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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): August 31, 2021
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator: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
(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)
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.

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