The dynamic behaviour of the chromosomal protein SmcHD1 during X chromosome inactivation

Thesis

Xist, the master regulator of X chromosome inactivation (XCI), employs a multitude of downstream factors to mediate chromosome-wide silencing. One of these factors is SmcHD1, which is a non-canonical member of the Smc family of chromosomal proteins. During XCI, SmcHD1 has been shown to play a role in gene repression, CpG islands methylation and remodelling of the chromosome architecture. Moreover, mutations in SmcHD1 have been associated with two genetic disorders: Facioscapulohumoral muscular dystrophy type 2 (FSHD2) and Bosma arrhinia and micropthalmia (BAMS). Nevertheless, the exact mechanism of action of SmcHD1 is not yet known in either physiological or pathological conditions.  

In this thesis I aimed to uncover the molecular mechanism employed by SmcHD1 to perform its function by investigating its dynamic behaviour. In this respect, I setup a methodology to enable tracking of single molecules of SmcHD1 on the inactive X (Xi) chromosome, during in vitro monolayer (ML) differentiation of mouse embryonic stem cells. This method was then used to show how the dynamics of SmcHD1 change as silencing of the Xi chromosome progresses. Subsequently, since live imaging of SmcHD1 revealed a population-wide enrichment of the protein at pericentric regions, the role of SmcHD1’s partner LRIF1 in this association was explored. Moreover, I observed that LRIF1 plays a role in the recruitment of SmcHD1 to the Xi in ML differentiation and, thus, investigated this further using the Xi-focused single-particle tracking methodology. Lastly, I have analysed the influence of three SmcHD1 mutations, including two disease-associated ones, on the dynamic behaviour of the protein as well as on its functionality in XCI. This has allowed me to dissect the details of how SmcHD1 loads and unloads from the underlying chromatin, highlighting a key role for the ATPase activity of the protein in this process. Overall, the findings presented here will be key for understanding better the role of SmcHD1 in XCI and, further on, in the pathogenesis of FSHD2 and BAMS.

Authors: Constantinescu, FA

• DOI: 10.5287/ora-0z4xg7yvz
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: single molecule tracking; x chromosome inactivation; smcHD1