How do RIF1 and Protein Phosphatase 1 regulate chromatin phosphorylation for genome maintenance?

Supervisors: Shin-ichiro Hiraga, Tony Ly (Edinburgh, Wellcome Centre for Cell Biology,  Institute of Cell Biology), Anne Donaldson

Project description:

Background and Introduction
Genomic mutations and rearrangements cause numerous diseases including cancer and birth defects.  Cells theregore deploy multiple DNA repair mechanisms to fix damaged DNA and maintain chromosomes stability throughout successive cell cycles. The RIF1 protein controls various chromosome maintenance pathways to preserve genome stability.  In particular RIF1 controls the cell's choice between two major damage repair pathways, homology-mediated repair (HR) and non-homologous end joining (NHEJ).  The molecular mechanism through which RIF1 regulates these pathways is currently unclear [1]: we suspect however that RIF1 ensures accurate chromosome maintenance by directing dephosphorylation of DNA repair proteins, as explained below.

Recently we discovered that the RIF1 protein controls DNA replication by acting as a substrate-targeting subunit for Protein Phosphatase 1 (PP1), with RIF1 directing PP1-mediated dephosphorylation of DNA replication components [2-3].  Our discovery raises the possibility that RIF1 also regulates DNA repair by directing dephosphorylation of specific targets.  A preliminary phospho-proteomic analysis by Drs. Hiraga and Ly suggests that RIF1 does indeed control the phosphorylation of multiple proteins implicated in damage response and damage repair (unpublished).  In particular, we found that RIF1 affects the phosphorylation of BRCA1 and 53BP1, master regulators of HR and NHEJ pathway choice.

Project

The aim of this project is to uncover the molecular mechanisms through which RIF1 ensures chromosome maintenance, by identifying and dissecting targets for dephosphorylation by RIF1-PP1.  This PhD offers an outstanding training opportunity with enthusiastic new group leaders in Aberdeen and Edinburgh. The student will address the following research questions:

1. Which chromatin proteins are dephosphorylated by RIF1-PP1 during DNA damage?

Substrates dephosphorylated by RIF1-PP1 will show increased phosphorylation when RIF1 is absent.  To identify proteins that mediate the effects of RIF1 following DNA damage, the phosphorylation status of chromatin proteins from damaged cells with or without RIF1 will be analysed.  The student will compare chromatin proteins from Control and RIF1-depleted cells treated with Bleomycin, which stimulates DNA damage.  Specifically, we will undertake a four-way quantitative proteomic comparison of chromatin isolated from cells with and without RIF1 and with and without Bleomycin, based on Tandem Mass Tag-based labelling of chromatin samples followed by high-resolution mass spectrometry.  These experiments will identify proteins whose phosphorylation levels change in response to DNA damage, pinpointing those being controlled by RIF1-PP1.

2. What is the importance of phosphorylation in response to replication stress?

The analysis in Part 1 will identify proteins whose phosphorylation is controlled by RIF1, and the specific phosphorylation sites affected.  Candidate targets will be chosen for further analysis based on known protein function and behaviour, including cell cycle localisation and involvement in DNA damage control.  For example, we expect that manipulating RIF1-controlled phosphorylation sites on the BRCA1 protein will affect whether the cell uses HR or NHEJ for subsequent damage repair.  We will utilise CRISPR/Cas9-based genome engineering to abolish or mimic particular phosphorylations, and assess the downstream effects.  Established repair assays (based on flow cytometry and microscopy) will be used to test repair pathway choice following DNA damage and the consequent effects on genome stability.

References:

[1] Kumar and Cheok 2014.  DNA Repair 15: 54-69.

[2] Hiraga et al. 2014.  Genes & Development 28: 372-383.

[3] Hiraga et al. 2017.  EMBO Reports 18: 403-419.

 

Other: