Academic literature on the topic 'Prmt4/carm1'

CARM1 (co-activator-associated arginine methyltransferase 1)/PRMT4 (protein arginine methyltransferase 4), functions as a co-activator for transcription factors that are regulators of muscle fibre type and oxidative metabolism, including PGC (peroxisome-proliferator-activated receptor γ co-activator)-1α and MEF2 (myocyte enhancer factor 2). We observed significantly higher Prmt4 mRNA expression in comparison with Prmt1–Prmt6 mRNA expression in mouse muscle (in vitro and in vivo). Transfection of Prmt4 siRNA (small interfering RNA) into mouse skeletal muscle C2C12 cells attenuated PRMT4 mRNA and protein expression. We subsequently performed additional qPCR (quantitative PCR) analysis (in the context of metabolism) to examine the effect of Prmt4 siRNA expression on >200 critical genes that control (and are involved in) lipid, glucose and energy homoeostasis, and circadian rhythm. This analysis revealed a strikingly specific metabolic expression footprint, and revealed that PRMT4 is necessary for the expression of genes involved in glycogen metabolism in skeletal muscle cells. Prmt4 siRNA expression selectively suppressed the mRNAs encoding Gys1 (glycogen synthase 1), Pgam2 (muscle phosphoglycerate mutase 2) and Pygm (muscle glycogen phosphorylase). Significantly, PGAM, PYGM and GYS1 deficiency in humans causes glycogen storage diseases type X, type V/McArdle's disease and type 0 respectively. Attenuation of PRMT4 was also associated with decreased expression of the mRNAs encoding AMPK (AMP-activated protein kinase) α2/γ3 (Prkaa2 and Prkag3) and p38 MAPK (mitogen-activated protein kinase), previously implicated in Wolff–Parkinson–White syndrome and Pompe Disease (glycogen storage disease type II). Furthermore, stable transfection of two PRMT4-site-specific (methyltransferase deficient) mutants (CARM1/PRMT4 VLD and CARM1E267Q) significantly repressed the expression of Gys1, Pgam2 and AMPKγ3. Finally, in concordance, we observed increased and decreased glycogen levels in PRMT4 (native)- and VLD (methylation deficient mutant)-transfected skeletal muscle cells respectively. This demonstrated that PRMT4 expression and the associated methyltransferase activity is necessary for the gene expression programme involved in glycogen metabolism and human glycogen storage diseases.

ABSTRACT Temporal regulation of gene expression is a hallmark of cellular differentiation pathways, yet the mechanisms controlling the timing of expression for different classes of differentiation-specific genes are not well understood. We previously demonstrated that the class II arginine methyltransferase Prmt5 was required for skeletal muscle differentiation at the early stages of myogenesis (C. S. Dacwag, Y. Ohkawa, S. Pal, S. Sif, and A. N. Imbalzano, Mol. Cell. Biol. 27:384-394, 2007). Specifically, when Prmt5 levels were reduced, the ATP-dependent SWI/SNF chromatin-remodeling enzymes could not interact with or remodel the promoter of myogenin, an essential early gene. Here we investigated the requirement for Prmt5 and the class I arginine methyltransferase Carm1/Prmt4 in the temporal control of myogenesis. Both arginine methyltransferases could bind to and modify histones at late-gene regulatory sequences. However, the two enzymes showed sequential requirements for gene expression. Prmt5 was required for early-gene expression but dispensable for late-gene expression. Carm1/Prmt4 was required for late- but not for early-gene expression. The reason for the requirement for Carm1/Prmt4 at late genes was to facilitate SWI/SNF chromatin-remodeling enzyme interaction and remodeling at late-gene loci. Thus, distinct arginine methyltransferases are employed at different times of skeletal muscle differentiation for the purpose of facilitating ATP-dependent chromatin-remodeling enzyme interaction and function at myogenic genes.

Attenuating the function of protein arginine methyltransferases (PRMTs) is an objective for the investigation and treatment of several diseases including cardiovascular disease and cancer. Bisubstrate inhibitors that simultaneously target binding sites for arginine substrate and the cofactor (S-adenosylmethionine (SAM)) have potential utility, but structural information on their binding is required for their development. Evaluation of bisubstrate inhibitors featuring an isosteric guanidine replacement with two prominent enzymes PRMT1 and CARM1 (PRMT4) by isothermal titration calorimetry (ITC), activity assays and crystallography are reported. Key findings are that 2-aminopyridine is a viable replacement for guanidine, providing an inhibitor that binds more strongly to CARM1 than PRMT1. Moreover, a residue around the active site that differs between CARM1 (Asn-265) and PRMT1 (Tyr-160) is identified that affects the side chain conformation of the catalytically important neighbouring glutamate in the crystal structures. Mutagenesis data supports its contribution to the difference in binding observed for this inhibitor. Structures of CARM1 in complex with a range of seven inhibitors reveal the binding modes and show that inhibitors with an amino acid terminus adopt a single conformation whereas the electron density for equivalent amine-bearing inhibitors is consistent with preferential binding in two conformations. These findings inform the molecular basis of CARM1 ligand binding and identify differences between CARM1 and PRMT1 that can inform drug discovery efforts.

Coactivator-associated arginine methyltransferase (CARM1/PRMT4)–mediated transcriptional coactivation and arginine methylation is known to regulate various tissue-specific differentiation events. Although CARM1 is expressed in the neural crest region in early development, coinciding with early neuronal progenitor specification, the role of CARM1 in any neuronal developmental pathways has been unexplored. Using a specific small-molecule inhibitor of CARM1-mediated H3R17 methylation in human embryonic stem cell line, we find that H3R17 methylation contributes to the maintenance of the astroglial cell population. A network of regulation was observed on the miR92a promoter by which H3R17-responsive Nanog bound to the miR92a promoter decreased upon inhibition, resulting in an abnormal gene expression program influencing the glial lineage. This was also true in zebrafish, in which, with the help of CARM1 inhibitor and CARM1 morpholinos, we show that inhibition of H3R17 methylation results in defective glial cell morphology and a sensory defect in a subpopulation. A gain-of-function strategy in which mCARM1 was introduced in the morpholino-treated embryos exhibited recovery of the sensory defect phenotype. This study thus establishes the functional cooperation between arginine methylation and microRNA expression in the neuronal developmental process, with potential implications in sensory development pathways.

Abstract Abstract 549 RUNX1 (also known as AML1) is the DNA binding component of the Core Binding Factor (CBF)-transcriptional regulatory complex, which plays an important role in hematopoiesis. Upon binding to the common binding sequence -PyGpyGGTPy (Py = pyrimidine) in the regulatory regions of promoters and enhancers of its target genes, RUNX1 acts either as an activator or a repressor, depending on promoter context and its interacting partners. Thus, modulation of the network of RUNX1 interactions can influence hematopoiesis. However, how RUNX1 selects one set of partners over another to assemble a functional complex is largely unknown. Posttranslational modifications, including ubiquitination, phosphorylation, acetylation and methylation, present a viable mean to fine-tune its functions. Here we shown that RUNX1 is arginine methylated at a specific residue, R223, by PRMT4, a type I arginine methyltransferase generally thought of as a co-activator molecule. We hypothesized that arginine methylation of RUNX1 by PRMT4 affects its protein-protein interactions, therefore, to identify proteins that specifically interact with unmethylated and/or methylated-R223 RUNX1, in an unbiased manner, we performed a peptide pull-down experiment, using a methyl-R223 RUNX1 peptide and an unmodified RUNX1 peptide as bait, following by mass spectrometry analysis. We identified several proteins that preferentially interacted with the R223 methyl peptide, but focused on a novel interacting protein, DPF2 (double PhD Finger 2), which is a widely expressed member of the d4 protein family, characterized by the presence of a tandem plant-homodomain (PHD domain). We confirmed the specific interaction between methylated-RUNX1 with DPF2 in vivo by immunoprecipitation. We generated an antibody specific for the R223 methylated-RUNX1 protein, and found that RUNX1 methylation decreases during the myeloid differentiation of human CD34+ haematopoietic stem/progenitor cells (HSPCs), without a change in the total level of RUNX1 protein, and this occurred co-incident with a downregulation of PRMT4 protein expression. Having determined that PRMT4 expression declines during myeloid differentiation, we examined the role of PRMT4 in this process, using short hairpin RNAs to knockdown PRMT4 expression in CD34+ cells. Knockdown of PRMT4 accelerates the myeloid differentiation of the cells, whereas overexpression of PRMT4 in human CD34+ cells blocked their myeloid differentiation. When analyzing the expression of several “master” regulators of myeloid differentiation, we identified microRNA-223, a myeloid specific microRNA, as a common target gene of PRMT4 and RUNX1. Furthermore, we have found that by promoting the assembly of a functional complex containing R223 methylRUNX1 and DPF2 at the transcriptional regulatory region of the microRNA-223 promoter, PRMT4 can control miR-223 expression and myeloid differentiation. We have verified the role of DPF2 in this process, as DPF2 represses miR-223 expression and loss of DPF2 promotes myeloid differentiation. Thus, DPF2 acts in a common pathway with PRMT4 to regulate myeloid differentiation. In conclusion, our study elucidates a novel mechanism, where the arginine methylation of RUNX1 regulates its recruitment of interacting partner(s). In addition to demonstrating that PRMT4 can trigger repression of gene expression, we have identified a novel role for PRMT4 (aka CARM1) in myeloid differentiation. Disclosures: No relevant conflicts of interest to declare.

Although “histone” methyltransferases and demethylases are well established to regulate transcriptional programs and to use nonhistone proteins as substrates, their possible roles in regulation of heat-shock proteins in the nucleus have not been investigated. Here, we report that a highly conserved arginine residue, R469, in HSP70 (heat-shock protein of 70 kDa) proteins, an evolutionarily conserved protein family of ATP-dependent molecular chaperone, was monomethylated (me1), at least partially, by coactivator-associated arginine methyltransferase 1/protein arginine methyltransferase 4 (CARM1/PRMT4) and demethylated by jumonji-domain–containing 6 (JMJD6), both in vitro and in cultured cells. Functional studies revealed that HSP70 could directly regulate retinoid acid (RA)-induced retinoid acid receptor β2 (RARβ2) gene transcription through its binding to chromatin, with R469me1 being essential in this process. HSP70’s function in gene transcriptional regulation appears to be distinct from its protein chaperon activity. R469me1 was shown to mediate the interaction between HSP70 and TFIIH, which involves in RNA polymerase II phosphorylation and thus transcriptional initiation. Our findings expand the repertoire of nonhistone substrates targeted by PRMT4 and JMJD6, and reveal a new function of HSP70 proteins in gene transcription at the chromatin level aside from its classic role in protein folding and quality control.

Abstract Small molecule protein arginine methyltransferase inhibitors (PRMTi) are being actively pursued for the treatment of a variety of cancers; however, the mechanisms of response to PRMTi remain poorly understood. CARM1, also known as PRMT4, is significantly overexpressed in AML, as well as many solid tumors, and regulates myeloid differentiation. We have shown the dependency of AML cells, but not normal blood cells, on CARM1 activity, based on CARM1 knockout, CARM1 knockdown, and chemical inhibition (Greenblatt et al. Cancer Cell 2018). These experiments showed that CARM1 regulates essential processes in leukemia cells, and is critical for leukemic transformation. Although several small molecule inhibitors of CARM1 have been reported recently, many display a lack of selectivity for CARM1 or fail to produce a biological response. The recent discovery of potent and selective CARM1 inhibitors (Drew et al., 2017), has made it possible to investigate the implications of pharmacological inhibition of CARM1 in vitro and in vivo. In vitro, a selective CARM1 inhibitor, EPZ025654, reduced the methylation of a CARM1 substrate, BAF155, in a time and concentration-dependent manner, while the specific histone targets of CARM1 remained unchanged. Translocation (8;21) AML samples in the Eastern Cooperative Oncology Group cohort, have significantly higher CARM1 expression compared to normal CD34+ controls. This led us to hypothesize that CARM1 is a direct target of the AML1-ETO fusion protein. Therefore, we assessed whether EPZ025654 could inhibit AML1-ETO driven gene expression. AML1-ETO specific target genes showed significant changes in expression following EPZ025654 treatment. AML1-ETO positive patient samples also displayed decreased colony formation in methylcellulose and increased myeloid differentiation in response to CARM1 inhibition. We next evaluated EZM2302, a compound structurally related to EPZ025654, that is highly orally bioavailable and is well tolerated in mice (Drew et al., 2017). We generated AE9a-GFP primary transplantation mice and treated them with 100 mg/kg of EZM2302 or vehicle twice-daily (BID). The inhibitor treated mice showed significantly improved survival as well as fewer GFP+ cells in the peripheral blood over time. GFP+ AE9a bone marrow cells also showed decreased colony formation in vitro and induced macrophage differentiation in methylcellulose. GFP+ cells were isolated by FACS and submitted for RNA-sequencing. Flow cytometry analysis post-treatment revealed a significant downregulation of c-Kit and increased differentiation of hematopoietic stem and progenitor cells. Resistance to epigenetic targeted therapeutics has been observed, often through the induction of kinase signaling pathways. Therefore, we explored synergistic combinations with CARM1 inhibition using RNA-sequencing and proteomics analysis in leukemia cell lines. We used L1000 profiling (Subramanian et al., 2017) to simultaneously profile the transcriptional response of 18 AML cell line and CD34+ cells after 6 days of treatment. The AML1-ETO positive cell lines exhibited an IC50 in the 0.4-3 μM range, while CD34+ cells and several AML cell lines appeared to be resistant to CARM1 inhibition. While gene expression changes resulting from alterations in RNA stability were observed, the most significant differences between sensitive and resistant cell lines were genes associated with the regulation of cell cycle progression. Gene expression changes were evaluated over time in an AML1-ETO positive cell line, SKNO-1. SKNO-1 cell lines showed an upregulation of a gene expression signature associated with PI3K/AKT/mTOR signaling, with the most significant gene expression changes occurring 7-14 days post treatment. We simultaneously profiled these cells using multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in response to CARM1 inhibition over time. A comparison of this response to chemical perturbation signatures in the L1000 database, identified several chemical inhibitors of the PI3K/AKT/mTOR axis that could reverse the gene expression changes induced by CARM1 inhibition. This finding elucidated a response mechanism for CARM inhibition and a synergistic therapeutic strategy that has the potential to improve CARM1 directed therapy. Disclosures No relevant conflicts of interest to declare.

Les enzymes de remodelage et de modification de la chromatine jouent un rôle majeur dans de nombreux processus biologiques et sont notamment primordiales au cours de la régulation de la transcription. L'arginine méthyltransférase CARM1/PRMT4 méthyle l'histone H3 ainsi que d'autres protéines et joue un rôle clé dans l'activation de la transcription dépendante des récepteurs nucléaires. Nous avons mis en évidence l'implication de CARM1 dans la régulation de la transcription dépendante d'autres facteurs de transcription tels que E2F-1 ou le transactivateur CIITA. Enfin, nos résultats indiquent que CARM1 régule également l'expression de gènes cibles du facteur de transcription c-Fos à un niveau transcriptionnel mais aussi post-transcriptionnel. Ainsi, nos résultats suggèrent un rôle majeur de CARM1 dans la régulation de l'expression génique dans différentes voies de signalisation
Chromatin remodelling and modifying enzymes play a major role in numerous biological processes including transcription regulation. The arginine methyltransfearse CARM1/PRMT4 methylates specifically histone H3 N-terminal tail as well as others proteins and plays a key role in nuclear receptors-dependent transcriptional regulation. Here we show that CARM1 is implicated in transcriptional regulation dependent upon others transcription factors, such as E2F-1 or the CIITA transactivator. Finally, we demonstrate that CARM1 also regulates c-Fos-dependent gene expression both at the transcriptional and post-transcriptional level. Hence, our results suggest a role for CARM1 in gene expression regulation implicated in different pathways

La méthylation des arginines est une modification post-traductionnelle catalysée par les protéines arginine-méthyltransférases (PRMT1-9). En méthylant leurs substrats, les PRMTs régulent les interactions protéines-protéines et protéines-acides nucléiques et sont impliquées dans diverses fonctions cellulaires telles que l’expression des gènes, le métabolisme des ARNs ou la réparation de l’ADN. Contrairement aux autres PRMTs, PRMT4 ou CARM1 (Co-activator Associated Arginine Methyltransferase 1), méthyle préférentiellement des arginines proches de prolines. CARM1 est surexprimée dans les cancers du sein par rapport aux tissus mammaires normaux et est localisée dans le noyau et dans le cytoplasme des cellules tumorales. Bien que ses fonctions nucléaires aient été largement étudiées, ses fonctions cytoplasmiques restent peu décrites. L’immunoprécipitation de CARM1, couplée à une analyse par spectrométrie de masse, a permis d’identifier ses partenaires dans plusieurs lignées cellulaires de cancer du sein et dans des cellules HeLa. Parmi ceux-ci, plusieurs sont impliqués dans le trafic vésiculaire, et nous avons confirmé l’interaction de CARM1 avec deux protéines cytoplasmiques : ArfGAP3 (protéine GTPase-activatrice de facteur ADP-ribosylation 3), et ALIX (protéine X interagissant avec ALG-2), une protéine accessoire des complexes de tri endosomaux nécessaires au transport (ESCRT) impliquée dans des processus de remodelage membranaire tels que la formation de corps multivésiculaires et la cytokinèse. Nous avons démontré que le domaine catalytique de CARM1 se lie au domaine riche en proline (PRD) d'ALIX et identifié les résidus clés impliqués dans cette interaction par microscopie électronique cryogénique. CARM1 méthyle in vitro et in vivo deux arginines du PRD d'ALIX et nous avons ensuite exploré les conséquences fonctionnelles de cette méthylation. La cytokinèse est la dernière étape de la division cellulaire, permettant la séparation physique des cellules filles. Au cours de ce processus, les cellules en division sont connectées par un pont intercellulaire, au milieu duquel une plateforme appelée "midbody" facilite le recrutement des protéines nécessaires à l'abscission, y compris ALIX et la machinerie ESCRT. Des analyses protéomiques de midbodies libérés dans le milieu extracellulaire après abscission ont identifié CARM1 dans cette structure, suggérant que CARM1 pourrait réguler la dernière étape de la division cellulaire. Nous avons démontré que CARM1 joue un rôle essentiel au cours de la cytokinèse, puisque sa déplétion, de manière similaire à celle d'ALIX, retarde la coupure des microtubules dans le pont intercellulaire —étape critique précédant l'abscission — et induit la formation de cellules multinucléées. Nous avons montré par une approche de pulldown peptidique que la méthylation d'ALIX empêche son interaction avec plusieurs partenaires impliqués dans la cytokinèse, dont CD2AP, les protéines CAPZ de coiffe de l’actine, et l’endophiline-A2. Un mutant d'ALIX incapable de lier ces protéines conduit à la formation de cellules multinucléées, mettant en évidence le rôle essentiel de ces interactions dans le déroulement normal de la cytokinèse. De plus, nos résultats indiquent que CARM1 régule la transcription de gènes codant pour des protéines impliquées dans la cytokinèse, ce qui suggère que CARM1 pourrait contrôler l’étape finale de la division cellulaire par le biais de plusieurs mécanismes moléculaires. En conclusion, cette étude révèle de nouvelles fonctions cytoplasmiques de CARM1, notamment dans le trafic vésiculaire et les processus de remodelage membranaire, ainsi qu’une nouvelle modification post-traductionnelle permettant de contrôler la cytokinèse. Ces travaux ouvrent aussi de nouvelles perspectives de recherche sur le rôle du complexe CARM1-ALIX dans d'autres fonctions cellulaires, telles que le recyclage des récepteurs, la réparation membranaire, le bourgeonnement des virus, et la biogenèse des exosomes
Arginine methylation is a post-translational modification catalyzed by the 9 members of the protein arginine methyltransferase family (PRMT1-9). By adding methyl marks on arginines, PRMTs regulate their substrates' ability to bind to other proteins or nucleic acids, thereby modulating various biological processes such as gene expression, RNA metabolism or DNA damage response. Contrary to the other PRMTs, PRMT4, also known as CARM1 (Co-activator Associated aRginine Methyltransferase 1), specifically methylates arginines within proline-rich motifs. CARM1 is over-expressed in breast tumors compared to normal breast tissue and is localized both in the nucleus and in the cytoplasm of tumors cells. While the nuclear substrates and functions of CARM1 have been well described and have underscored its critical role in the nucleus, its cytoplasmic functions remain largely uncharacterized. Therefore, we investigated the CARM1 interactome by immunoprecipitating CARM1 from several breast cancer cell and HeLa cell lysates followed by mass-spectrometry analysis, and retrieved several proteins involved in vesicular trafficking, including endocytosis, vesicle transport and sorting. We showed that CARM1 interacts with two cytoplasmic proteins: ArfGAP3 (ADP-ribosylation factor GTPase-activating protein 3), a crucial modulator of vesicular trafficking, and ALIX (ALG-2 interacting protein X), an accessory protein of the Endosomal Sorting Complexes Required for Transport (ESCRT) that is involved in membrane remodeling processes such as virus budding, multivesicular body formation and cytokinesis. We demonstrated that the catalytic domain of CARM1 binds to the C-terminus proline-rich domain (PRD) of ALIX and identified the key residues involved in the interaction by cryogenic electron microscopy. We showed that CARM1 methylates two arginine residues in ALIX PRD in vitro and in cells, and we explored the functional consequence of ALIX methylation in its cellular functions. Cytokinesis is the final step of cell division leading to the physical separation of the daughter cells. During this process, the dividing cells are connected by a thin intercellular bridge, within which a platform called “midbody” facilitates the recruitment of proteins required for the abscission, including ALIX and the ESCRT machinery. Several proteomic analyses of midbody remnants, released in the extracellular medium following abscission, identified CARM1 in this structure, suggesting that CARM1 may regulate the last step of cell division. We demonstrated that CARM1 was essential during cytokinesis, as its depletion delayed the severing of microtubules in the intercellular bridge - a critical step prior to abscission - and induced the formation of multinucleated cells, mirroring the effects observed upon ALIX depletion. Mechanistically, we showed by a peptide pulldown approach that ALIX methylation impaired its interaction with several partners involved in cytokinesis, including CD2AP, CIN85, capping proteins and endophilin-A2. Further, a mutant of ALIX that is unable to bind these proteins promoted the formation of multinucleated cells, highlighting the requirement for ALIX to bind to these partners, for proper cytokinesis. Additionally, CARM1 regulated the transcription of genes coding for proteins involved in cytokinesis, suggesting that CARM1 could control the last step of cell division through several molecular mechanisms. Altogether, this study uncovered new cytoplasmic functions of CARM1 in vesicular trafficking and membrane remodeling processes and unveiled a novel post-translational modification controlling cytokinesis. This work also opens new areas of research regarding the relevance of CARM1-mediated ALIX methylation in other ALIX-dependent cellular functions such as receptor recycling, membrane repair, virus budding, and exosome biogenesis

Les protéines arginine méthyl transférases ("PRMTs") sont impliquées dans de nombreux processus cellulaires essentiels. La protéine CARMI ("Coactivator-associated arginine methyltransferase 1", appelée aussi "PRMT4") a été initialement identifiée par sa fonction co-activatrice de la transcription impliquantplusieurs récepteurs nucléaires des hormones. CARMI est une enzyme qui catalyse la réaction de méthylation sur les histones via un donneur de méthyl naturel, la S-adénosY-L -méthionine (SAM). De nombreux travaux ont montré que CARMI est surexprimée dans les cancers du sein et de la prostate. L' objectif de ce travail est la compréhension à l'échelle moléculaire du mode d'action de CARMI et l'étude du mécanisme de reconnaissances moléculaires et de transferts d' informations gouvernés par la protéine CARMI. La structure cristallographique obtenue de cette enzyme en présence de cofacteur, la S-AdénosyhHomocystéine ou la Sinefungine a eu un effet stabilisant. Ainsi, notre stratégie a été de créer des molécules hameçons basées sur le motif de la SAM capables d' ancrer un peptide mimant la séquence de l' histone H3, pour ensuite les tester en co-cristallisation avec CARMI. Ainsi, grâce à la diffraction aux rayons X, les interactions mises en jeu dans le complexe CARMlImolécules hameçons/peptide pourront être déterminées. Cette stratégie s'est effectuée en trois étapes : la première étape, décrite dans le chapitre 2, a consisté en la synthèse d'analogues de la SAM obtenus grâce à des modifications réalisées autour de l'atome de soufre. Ces composés nous ont permis d' explorer la " poche du sulfonium ". Puis la seconde étape, décrite dans le chapitre 3, a été la synthèse * d'analogues de bisubstrats nécessaires pour l'exploration de la " poche de l'arginine ". Dans une dernière étape, décrite dans les chapitres 4 et 5, nous avons abordé la synthèse d'adduits SAM-peptide pouf pouvoir étudier le " domaine de fixation du peptide ". Dans le quatrième chapitre, la méthode de choix est la création d'un lien covalent entre une molécule hameçon électrophile etun peptide par chimie de click in-situ : par réaction de cycloaddition de Huisgen; par réaction entre des molécules hameçons électrophiles capables de piéger un peptide cystéine ou un peptide arginine. Ces essais se sont révélés infructueux et une nouvelle stratégie a été employée en utilisant des molécules ancres. Dansle cinquième chapitre des molécules ancres ont donc été préformés pour ensuite être testés en cocristallisation dans CARMl.

p300/CBP is involved in the expression of a wide range of genes, both as a histone acetyltransferase (HAT) and as a coactivator of transcription factors. p300/CBP is the specific substrate of CARM1, and its KIX domain and GBD domain are the main sites methylated by arginine methyltransferase 4 (PRMT4/CARM1). p300/CBP plays an important role in lung cancer, which is a cell cycle disease. More importantly, the methylation of p300/CBP by CARM1 affects the progression of lung cancer through the cAMP-PKA pathway, p53 pathway and ER pathway. The structure, function, methylation modification sites, methylation-related enzymes, genes associated with lung cancer and the possible mechanisms of p300/CBP action are reviewed.