A mass spectrometry-based assay using metabolic labeling to rapidly monitor chromatin accessibility of modified histone proteins

Autor(es)

Sidoli, Simone ^{[1, 2]} ; Lopes, Mariana ^{[2, 3]} ; Lund, Peder J. ^[2] ; Goldman, Naomi ^{[4, 5]} ; Fasolino, Maria ^[4] ; Coradin, Mariel ^{[6, 2]} ; Kulej, Katarzyna ^[2] ; Bhanu, V, Natarajan ; Vahedi, Golnaz ^[4] ; Garcia, Benjamin A. ^[7]

Afiliação do(s) autor(es)

^[1] Albert Einstein Coll Med, Dept Biochem, Bronx, NY 10461 - USA

^[2] Univ Penn, Perelman Sch Med, Epigenet Inst, Dept Biochem & Biophys, Philadelphia, PA 19104 - USA

^[3] Inst Butantan, Ctr Toxins Immune Response & Cell Signaling CeT, Lab Especial Ciclo Celular, BR-05503900 Sao Paulo - Brazil

^[4] Univ Penn, Perelman Sch Med, Epigenet Inst, Inst Immunol, Dept Genet, Philadelphia, PA 19104 - USA

^[5] Univ Penn, Perelman Sch Med, Immunol Grad Grp, Philadelphia, PA 19104 - USA

^[6] Univ Penn, Perelman Sch Med, Biochem & Mol Biophys Grad Grp, Philadelphia, PA 19104 - USA

^[7] Bhanu, Natarajan, V, Univ Penn, Perelman Sch Med, Epigenet Inst, Dept Biochem & Biophys, Philadelphia, PA 19104 - USA

• Tipo de documento: Artigo Científico
• Fonte: SCIENTIFIC REPORTS; v. 9, SEP 20 2019.
• Citações Web of Science: 0

Resumo

Histone post-translational modifications (PTMs) contribute to chromatin accessibility due to their chemical properties and their ability to recruit enzymes responsible for DNA readout and chromatin remodeling. To date, more than 400 different histone PTMs and thousands of combinations of PTMs have been identified, the vast majority with still unknown biological function. Identification and quantification of histone PTMs has become routine in mass spectrometry (MS) but, since raising antibodies for each PTM in a study can be prohibitive, lots of potential is lost from MS datasets when uncharacterized PTMs are found to be significantly regulated. We developed an assay that uses metabolic labeling and MS to associate chromatin accessibility with histone PTMs and their combinations. The labeling is achieved by spiking in the cell media a 5x concentration of stable isotope labeled arginine and allow cells to grow for at least one cell cycle. We quantified the labeling incorporation of about 200 histone peptides with a proteomics workflow, and we confirmed that peptides carrying PTMs with extensively characterized roles in active transcription or gene silencing were in highly or poorly labeled forms, respectively. Data were further validated using next-generation sequencing to assess the transcription rate of chromatin regions modified with five selected PTMs. Furthermore, we quantified the labeling rate of peptides carrying co-existing PTMs, proving that this method is suitable for combinatorial PTMs. We focus on the abundant bivalent mark H3K27me3K36me2, showing that H3K27me3 dominantly represses histone swapping rate even in the presence of the more permissive PTM H3K36me2. Together, we envision this method will help to generate hypotheses regarding histone PTM functions and, potentially, elucidate the role of combinatorial histone codes. (AU)

• Processo FAPESP: 17/15835-1 - Integração entre vias de sinalização e modificações pós traducionais em histonas em resposta a fator de crescimento
• Beneficiário: Mariana de Camargo Lopes
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Doutorado Direto