Understanding the spatial distribution of cohesin on chromosomes
Project Details - Understanding the spatial distribution of cohesin on chromosomes
|Supervisor(s): Prof Adele Marston & Dr Laura Spagnolo|
|Centre/Institute: Institute of Cell Biology|
Cohesin is a ring-shaped protein complex that plays key roles in chromosome segregation, DNA repair and gene expression1,2. Cohesin loading onto chromosomes depends on a separate complex, called Scc2/Scc4 in yeast or Nipbl/Mau2 in humans. Importantly, mutations in Nipbl are known to cause a severe developmental disorder in humans, called Cornelia de Lange syndrome, however the molecular basis of the pathology remains unknown.
Recent structural and molecular analysis revealed a conserved patch on the surface of the Scc4/Mau2 component of the cohesin loader3. Mutation of this patch prevented cohesin loading specifically onto centromeres in budding yeast, while cohesin loading elsewhere remained unaffected. Therefore, cohesin loading by Scc2/4 can occur in two modes: targeted to centromeres, which depends on the conserved patch; and general, which does not. Importantly, the identity of cohesin loading sites other than centromeres remain undefined so that the general mode by which cohesin binding sites are established remains to be uncovered. In this project, this fundamental question will be addressed using budding yeast, where established tools and reagents will allow for a detailed dissection of the cohesin loading mechanism.
1. What is the identity of cohesin loading sites on chromosome arms?
To determine the identity of cohesin loading sites that are not centromeres, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) will be used to determine the localization of the Scc2/Scc4 cohesin loader and cohesin on chromosomes in conditions where centromere loading has been abolished. Following recent advances in ChIP-Seq, methods will be developed to use heterologous species to achieve quantifiable ChIP-Seq data. Experimental design and data analysis will be carried out with the assistance and training of the core bioinformatics facility at the Wellcome Trust Centre for Cell Biology.
2. What are the factors required for cohesin loading on chromosome arms?
In a parallel genomics approach, systematic high throughput synthetic lethal screens will be carried out to identify genes that are required for cohesin loading on chromosome arms. A collection of yeast strains (~6000) in which essentially every gene has been mutated individually will be used together to identify synthetic lethal interactions with characterized mutations that affect cohesin loading either specifically at centromeres, or globally. Identified candidates will be characterized in detail using cell biological and biochemical methods.
3. How do the structure and interactions of the cohesin loader differ in the targeted and general cohesin-loading modes?
It is likely that centromere and chromosomal arm-associated cohesin loader complexes dock onto chromosome-bound factors and adopt different conformations. To begin to understand the mechanistic basis of these distinct modes of cohesin loading, the fully functional Scc2/Scc4 cohesin loader, or the version that fails to associate with centromeres, will be purified from yeast cells using established methods4. Alterations in associated proteins will be identified by quantitative mass spectrometry. Ultimately, understanding distinct modes of cohesin loading will require structural analysis. Towards this goal, the student will work in the laboratory of Dr. Laura Spagnolo (second supervisor) to establish the use of electron microscopy in determining the overall conformation of the Scc2/4 cohesin loader. For more information about research in the Marston lab see: http://marston.bio.ed.ac.uk/
The student will receive state-of-the-art training in several generally applicable “wet” lab and computational methods. Several data-rich outputs will be generated, including high throughput sequencing data, genomic screen outcomes, proteomic data requiring quantitative analysis and electron microscopy images. Importantly, available expertise in all techniques is available either in Edinburgh (Marston, Wellcome Trust Centre for Cell Biology) or in Glasgow (Spagnolo). This broad experience will provide a unique combination of highly desirable skills for future employment.
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