Full text
| |
| Author(s): |
Francisca Nathália de Luna Vitorino
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2022-04-06 |
| Examining board members: |
Júlia Pinheiro Chagas da Cunha;
Erico Tosoni Costa;
Adriana Franco Paes Leme;
Carla Columbano de Oliveira;
Patrícia Severino;
Mari Cleide Sogayar
|
| Advisor: | Júlia Pinheiro Chagas da Cunha |
| Abstract | |
Fibroblast growth factor 2 (FGF2), despite inducing cell proliferation in several contexts, irreversibly inhibits the proliferation of tumor cells with the k-ras oncogene overexpressed, as in Y1 cells, in the G2/M transition of the cell cycle. To better understand the molecular mechanism induced by this factor, we aim to understand how the cell\'s proteome is responding to this stimulus. We proposed to analyze by quantitative proteomics the alterations induced by FGF2, in the total extract, in the chromatin, nucleolus and in the rDNA loci. In the total proteome, more than 2900 proteins were quantified, indicating that the terms associated with metabolism, RNA processing, replication and transcription are enriched among the differentially expressed proteins in the FGF2 stimulus. Analysis of the transcriptional regulatory network indicates members of AP-1 (mainly FosB) as major regulators of FGF2 signaling. JunB expression starts after 1 h of FGF2 stimulation and peaks after 5 h, FosB expression after 3 h to 24 h, peaking at 3 h to 8 h. Both levels of expression of these proteins depend on fibroblast growth factor receptor (FGFR) and src signaling. JUNB and FOSB knockdown does not rescue cells from FGF2-induced growth arrest; however, FOSB knockdown rescues cells from delayed DNA replication, indicating that FOSB expression underlies the delay in S-phase progression. In the chromatin proteome, more than 5000 proteins have been identified, with more than 300 proteins downregulated after stimulation with FGF2 for 24 h, including several proteins associated with RNA polymerase II. A modulation of global transcription was demonstrated by stimulation with FGF2, through a run-on assay, showing opposite effects in short and long stimulation times. In the nucleolar proteome, more than 1200 proteins were identified, indicating that terms associated with transcription, rRNA processing and ribosome biogenesis are enriched among the proteins differentially expressed by FGF2. Several proteins belonging to chromatin remodeling complexes are differentially expressed in the nucleolus after stimulation with FGF2, suggesting a modulation of the transcriptional state of rDNA. An increase in immature rRNA transcripts was identified after stimulation with FGF2 for 24 h, which may be an indication of increased transcription or RNA Polymerase I activity. However, mature ribosomes seem not to be affected and there is nucleolar disorganization, observed through the dispersion of the fibrillarin label, after stimulation with FGF2. In the rDNA loci proteome, 556 proteins were identified, 27 of which were differentially expressed after stimulation with FGF2. Among these proteins, we highlight the Nolc1 and Tcof1 proteins, which, among other functions, participate in the processing of rRNA, mainly regarding post-transcriptional modifications of rRNA, and modulation of ribosomes. Taken together, these results demonstrate that the antiproliferative stimulus of FGF2 triggers important transcriptional changes, from the overactivation of immediate response genes, to the modulation of the main cell transcription site, the nucleolus, directly modulating rDNA. (AU) | |
| FAPESP's process: | 17/18344-9 - Quantitative chromatin proteomics upon FGF2 treatment: analysis of transcriptional regulation and involvement of cdc42 |
| Grantee: | Francisca Nathália de Luna Vitorino |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |