11 research outputs found
Assemblage et spécificité des complexes acétyltransférases de la famille MYST
Tableau d'honneur de la FacultĂ© des Ă©tudes supĂ©rieures et postdorales, 2014-2015La chromatine est une structure nuclĂ©aire composĂ©e des histones, autour desquelles lâADN sâenroule pour ĂȘtre empaquetĂ© dans le noyau. La dynamique de cette structure permet de rĂ©guler plusieurs procĂ©dĂ©s nuclĂ©aires, tels que la transcription, la rĂ©plication et la rĂ©paration de lâADN. Il existe, entre autre, des complexes de modifications de la chromatine qui collaborent Ă la rĂ©gulation de ces diffĂ©rentes fonctions nuclĂ©aires. Les acĂ©tyltransfĂ©rases de la famille MYST participent Ă lâacĂ©tylation des queues N-term des histones. TrĂšs conservĂ©es de la levure Ă lâhumain, elles possĂšdent des rĂŽles importants dans plusieurs processus cellulaires. Deux des complexes MYST ont Ă©tĂ© au cĆur de mes Ă©tudes doctorales, soit le complexe HBO1 et MOZ/MORF. Mon projet de doctorat avait comme premier objectif de dissĂ©quer les diffĂ©rents domaines protĂ©iques prĂ©sents au sein de ces deux complexes et de caractĂ©riser leurs interactions soit avec les autres sous-unitĂ©s, soit avec la chromatine. Par des analyses biochimiques, nous avons dĂ©terminĂ© le mode dâassemblage des complexes MYST. Nous avons Ă©galement caractĂ©risĂ© leurs diffĂ©rents domaines de reconnaissance de modifications post-traductionnelles des histones, afin de dĂ©terminer leur mode de recrutement. Des analyses Ă lâĂ©chelle du gĂ©nome entier nous ont aussi permis de localiser ces protĂ©ines Ă des loci bien prĂ©cis. De plus, il nous a Ă©tĂ© possible de constater lâimportance de lâassociation des protĂ©ines INGs sur la fonction suppresseur de tumeur du complexe HBO1-JADE. Suite Ă une purification de la protĂ©ine BRPF1, jâai pu constater lâassociation de HBO1 avec cette protĂ©ine. Comme deuxiĂšme objectif de thĂšse, jâai donc eu Ă caractĂ©riser le nouveau complexe HBO1-BRPF1 et Ă dĂ©montrer sa spĂ©cificitĂ© dâacĂ©tylation. En utilisant des essais dâacĂ©tylation in vitro combinĂ©s Ă des expĂ©riences dâimmunoprĂ©cipitation de la chromatine, jâai pu Ă©tablir un nouveau mode de rĂ©gulation de lâactivitĂ© acĂ©tyltransfĂ©rase de la protĂ©ine HBO1. Ce mĂ©canisme Ă©tonnant dĂ©montre un changement de spĂ©cificitĂ© de lâactivitĂ© catalytique des MYST en fonction de leur association aux protĂ©ines dâĂ©chafaudage. Tous ces rĂ©sultats dĂ©montrent donc quâil est important de considĂ©rer lâensemble des sous-unitĂ©s des complexes MYST, car elles sont toutes aussi importantes que lâenzyme pour la reconnaissance, la spĂ©cificitĂ© dâacĂ©tylation ainsi que les fonctions cellulaires de ces complexes.Chromatin is a nuclear structure formed by DNA that is wrapped around histone octamers, allowing for its compaction in the nucleus. This structure is dynamic and regulates many nuclear processes, such as transcription, replication and DNA repair. Among other factors, complexes that modify chromatin collaborate for the regulation of these nuclear functions. The MYST acetyltransferase family participate in the acetylation of histone N-term tails. Highly conserved from yeast to human, they play various roles in many cellular pathways. During my PhD, I have focused on two of these MYST acetyltransferases, HBO1 and MOZ/MORF. The first objective of my project was to dissect the different protein domains comprised within these complexes and define their interactions either with other subunits or with chromatin. Using biochemical experiments, we brought to the forefront the assembly mechanism of the MYST complexes. Additionally, we characterized their chromatin recognition domains, which helped us determine their recruitment mechanism. Genome-wide analysis also gave us the precise localisation of these proteins on many loci. Moreover, we could determine that the association with ING subunits is essential for the tumor suppressor function of these complexes. Following purification of the BRPF1 protein, we could detect binding of the HBO1 protein. Thus, the second objective of my PhD project was to characterize the newly identified HBO1-BRPF1 complex and determine its acetylation specificity. Using in vitro acetylation assays combined with chromatin immunoprecipitation experiments, we unravelled a new regulation mechanism of the HBO1 acetyltransferase activity. This surprising mechanism shows a switch of histone tail acetylation specificity depending of the associated scaffold proteins, an activity previously thought to be intrinsic to the catalytic subunit. These data highlight a new role of the associated scaffold subunits within MYST-ING acetyltransferase complexes in directing the acetylation of specific histone tails. Altogether, these results demonstrate that it is important to consider MYST acetyltransferases as complexes, since their different subunits contribute to chromatin recognition, acetylation specificity and cellular functions
Impacts fonctionnels des polymorphismes dans les promoteurs des gĂšnes de lâapoptose
La susceptibilitĂ© ou la rĂ©sistance aux cancers peuvent impliquer plusieurs mĂ©canismes, incluant lâapoptose, la croissance cellulaire et la diffĂ©renciation, la rĂ©plication et la rĂ©paration de lâADN. Mon projet porte plus particuliĂšrement sur lâapoptose. Une dĂ©rĂ©gulation dans les voies dâactivation de lâapoptose entraĂźne une accumulation de cellules dĂ©rĂ©glĂ©es, crĂ©ant ainsi un environnement propice Ă lâinstabilitĂ© gĂ©nĂ©tique et au dĂ©veloppement du cancer. Comme lâapoptose est une voie biologique hautement rĂ©gulĂ©e, nous proposons lâhypothĂšse que des polymorphismes « fonctionnels » dans les rĂ©gions de rĂ©gulations des gĂšnes (rSNPs) perturberaient cette voie Ă cause de taux variables de transcrits et des protĂ©ines correspondantes dĂ» Ă la modification des sites de reconnaissances des facteurs de transcription. Les principaux objectifs de mon projet sont : (i) identifier les SNPs prĂ©sents dans la rĂ©gion promotrice des gĂšnes dâapoptose; (ii) dĂ©terminer les haplotypes de promoteurs les plus frĂ©quents prĂ©sents dans la population gĂ©nĂ©rale; (rHaps) (iii) vĂ©rifier leurs impacts fonctionnels sur lâexpression gĂ©nique par des essais in vitro (gĂšne rapporteur et retard sur gel). Cette Ă©tude permettra dâidentifier des rSNPs et rHaps ayant un impact sur le niveau dâexpression des gĂšnes dâapoptose, au moins dans un contexte in vitro. Ces diffĂ©rences allĂ©liques au niveau de lâexpression de ces gĂšnes dâapoptose pourraient contribuer Ă la susceptibilitĂ© interindividuelle de dĂ©velopper un cancer.Cancer susceptibility can involve many different mechanisms, including apoptosis, cell growth, cell differentiation, DNA replication and DNA repair. My project focuses on the apoptosis pathway. Decreased apoptosis level can lead to the accumulation of dysregulated cells, creating a favourable environment for genetic instability and tumorigenesis. Since apoptosis is a highly regulated pathway, the hypothesis of this project is that functional polymorphisms (rSNPs) in the regulatory regions of apoptosis genes such as promoters could create or disrupt transcription factor binding sites and modify their transcription levels. The main objectives of my project are: (i) Identify rSNPs in promoter regions of apoptosis genes; (ii) Calculate the frequent promoter haplotypes (rHaps) in general population; (iii) Validate their functional impact on gene expression with in vitro assays (gene reporter and gel shift). This study will allow identification of rSNPs and rHaps that could influence apoptosis gene expression levels, at least in the in vitro context. These allelic differences of expression in apoptosis genes could contribute to interindividual cancer susceptibility
Tandem PHD Fingers of MORF/MOZ Acetyltransferases Display Selectivity for Acetylated Histone H3 and Are Required for the Association with Chromatin
MORF (monocytic leukemia zinc-finger protein (MOZ)-related factor) and MOZ are catalytic subunits of histone acetyltransferase (HAT) complexes essential in hematopoiesis, neurogenesis, skeletogenesis and other developmental programs and implicated in human leukemias. The canonical HAT domain of MORF/MOZ is preceded by a tandem of plant homeodomain (PHD) fingers whose biological roles and requirements for MORF/MOZ activity are unknown. Here we demonstrate that the tandem PHD1/2 fingers of MORF recognize the N-terminal tail of histone H3. Acetylation of Lys9 (H3K9ac) or Lys14 (H3K14ac) enhances binding of MORF PHD1/2 to unmodified H3 peptides two to three fold. The selectivity for acetylated H3 tail is conserved in the double PHD1/2 fingers of MOZ. This interaction requires the intact N-terminus of histone H3 and is inhibited by trimethylation of Lys4. Biochemical analysis using NMR, fluorescence spectroscopy and mutagenesis identified key amino acids of MORF PHD1/2 necessary for the interaction with histones. Fluorescence microscopy and immunoprecipitation experiments reveal that both PHD fingers are required for binding to H3K14ac in vivo and localization to chromatin. The HAT assays indicate that the interaction with H3K14ac may promote enzymatic activity in trans. Together, our data suggest that the PHD1/2 fingers play a role in MOZ/MORF HATs association with the chromatic regions enriched in acetylated marks
The oncometabolite 2-hydroxyglutarate activates the mTOR signalling pathway
The identification of cancer-associated mutations in the tricarboxylic acid (TCA) cycle enzymes isocitrate dehydrogenases 1 and 2 (IDH1/2) highlights the prevailing notion that aberrant metabolic function can contribute to carcinogenesis. IDH1/2 normally catalyse the oxidative decarboxylation of isocitrate into α-ketoglutarate (αKG). In gliomas and acute myeloid leukaemias, IDH1/2 mutations confer gain-of-function leading to production of the oncometabolite R-2-hydroxyglutarate (2HG) from αKG. Here we show that generation of 2HG by mutated IDH1/2 leads to the activation of mTOR by inhibiting KDM4A, an αKG-dependent enzyme of the Jumonji family of lysine demethylases. Furthermore, KDM4A associates with the DEP domain-containing mTOR-interacting protein (DEPTOR), a negative regulator of mTORC1/2. Depletion of KDM4A decreases DEPTOR protein stability. Our results provide an additional molecular mechanism for the oncogenic activity of mutant IDH1/2 by revealing an unprecedented link between TCA cycle defects and positive modulation of mTOR function downstream of the canonical PI3K/AKT/TSC1-2 pathway
Identification of functional DNA variants in the constitutive promoter region of <it>MDM2</it>
Abstract Although mutations in the oncoprotein murine double minute 2 (MDM2) are rare, MDM2 gene overexpression has been observed in several human tumors. Given that even modest changes in MDM2 levels might influence the p53 tumor suppressor signaling pathway, we postulated that sequence variation in the promoter region of MDM2 could lead to disregulated expression and variation in gene dosage. Two promoters have been reported for MDM2; an internal promoter (P2), which is located near the end of intron 1 and is p53-responsive, and an upstream constitutive promoter (P1), which is p53-independent. Both promoter regions contain DNA variants that could influence the expression levels of MDM2, including the well-studied single nucleotide polymorphism (SNP) SNP309, which is located in the promoter P2; i.e., upstream of exon 2. In this report, we screened the promoter P1 for DNA variants and assessed the functional impact of the corresponding SNPs. Using the dbSNP database and genotyping validation in individuals of European descent, we identified three common SNPs (â1494âGâ>âA; indel 40âbp; and â182âCâ>âG). Three major promoter haplotypes were inferred by using these three promoter SNPs together with rs2279744 (SNP309). Following subcloning into a gene reporter system, we found that two of the haplotypes significantly influenced MDM2 promoter activity in a haplotype-specific manner. Site-directed mutagenesis experiments indicated that the 40âbp insertion/deletion variation is causing the observed allelic promoter activity. This study suggests that part of the variability in the MDM2 expression levels could be explained by allelic p53-independent P1 promoter activity.</p
BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation
During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD fingerâcontaining protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1 that specifically acetylates histone H3K14, and genomewide analysis shows high enrichment of BRPF3, HBO1 and H3K14ac at ORC1âbinding sites and replication origins found in the vicinity of TSSs. Consistent with this, BRPF3 is necessary for H3K14ac at selected origins and efficient origin activation. CDC45 recruitment, but not MCM2â7 loading, is impaired in BRPF3âdepleted cells, identifying a BRPF3âdependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3âHBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of nearby replication origins
Tandem PHD Fingers of MORF/MOZ Acetyltransferases Display Selectivity for Acetylated Histone H3 and Are Required for the Association with Chromatin
MORF (monocytic leukemia zinc-finger protein (MOZ)-related factor) and MOZ are catalytic subunits of histone acetyltransferase (HAT) complexes essential in hematopoiesis, neurogenesis, skeletogenesis and other developmental programs and implicated in human leukemias. The canonical HAT domain of MORF/MOZ is preceded by a tandem of plant homeodomain (PHD) fingers whose biological roles and requirements for MORF/MOZ activity are unknown. Here we demonstrate that the tandem PHD1/2 fingers of MORF recognize the N-terminal tail of histone H3. Acetylation of Lys9 (H3K9ac) or Lys14 (H3K14ac) enhances binding of MORF PHD1/2 to unmodified H3 peptides two to three fold. The selectivity for acetylated H3 tail is conserved in the double PHD1/2 fingers of MOZ. This interaction requires the intact N-terminus of histone H3 and is inhibited by trimethylation of Lys4. Biochemical analysis using NMR, fluorescence spectroscopy and mutagenesis identified key amino acids of MORF PHD1/2 necessary for the interaction with histones. Fluorescence microscopy and immunoprecipitation experiments reveal that both PHD fingers are required for binding to H3K14ac in vivo and localization to chromatin. The HAT assays indicate that the interaction with H3K14ac may promote enzymatic activity in trans. Together, our data suggest that the PHD1/2 fingers play a role in MOZ/MORF HATs association with the chromatic regions enriched in acetylated marks