated with the HDAC3 inhibitor, apicidin, or shRNA-HDAC3 knockdown, IFN-?-induced galectin-9 expression was abolished. Overexpression of HDAC3 induced the interaction between phosphoinositol 3-kinase (PI3K) and IFN response factor 3 (IRF3), leading to IRF3 phosphorylation, nuclear translocation, and galectin-9 expression. HDAC3 functioned as a scaffold protein for PI3K/IRF3 interaction. In addition to galectin-9 expression, IFN-? also induced galectin-9 location onto plasma membrane, which was HDAC3-independent. Importantly, HDAC3 was essential for the constitutive transcription of PI3K and IRF3, which might be responsible for the basal level of galectin-9 expression. The phosphorylation of IRF3 was essential for galectin-9 expression. This study provides new evidence that HDAC3 regulates galectin-9 expression in endothelial cells via interaction with PI3K-IRF3 signal pathway.

Histone H2A exogène induit à différenciation et la sénescence des cellules cancéreuses

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Rôle de l’acétylation/déacétylation des histones dans la régulation de l’expression des gènes de la COX-2, iNOS et mPGES-1 dans les tissus articulaires.

L’arthrose ou ostéoarthrose (OA) est l’affection rhumatologique la plus fréquente au monde. Elle est caractérisée principalement par une perte du cartilage articulaire et l’inflammation de la membrane synoviale. L’interleukine (IL)-1ß, une cytokine pro-inflammatoire, joue un rôle très important dans la pathogenèse de l’OA. Elle exerce son action en induisant l’expression des enzymes cyclo-oxygénase 2 (COX-2), prostaglandine E synthétase microsomale 1 (mPGES-1) et l’oxyde nitrique synthétase inductible (iNOS) ainsi que la production de la prostaglandine E2 (PGE2) et de l’oxyde nitrique (NO). Ces derniers (PGE2 et NO) contribuent à la synovite et la destruction du cartilage articulaire par leurs effets pro-inflammatoires, pro-cataboliques, anti-anaboliques, pro-angiogéniques et pro-apoptotiques. Les modifications épigénétiques, telles que la méthylation de l’ADN, et l’acétylation et la méthylation des histones, jouent un rôle crucial dans la régulation de l’expression des gènes. Parmi ces modifications, l’acétylation des histones est la plus documentée. Ce processus est contrôlé par deux types d’enzymes : les histones acétyltransférases (HAT) qui favorisent la transcription et les histones déacétylases (HDAC) qui l’inhibent. L’objectif de ce travail est d’examiner le rôle des enzymes HDAC dans la régulation de l’expression de la COX-2, mPGES-1 et iNOS. Nous avons montré qu’au niveau des chondrocytes, les inhibiteurs des HDAC (iHDAC), trichostatine A (TSA) et butyrate de sodium (NaBu), suppriment l’expression de la COX-2 et iNOS au niveau de l’ARNm et protéique, ainsi que la production de la PGE2 et du NO, induites par l’IL-1ß. L’effet inhibiteur à lieu sans affecter l’activité de liaison à l’ADN du facteur de transcription NF-κB (nuclear factor κ B). La TSA et le NaBu inhibent également la dégradation induite par l’IL-1ß des protéoglycanes au niveau du cartilage. Nous avons également montré, qu’au niveau des fibroblastes synoviaux, les iHDAC, TSA, NaBu et acide valproïque (VA), suppriment l’expression de la mPGES-1 ainsi que la production de la PGE2 induites par l’IL-1ß. En utilisant diverses approches expérimentales, nous avons montré que HDAC4 est impliquée dans l’induction de l’expression de la mPGES-1 par l’IL-1ß. HDAC4 exerce son action, via son activité déacétylase, en augmentant l’activité transcriptionnelle de Egr-1 (early growth factor 1), facteur de transcription principal de l’expression de la mPGES-1. L’ensemble de ces résultats suggère que les inhibiteurs des HDAC pourraient être utilisés dans le traitement de l’OA.

Étude de la localisation génomique du complexe HDAC Set3

La chromatine est un système de compaction de l’ADN jouant un rôle important dans la régulation de l’expression génique. L’acétylation de la chromatine provoque un relâchement de sa structure, facilitant le recrutement de facteurs de transcription. Inversement, des complexes histones déacétylases favorisent une structure compacte, réprimant l’expression de gènes. Un complexe HDAC, Rpd3S, est recruté par l’ARN polymérase II phosphorylée sur les régions codantes transcrites. Cette activité HDAC est stimulée par la déposition de la marque H3K36me générée par l’histone méthyltransférase Set2. Par approche génomique, en utilisant comme organisme modèle Saccharomyces cerevisiae, j’ai optimisé la méthode de ChIP-chip puis démontré que les sous unités Hos2 et Set3 d’un autre complexe HDAC, Set3C, étaient recrutées sur des régions codantes de gènes transcrits. De plus, Set3C est connu pour être recruté soit par H3K4me ou par la Pol II. La suite du projet portera sur le recrutement de Set3C qui semble similaire à Rpd3S.

Modulation de l'expression de Sirt-1 induite par l'endothéline-1 dans les cellules musculaires lisses vasculaires

Au cours des maladies cardiovasculaires (MCV), il peut se produire divers problèmes de santé, telle que l’insuffisance cardiaque ou encore l’HTA. Ces phénomènes se caractérisent, entre autres, par une augmentation de synthèse d’endotheline-1 (ET-1), un neuropeptide synthétisé par les cellules endothéliales ayant un effet vasoconstricteur sur les cellules musculaires lisses vasculaires (CMLV). Ainsi, la surexpression de ce vasopeptide, mène à terme, au maintien de l’HTA aggravée des sujets, précédée ou concomitante à l’athérosclérose ou à la resténose, cliniquement illustrées par une prolifération et une migration anormale des CMLV de la media vers l’intima des vaisseaux sanguins. Parallèlement, il a été observé que la protéine sirtuine-1 (Sirt-1), membre de la famille des protéines histones déacétylases (HDAC), présente des propriétés anti-athérosclérotiques par sa capacité d’atténuer la prolifération et la migration des CMLV. Des travaux récents ont aussi montré qu’au cours de l’HTA la protéine Sirt-1 est faiblement exprimée dans les CMLV. Son implication dans le développement des pathologies vasculaires semble apparente, mais des études demeurent nécessaires pour décrire son rôle exact dans la pathogenèse des MCV. Dans cette optique, l’objectif de cette étude a été d’observer la variation d’expression de Sirt-1 dans les CMLV, isolées de l’aorte ascendante de rat, en réponse à l’ET-1. On a remarqué qu’une heure de stimulation des CMLV avec l’ET-1 induit une diminution de l’expression de Sirt-1 via l’activation des récepteurs ETA. Ces résultats suggèrent que la capacité d’ET-1 à atténuer l’expression de Sirt-1 serait un éventuel mécanisme d’action avec des effets favorisant les MCV.

Regulation of Nox4 expression by histone deacetylases in human endothelial cells : involvement of epigenetic mechanisms

Nox4 is a member of the NADPH oxidase family, which represents a major source of reactive oxygen species (ROS) in the vascular wall. Nox4-mediated ROS production mainly depends on the expression levels of the enzyme. The aim of my study was to investigate the mechanisms of Nox4 transcription regulation by histone deacetylases (HDAC). Treatment of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy926 cells with the pan-HDAC inhibitor scriptaid led to a marked decrease in Nox4 mRNA expression. A similar down-regulation of Nox4 mRNA expression was observed by siRNA-mediated knockdown of HDAC3. HDAC inhibition in endothelial cells was associated with enhanced histone acetylation, increased chromatin accessibility in the human Nox4 promoter region, with no significant changes in DNA methylation. In addition, the present study provided evidence that c-Jun played an important role in controlling Nox4 transcription. Knockdown of c-Jun with siRNA led to a down-regulation of Nox4 mRNA expression. In response to scriptaid treatment, the binding of c-Jun to the Nox4 promoter region was reduced despite the open chromatin structure. In parallel, the binding of RNA polymerase IIa to the Nox4 promoter was significantly inhibited as well, which may explain the reduction in Nox4 transcription. In conclusion, HDAC inhibition decreases Nox4 transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the Nox4 promoter, most likely because of a hyperacetylation-mediated steric inhibition. In addition, HDAC inhibition-induced Nox4 downregulation may also involves microRNA-mediated mRNA destabilization, because the effect of the scriptaid could be partially blocked by DICER1 knockdown or by transcription inhibition.

Pan-histone deacetylase inhibitor panobinostat sensitizes gastric cancer cells to anthracyclines via induction of CITED2

Chemotherapy modestly prolongs survival of patients with advanced gastric cancer, but strategies are needed to increase its efficacy. Histone deacetylase (HDAC) inhibitors modify chromatin and can block cancer cell proliferation and promote apoptosis.

Alteration of CEACAM1 gene transcription in prostate cancer cells: The role of AR, Sp2, and HDAC

Cell to cell adhesion molecule (CEACAM1), a type II tumor suppressor, has been found to be down-regulated in prostate cancer cells. The mechanism that causes CEACAM1's down-regulation in tumorigenesis is unknown. Here we show that the transcriptional activity of CEACAM1 is decreased in prostate cancer cells. This decrease is not due to methylation of the CEACAM1's promoter, but rather to the alteration of transcription factors regulating CEACAM1 expression. ^ Since androgen/androgen receptors (AR) are potent regulators of prostate growth and differentiation, their role on CEACAM1 gene transcription was examined. The androgen receptor could directly increase CEACAM1 transcriptional activity in a ligand dependent manner by interacting with an AR consensus element that resides in the CEACAM1 promoter. However, AR binding to the CEACAM1 promoter is not related to the loss of CEACAM1 during prostate cancer progression. ^ Further analysis enabled us to determine the particular region in the CEACAM1 promoter that mediates a decrease in CEACAM1 transcriptional activity in prostate cancer cells. Upon further examination, we found that this CEACAM1 promoter region interacts with the Sp1, Sp2, and Sp3 transcription factors. However, only Sp2 expression was found to increase in prostate cancer cells. Inhibiting Sp2 from binding to the CEACAM1 promoter caused an increase in CEACAM1 transcriptional activity in prostate cancer cells. In addition, over-expressing Sp2 in normal prostate cells resulted in a decrease in CEACAM1 transcriptional activity and endogenous protein expression. These observations suggest that Sp2 is a transcription repressor of CEACAM1. Furthermore, prostate cancer cells treated with trichostatin A (TSA), a specific histone deacetylase (HDAC) inhibitor, activated CEACAM1 transcriptional activity. This implies that HDACs are involved in CEACAM1 transcriptional activity. Mutation of the Sp2 DNA binding region on the CEACAM1 promoter inhibited TSA activation of CEACAM1 transcriptional activity. This indicates that HDACs inhibit CEACAM1 transcriptional activity through Sp2. Base on these results, we propose that Sp2 is critical for down-regulating CEACAM1 expression, and one mechanism by which Sp2 represses CEACAM1 expression is by recruiting HDAC to the CEACAM1 promoter in prostate cancer cells. Collectively, these findings provide novel insights into mechanisms that cause the down-regulation of CEACAM1 expression in prostate cancer cells. ^

Recruitment of SMRT/N-CoR-mSin3A-HDAC-repressing complexes is not a general mechanism for BTB/POZ transcriptional repressors: The case of HIC-1 and γFBP-B

Hypermethylated in cancer (HIC-1), a new candidate tumor suppressor gene located in 17p13.3, encodes a protein with five C2H2 zinc fingers and an N-terminal broad complex, tramtrack, and bric à brac/poxviruses and zinc-finger (BTB/POZ) domain found in actin binding proteins or transcriptional regulators involved in chromatin modeling. In the human B cell lymphoma (BCL-6) and promyelocityc leukemia (PLZF) oncoproteins, this domain mediates transcriptional repression through its ability to recruit a silencing mediator of retinoid and thyroid hormone receptor (SMRT)/nuclear receptor corepressor (N-CoR)-mSin3A-histone deacetylase (HDAC) complex, a mechanism shared with numerous transcription factors. HIC-1 appears unique because it contains a 13-aa insertion acquired late in evolution, because it is not found in its avian homologue, γF1-binding protein isoform B (γFBP-B), a transcriptional repressor of the γF-crystallin gene. This insertion, located in a conserved region involved in the dimerization and scaffolding of the BTB/POZ domain, mainly affects slightly the ability of the HIC-1 and γFBP-B BTB/POZ domains to homo- and heterodimerize in vivo, as shown by mammalian two-hybrid experiments. Both the HIC-1 and γFBP-B BTB/POZ domains behave as autonomous transcriptional repression domains. However, in striking contrast with BCL-6 and PLZF, both HIC-1 and γFBP-B similarly fail to interact with members of the HDAC complexes (SMRT/N-CoR, mSin3A or HDAC-1) in vivo and in vitro. In addition, a general and specific inhibitor of HDACs, trichostatin A, did not alleviate the HIC-1- and γFBP-B-mediated transcriptional repression, as previously shown for BCL-6. Taken together, our studies show that the recruitment onto target promoters of an HDAC complex is not a general property of transcriptional repressors containing a conserved BTB/POZ domain.