Use this URL to cite or link to this record in EThOS:
Title: The role of Roma in mouse mammary epithelial cells
Author: Alenazi, Ala
ISNI:       0000 0004 7961 8507
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
Epithelial to Mesenchymal Transition (EMT) and its reversal, via Mesenchymal to Epithelial Transition (MET), describes the highly coordinated cycle of epithelial plasticity by which normal tissues are remodelled and repositioned during development. Previous studies have characterised the role of EMT regulators in epithelial plasticity observed during branching morphogenesis in the mammary gland. For example, a microarray analysis of gene expression in terminal end buds identified elevated levels of EMT regulators, Snail and Twist, in the mammary gland. Additionally, a transgenic mouse model overexpressing a regulator of EMT, Mta3, resulted in hypo-proliferation in the mammary gland, showing a link between EMT regulators and mammary gland development. In a similar pattern to Mta3, mice deficient in Roma (Zfp157), a newly characterised Krüppel-associated box zinc finger protein, exhibited hyperproliferation in mammary epithelial cells (MECs) during early pregnancy. Roma/Zfp157 (Regulator Of Mammary Alveologenesis), has been shown by the Watson laboratory to be a critical regulator of alveolar lineage commitment in the mammary gland, with its depletion also resulting in accelerated alveologenesis during pregnancy coupled with DNA damage and cell cycle dysregulation. Whilst these developmental phenotypes have been described, a complete molecular characterisation of Roma and its interacting partners is yet to be achieved. This body of work aimed to expand our current understanding of Roma's role in the mammary gland by elucidating its interactome. To this end, Roma's binding partners were investigated by Rapid Immunoprecipitation of Endogenous proteins by mass spectrometry (RIME), a novel approach for identifying proteins in transcription factor complexes. Further in silico analysis of RIME data, coupled with co-immunoprecipitation experiments, helped refine several putative binding partners of Roma, identifying Roma's binding to Mta3, a regulator of the cell cycle with a role in EMT. This thesis also shows that Roma overexpression led to the downregulation of key EMT regulators, Snail and Twist, in EpH4s MECs, which is interesting given previous studies have shown Mta3 represses Snail expression in transgenic-overexpression mouse models. Moreover, a Roma-knockout mouse model was probed to investigate the loss of Roma on EMT factors in the mammary gland, which concurrently showed elevated levels of Snail and Twist, and the downregulation of Slug and Zeb1 during early pregnancy. Additionally, a novel optical-clearing and confocal imaging technique (CUBIC R1a) was used to clear the mammary gland and visualise the extensive disorganisation of MECs in the Roma-knockout mice during lactation. This finding may suggest a potential role for Roma in epithelial plasticity through EMT regulators and architectural organisation in the mammary gland. Finally, this thesis has undertaken important steps towards identifying the human orthologue of Roma, using several in silico approaches. In particular, online microarray databases, phylogenetics, BLAST sequence alignments of conserved domains and breast cancer signatures were analysed and cross- referenced, generating a list of candidates for the human orthologue of Roma. Overall, the findings of this thesis suggest that Roma may regulate mammary gland development through manipulation of key EMT factors via Mta3. Further characterisation of this protein will provide important information on mechanisms driving extensive tissue remodelling in the mammary gland, which is strictly regulated due to implications for both normal and malignant development.
Supervisor: Watson, Christine Sponsor: Saudi Arabian Culture Bureau
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Roma ; MTA3 ; KRAB-ZNF ; KRAB-ZFP ; ZFP157 ; Mouse mammary gland ; EMT ; RIME