| Grant number: | 17/18344-9 |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |
| Start date: | January 01, 2018 |
| End date: | March 31, 2022 |
| Field of knowledge: | Biological Sciences - Biochemistry |
| Principal Investigator: | Julia Pinheiro Chagas da Cunha |
| Grantee: | Francisca Nathália de Luna Vitorino |
| Host Institution: | Instituto Butantan. 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) | |
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