Quantitative chromatin proteomics upon FGF2 treatment: analysis of transcriptional regulation and involvement of cdc42

• Grant number: 17/18344-9
• Support type: Scholarships in Brazil - Doctorate (Direct)
• Effective date (Start): January 01, 2018
• Effective date (End): March 31, 2022
• Field of knowledge: Biological Sciences - Biochemistry
• Principal researcher: Julia Pinheiro Chagas da Cunha
• Grantee: Francisca Nathália de Luna Vitorino
• Home Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 13/07467-1 - CeTICS - Center of Toxins, Immune-Response and Cell Signaling, AP.CEPID
• Associated scholarship(s): 19/17675-7 - DCas9-targeted rDNA loci-specific protein isolation of tumor cells stimulated with FGF2, BE.EP.DD

Abstract

Chromatin has an important function in essential cellular processes such as mitosis, transcription, replication, repair, among others. Fibroblast Growth Factor 2 (FGF2), despite inducing cell proliferation in several contexts, irreversibly inhibits the proliferation of mouse tumor cells (Y1 cell line) at the G2/M. To better understand the molecular mechanism induced by this factor, our group performed several proteomic analyzes using total or chromatin extracts after FGF2 stimulation. In chromatin we identified about 1660 proteins while 169 of them were found differentially expressed upon FGF2. Several proteins associated with transcriptional regulation were found down regulated whereas the cdc42 protein, a Rho GTPase, was found more abundant. As direct and indirect associations between RhoGTPases and transcriptional regulation are described, particularly through the regulation of Carboxy Terminal Portion (CTD) of RNA Pol II, we intend to further explore the effects of FGF2 on chromatin by modulating transcriptional levels and to investigate whether cdc42 participates in this modulation. To our knowledge, the chromatin localization of this protein is a new finding, thus this project also aims to investigate it in more detail. The transcriptional levels will be evaluated by Run-on (EU and BrUTP) assays using or not specific inhibitors against RNA Polymerases I and II while the CTD phosphorylation status will be evaluated by western blotting assays. The location of cdc42 in chromatin will be assessed by confocal microscopy, ChIP and western blotting assays using different chromatin protocols. Y1 cells expressing constitutive active cdc42 (cdc42-V12) or negative dominant (cdc42-N17) as well as knockdown for cdc42 will be obtained and the transcriptional and phosphorylation levels of CTD will be evaluated. Finally, these cells will be also evaluated regarding resistance or sensitivity to FGF2 stimulation. (AU)

Scientific publications

(4)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

LUND, PEDER J.; LOPES, MARIANA; SIDOLI, SIMONE; CORADIN, MARIEL; DE LUNA VITORINO, FRANCISCA NATHALIA; CHAGAS DA CUNHA, JULIA PINHEIRO; GARCIA, BENJAMIN A. FGF-2 induces a failure of cell cycle progression in cells harboring amplified K-Ras, revealing new insights into oncogene-induced senescence. MOLECULAR OMICS, v. 17, n. 5, p. 725-739, OCT 11 2021. Web of Science Citations: 0.

LONGHI, M. T.; SILVA, L. E.; PEREIRA, M.; MAGALHAES, M.; REINA, J.; VITORINO, F. N. L.; GUMBINER, B. M.; DA CUNHA, J. P. C.; CELLA, N. PI3K-AKT, JAK2-STAT3 pathways and cell-cell contact regulate maspin subcellular localization. CELL COMMUNICATION AND SIGNALING, v. 19, n. 1 AUG 14 2021. Web of Science Citations: 0.

AZEVEDO, HATYLAS; PESSOA, GUILHERME CAVALCANTE; DE LUNA VITORINO, FRANCISCA NATHALIA; NSENGIMANA, JEREMIE; NEWTON-BISHOP, JULIA; REIS, EDUARDO MORAES; DA CUNHA, JULIA PINHEIRO CHAGAS; JASIULIONIS, MIRIAM GALVONAS. Gene co-expression and histone modification signatures are associated with melanoma progression, epithelial-to-mesenchymal transition, and metastasis. CLINICAL EPIGENETICS, v. 12, n. 1 AUG 24 2020. Web of Science Citations: 0.

DE LIMA, LOYZE P.; POUBEL, SALOE BISPO; YUAN, ZUO-FEI; ROSON, JULIANA NUNES; DE LUNA VITORINO, FRANCISCA NATHALIA; HOLETZ, FABIOLA BARBIERI; GARCIA, BENJAMIN A.; CHAGAS DA CUNHA, JULIA PINHEIRO. Improvements on the quantitative analysis of Trypanosoma cruzi histone post translational modifications: Study of changes in epigenetic marks through the parasite's metacyclogenesis and life cycle. JOURNAL OF PROTEOMICS, v. 225, APR 15 2020. Web of Science Citations: 0.