The TOR pathway is the master controller of metabolism, proliferation and survival in response to nutrients, energy status, growth factors and hormones. In our lab we study how this highly conserved pathway conveys extracellular signals to chromatin regulators and transcription factors to ultimately elicit an adequate transcriptional response (Martin et al., 2017, Curr Bio). We have found that transcriptional co-repressors belonging to the Sin3/HDAC I family and members of the Tup/TLE/Grg groucho family control the expression of genes induced upon TOR inhibition and that they physically interact with members of the TOR-PP2A pathway. However, the underlying molecular mechanisms behind their regulation by the TOR pathway requires further investigation. Based on this premises, this project aims at delving into the mechanistic link between TOR signaling and these chromatin factors. The project will entail: (1) the identification of post-transcriptional modifications affecting these factors in response to changes in TOR activity and (2) the generation of specific mutants (lacking the modified residues identified in the first aim) and their phenotypic and genomic characterization.
Finding direct targets of signaling pathways in chromatin control is important to understand how the environment dictates cell-fate decisions in healthy cells and how this could be targeted in disease.
The master student that will participate in this project will learn the following techniques:
- Cell culturing and manipulation of the yeast model organism: Schizosaccharomyces pombe
- In vitro cell culturing of mammalian cells (breast cancer cell lines MCF7, BT474 and MDA-MB-231)
- Genetic tools for creating mutants and gene modifications (genetic crosses, transformation, RNAi, CRISPR/Cas9)
- Protein purification (IP) and detection (WB)
- RNA extraction and expression analysis (RT-PCR)
- Chromatin Immunopurification (ChIP-PCR)
- live-cell imaging