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How to silence a chromosome: Molecular organization of epigenetic complexes that control X inactivation


   Molecular and Cell Biology

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  Dr Yolanda Markaki, Prof John Schwabe  No more applications being accepted  Funded PhD Project (Students Worldwide)

About the Project

In early embryonic development, XX female mammals will switch off nearly 1000 genes on one of their two X chromosomes (X chromosome inactivation, XCI) to balance gene expression with XY males. The silenced state of the inactive X (Xi) will be remembered by daughter cells and play a crucial role in cell physiology. Exit from pluripotency requires XCI, which is essential for the survival and development of female embryos. Dysregulation of XCI is involved in over 500 X-linked diseases including immune diseases, cancer and during ageing. Importantly, maintenance of XCI is necessary for the epigenetic stability of human pluripotent stem cells, which often reactivate the Xi in culture, and thus their applicability to cell-based therapies. 

The non-coding (nc) RNA Xist regulates XCI by recruiting silencing proteins to the X. Many Xist-interacting proteins contain intrinsically disordered regions (IDRs) which are involved in protein-protein interactions and the formation of nuclear condensates. Using super-resolution microscopy and biochemical assays in stem cell models, we recently discovered that Xist does not bind each gene on the X chromosome subject to silencing. Instead, only 100 Xist molecules form hubs for the binding of hundreds of identified protein molecules generating a nuclear compartment where proteins interact to form supercomplexes (SMACs). Through CRISPR/Cas9 deletions we showed that the essential transcriptional repressor SPEN (SHARP) which is recruited to SMACs, functions in a concentration-dependent manner through its IDRs. Therefore, protein-protein interactions within SMACs play an essential role in epigenetic regulation.  

To understand how RNA-guided molecular machines regulate gene expression it is critical to dissect their molecular organization. This project will form a unique opportunity for interdisciplinary work integrating stem cell biology, bioengineering, quantitative super-resolution microscopy and structural biology, such as small angle X-ray scattering and cryo-electron microscopy. It will span scales addressing the function of individual proteins and protein domains, the molecular organization of supercomplexes and their in situ distribution within cells at subdiffraction resolution. We will use CRISPR/Cas9-based methods to bioengineer minimal RNA-guided molecular assemblies within cells, coupled with downstream cross-linking mass spectrometry and spatial gene expression analyses with small molecule FISH probes. This platform will allow the study of RNA-protein complexes within their native environment with minimal intervention. 

Ultimately, understanding the molecular mechanisms underpinning formation of Xist-complexes will allow the development of therapeutic applications to tackle dysregulation of XCI in disease or the production of epigenetically stable human pluripotent stem cells. Moreover, unravelling the function of Xist will provide insights into mechanisms of gene regulation and the role of ncRNAs implicated in embryonic development or cancer that share the same protein interactome with Xist. 

Techniques that will be undertaken during the project:

·       Embryonic stem cell culturing and differentiation 

·       Cloning and other molecular biology methods 

·       Gene editing and bioengineering techniques using CRISPR/Cas9 

·       RNA/DNA Fluorescence In Situ Hybridization (FISH), immunofluorescence 

·       Super-Resolution and Confocal Laser Scanning Microscopy  

·       Biochemical protein-RNA/protein-protein interaction assays and affinity purification 

·       High-resolution structural studies: small angle X-ray scattering, cryo-electron microscopy and other structural biology methods 

·       Data analysis and visualization in Fiji, R and Python 

BBSRC Strategic Research Priority: Understanding the Rules of Life – Stem Cells, Structural Biology

Start date: 25 September 2023

 

Entry requirements

  • Those who have a 1st or a 2.1 undergraduate degree in a relevant field are eligible.
  • Evidence of quantitative training is required. For example, AS or A level Maths, IB Standard or Higher Maths, or university level maths/statistics course.
  • Those who have a 2.2 and an additional Masters degree in a relevant field may be eligible.
  • Those who have a 2.2 and at least three years post-graduate experience in a relevant field may be eligible.
  • Those with degrees abroad (perhaps as well as postgraduate experience) may be eligible if their qualifications are deemed equivalent to any of the above.
  • University English language requirements apply

To apply

Carefully read the application advice on our website and submit your PhD application and MIBTP funding form. 

https://le.ac.uk/study/research-degrees/funded-opportunities/bbsrc-mibtp


Funding Notes

All MIBTP students will be provided with a 4 years studentship.
Studentships include:
• Tuition fees at the UK fee rate*
• a tax free stipend which for 2022/3 was £17,668 (2023/24 stipend to be confirmed)
• a travel allowance in year 1
• a travel/conference budget
• a generous consumables budget
• use of a laptop for the duration of the programme.
* International students are welcome to apply but applicants must be able to fund the difference between UK and International fees for the duration of their studies.

References

1. Markaki Y*, Chong JG, Wang Y, Jacobson EC, Luong C, Tan SYX, Jachowicz JW, Strehle M, Maestrini D, Dror I, Mistry BA, Schöneberg J, Banerjee A, Guttman M, Chou T*, Plath K*. Xist nucleates local protein gradients to propagate silencing across the X chromosome. Cell. 2021.
2. Pandya-Jones A, Markaki Y, Serizay J, Chitiashvili T, Mancia Leon WR, Damianov A, Chronis C, Papp B, Chen CK, McKee R, Wang XJ, Chau A, Sabri S, Leonhardt H, Zheng S, Guttman M, Black DL, Plath K. A protein assembly mediates Xist localization and gene silencing. Nature. 2020;587(7832):145-51.doi:10.1038/s41586-020-2703-0.
3. 3. Kraus F, Miron E, Demmerle J, Chitiashvili T, Budco A, Alle Q, Matsuda A, Leonhardt H, Schermelleh L, Markaki Y. Quantitative 3D structured illumination microscopy of nuclear structures. Nat Protoc. 2017;12(5):1011-28.
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