How does the chromosome maintenance factor Rif1 control centromere function?

Supervisors: Dr Shin-ichiro HiragaProfessor Adèle MarstonProfessor Anne Donaldson

Project Description:

Rif1 protein is an evolutionarily conserved protein with multiple roles in chromosome maintenance. It controls DNA replication origin licensing and initiation, choice of repair mechanism for broken DNA, and resolution of entangled chromosomes at mitosis [Fontana et al. 2018].  We and others showed that Rif1 directs Protein Phosphatase 1 (PP1) to dephosphorylate specific substrates, but we do not fully understand which of its functions Rif1 executes through this mechanism.  Our very recent chromosomal binding site analysis showed that budding yeast Rif1 binds directly to DNA replication origins and stalled replication forks [Hiraga et al. 2018].  Unexpectedly, we also found Rif1 associates with centromeres, particularly during S phase of the cell cycle.  The aim of this PhD project is to examine how Rif1 binds centromeres and whether it controls centromere function.   
This PhD position provides an outstanding opportunity to train in two leading labs.  The lab of Dr Hiraga and Prof Donaldson has an established track record in analysing Rif1 chromosome maintenance functions, with cutting-edge investigations of DNA replication and nuclear structure. University of Edinburgh co-supervisor Prof Adele Marston provides complementary expertise as a world leader in centromere biology. 

Research project
This 4-year PhD project will address four main avenues:

1.  Identify structural domain(s) of Rif1 required for centromere association.
We already know that the C-terminal region of Rif1 is needed for centromere association.  We will dissect the precise domain required by testing the association of a series of yeast Rif1 truncated versions with centromeres, by ChIP-Seq analysis.  We will test also test whether checkpoint-mediated phosphorylation mediates Rif1 centromere association.

2.  Identify the mechanism of recruitment of Rif1 to these sites
We will examine how Rif1 is recruited to centromeres, by examining the requirement for centromere sequences and known centromere binding proteins.

3.  Understand how Rif1 affects centromere function
Using established microscopic assays for centromere function, we will investigate the role of Rif1 in centromere function.  We will test the effect of Rif1 removal on the distribution of cohesin and its loader proteins at centromeres and on chromosome arms by ChIP-Seq.  We will also test whether constitutive tethering of Rif1 alters centromere function..

4.  Test if Rif1 function at centromeres requires Protein Phosphatase 1 
By analysing a RIF1 mutant unable to interact with PP1, we will test whether Rif1 function at centromeres involves targeting of PP1 activity.  The Marston lab recently discovered that cohesin loading near centromere is promoted by phosphorylation of the centromeric Ctf19 protein [Hinshaw et al. 2017].  We will directly test the intriguing idea that Rif1-PP1 counteracts this function by dephosphorylating Ctf19, hence regulating the balance of cohesin loading at centromeres and chromosome arms.  Using phosphoproteomics we will test more generally for additional centromeric substrates of RIF1-PP1 dephosphorylation.

Overall this PhD project will provide important insight into a new role for the evolutionarily conserved Rif1 protein.  Since accurate chromosome maintenance is crucial for cancer prevention, the findings will be of significance for understanding and preventing carcinogenesis. 

Fontana et al. 2018. Microb. Cell 5: 327-343
Hiraga et al. 2018. EMBO Rep. e46222
Hinshaw et al. 2017 Cell 172:71-84

If you wish to apply for this project, pleadse go to this link.