DCas9-targeted rDNA loci-specific protein isolation of tumor cells stimulated with FGF2

Abstract

Despite being a growth factor, FGF2 has anti-proliferative and tumor suppressive functions in some cellular contexts. In Y1 murine adrenocortical carcinoma cell line, the FGF2 promotes G0-G1 transition but delays S-phase and permanently blocks cells in G2/M. To better understand the chromatin response to proliferative and anti-proliferative stimuli induced, respectively, by Fetal Bovine Serum (FBS) and FGF2, we performed mass spectrometry-based quantitative proteomics (Label-free) focusing on chromatin-associated proteins. We found global transcription alterations (by run-on assays), nucleolar disorganization (by fibrillarin immunofluorescence and transmission electron microscopy assays), and pre- rRNA accumulation (by northern blot assays) after 1, 5, and 24 h upon FGF2 treatment. The rRNA processing and transcription are largely studied however, due to technical limitations, the proteomics content of rDNA locus have not yet been explored. Here, we propose to identify the proteins associated to these loci by using the CLASP (Cas9 locus-associated proteome) technique, a cutting-edge technology developed in collaboration with Dr. Washburn's group. The identification of the protein content at the rDNA loci will be explored in the context of FGF2 stimuli in a mice tumor cell line and a human cell (Y1 and HEK293, respectively) expressing inducible oncogenic HRAS (HEK/ER:H-rasV12). These data could indicate important mechanisms held at nucleolus by which FGF2 induces cell cycle arrest and also the unexplored proteome of rDNA loci possibly identifying new players of the intricate biology of nucleolus/rDNA loci. (AU)