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dc.contributor.authorBertero, Alessandro
dc.date.accessioned2018-03-20T12:36:24Z
dc.date.available2018-03-20T12:36:24Z
dc.date.issued2016-10-22
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/274120
dc.description.abstractHuman pluripotent stem cells (hPSCs) are an invaluable model for cellular and developmental biology, and hold great potential for translational applications. While great progress has been made in elucidating the signalling pathways regulating pluripotency and differentiation, our mechanistic understanding of the downstream regulations is still incomplete. Moreover, studies aimed at clarifying these aspects are severely impeded by the lack of efficient methods to conditionally modulate gene expression in hPSCs and hPSC-derived cells. In this dissertation I provide new insights into the molecular mechanisms controlled by the Activin/Nodal-SMAD2/3 signalling pathway, whose activity dictates the balance between hPSC pluripotency and differentiation. First, I show that SMAD2/3 modulates the chromatin epigenetic landscape of hPSCs by cooperating with the pluripotency factor NANOG to recruit the DPY30-COMPASS complex and promote histone 3 lysine 4 trimethylation (H3K4me3). This regulation promotes expression of pluripotency genes, while poising developmental regulators for activation during differentiation. Secondly, I describe a novel efficient approach for inducible gene knockdown in hPSCs and hPSC-derived cells. By taking advantage of this technology, I demonstrate that DPY30 is required for early differentiation of hPSCs into certain mesoderm and endoderm derivatives. Finally, I report the first large-scale proteomic identification of SMAD2/3 interacting proteins in both undifferentiated and differentiating hPSCs. This analysis not only confirms that SMAD2/3 interacts with multiple epigenetic modifiers involved in hPSC fate choices, but also implicates SMAD2/3 in several functions other than transcriptional regulation. In particular, I describe how SMAD2/3 physically and functionally interacts with the METTL3-METTL14-WTAP complex to promote the formation of N6-methyladenosine (m6A). This epitranscriptional modification antagonizes the expression of selected mRNAs, including pluripotency factors whose transcription is promoted by SMAD2/3. Therefore, this provides a negative feedback that facilitates rapid exit from pluripotency upon inhibition of Activin/Nodal signalling. Overall, the work presented in this dissertation advances the stem cell field in two ways. First, it demonstrates that the Activin/Nodal-SMAD2/3 pathway finely orchestrates the balance between pluripotency and differentiation by shaping both the epigenome and the epitranscriptome of hPSCs. Secondly, it provides a novel powerful technology to facilitate further studies of the mechanisms that regulate cell fate decisions.
dc.description.sponsorshipBritish Heart Foundation PhD Studentship (FS/11/77/39327)
dc.language.isoen
dc.rightsAttribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subjectstem cells
dc.subjectsmad
dc.subjectDPY30
dc.subjectTGF beta
dc.subjectm6A
dc.subjectepigenetics
dc.subjectepitranscriptome
dc.subjectshRNA
dc.subjectknockdown
dc.titleActivin/Nodal Signalling Controls the Epigenome and Epitranscriptome of Human Pluripotent Stem Cells
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.publisher.departmentSurgery
dc.date.updated2018-03-19T19:17:00Z
dc.identifier.doi10.17863/CAM.21202
dc.contributor.orcidBertero, Alessandro [0000-0002-4919-9087]
dc.publisher.collegeSt Catharine's
dc.type.qualificationtitlePhD in Surgery
cam.supervisorVallier, Ludovic
cam.supervisor.orcidVallier, Ludovic [0000-0002-3848-2602]
cam.thesis.fundingfalse
rioxxterms.freetoread.startdate2018-03-19


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Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
Except where otherwise noted, this item's licence is described as Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)