RESUMEN
Mutations in chromatin regulators are widespread in cancer. Among them, the histone H3 lysine 27 methyltransferase Polycomb Repressive Complex 2 (PRC2) shows distinct alterations according to tumor type. This specificity is poorly understood. Here, we model several PRC2 alterations in one isogenic system to reveal their comparative effects. Focusing then on lymphoma-associated EZH2 mutations, we show that Ezh2Y641F induces aberrant H3K27 methylation patterns even without wild-type Ezh2, which are alleviated by partial PRC2 inhibition. Remarkably, Ezh2Y641F rewires the response to PRC2 inhibition, leading to induction of antigen presentation genes. Using a unique longitudinal follicular lymphoma cohort, we further link EZH2 status to abnormal H3K27 methylation. We also uncover unexpected variability in the mutational landscape of successive biopsies, pointing to frequent co-existence of different clones and cautioning against stratifying patients based on single sampling. Our results clarify how oncogenic PRC2 mutations disrupt chromatin and transcription, and the therapeutic vulnerabilities this creates.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2 , Histonas , Linfoma Folicular , Mutación , Complejo Represivo Polycomb 2 , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Humanos , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Histonas/metabolismo , Histonas/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Metilación , Cromatina/metabolismo , Cromatina/genética , Transcripción GenéticaRESUMEN
Identification of histone lysine methyltransferase (HKMT) substrates has recently benefited from chemical-biology-based strategies in which artificial S-adenosyl-L-methionine (SAM) cofactors are engineered to allow substrate labeling using either the wild-type target enzyme or designed mutants. Once labeled, substrates can be selectively functionalized with an affinity tag, using a bioorthogonal ligation reaction, to allow their recovery from cell extracts and subsequent identification. In this chapter, we describe steps on how to proceed to set up such an approach to characterize substrates of specific HKMTs of the SET domain superfamily, from the characterization of the HKMT able to accommodate a SAM surrogate containing a bioorthogonal moiety, to the proteomic analysis conducted on a cell extract. We focus in particular on the controls that are necessary to ensure reliable proteomic data analysis. The example of PR-Set7 on which we have implemented this approach is shown.
Asunto(s)
Metionina , S-Adenosilmetionina , N-Metiltransferasa de Histona-Lisina/química , Dominios PR-SET , Proteómica , S-Adenosilmetionina/químicaRESUMEN
The polycomb repressive complex 2 (PRC2) consists of core subunits SUZ12, EED, RBBP4/7, and EZH1/2 and is responsible for mono-, di-, and tri-methylation of lysine 27 on histone H3. Whereas two distinct forms exist, PRC2.1 (containing one polycomb-like protein) and PRC2.2 (containing AEBP2 and JARID2), little is known about their differential functions. Here, we report the discovery of a family of vertebrate-specific PRC2.1 proteins, "PRC2 associated LCOR isoform 1" (PALI1) and PALI2, encoded by the LCOR and LCORL gene loci, respectively. PALI1 promotes PRC2 methyltransferase activity in vitro and in vivo and is essential for mouse development. Pali1 and Aebp2 define mutually exclusive, antagonistic PRC2 subtypes that exhibit divergent H3K27-tri-methylation activities. The balance of these PRC2.1/PRC2.2 activities is required for the appropriate regulation of polycomb target genes during differentiation. PALI1/2 potentially link polycombs with transcriptional co-repressors in the regulation of cellular identity during development and in cancer.
Asunto(s)
Complejo Represivo Polycomb 2/genética , Proteínas Represoras/genética , Vertebrados/genética , Secuencia de Aminoácidos , Animales , Diferenciación Celular/genética , Línea Celular , Células HEK293 , Histonas/genética , Humanos , Metilación , Metiltransferasas/genética , Ratones , Neoplasias/genética , Alineación de SecuenciaRESUMEN
MicroRNAs are small regulatory RNAs with many biological functions and disease associations. We showed that in situ hybridization (ISH) using conventional formaldehyde fixation results in substantial microRNA loss from mouse tissue sections, which can be prevented by fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide that irreversibly immobilizes the microRNA at its 5' phosphate. We determined optimal hybridization parameters for 130 locked nucleic acid probes by recording nucleic acid melting temperature during ISH.