Investigating the Role of KAP1 in Mammary Gland Development

Govindrajan, Nayantara

Investigating the Role of KAP1 in Mammary Gland Development

dc.contributor.advisor	Shemanko, Carrie Simone
dc.contributor.author	Govindrajan, Nayantara
dc.contributor.committeemember	Shemanko, Carrie Simone
dc.contributor.committeemember	Cobb, John
dc.contributor.committeemember	Chua, Gordon
dc.contributor.committeemember	Jones, Nina
dc.contributor.committeemember	Grewal, Savraj
dc.date	2024-11
dc.date.accessioned	2024-09-16T20:05:43Z
dc.date.available	2024-09-16T20:05:43Z
dc.date.issued	2024-09-11
dc.description.abstract	KAP1 controls cell fate specification of spermatogonial, skeletal, and hematopoietic progenitors, and promotes embryonic and breast cancer stem cell renewal. It regulates cell proliferation and DNA damage repair, with the latter further linking it to mammary progenitor specification. KAP1 could thus act as a master regulator of mammary lactogenic specification. My studies aimed to test the hypothesis that KAP1 is required for proliferation and lactogenic differentiation during mammary gland development. I generated novel, mammary epithelial-specific KAP1 knockout models using MMTV-cre in mice and CRISPR-Cas9 in the mouse mammary epithelial cell line HC11. KAP1 loss was heterogeneous due to cre mosaicism in knockout mice, but there were strong morphological defects like decreased tertiary branching and alveolar formation during pregnancy. Alveolar defects persisted in lactation. HC11 acted as a complementary system and lacked KAP1 in every cell. Knockout HC11 cells had decreased proliferation and migration, and blocked lactogenic differentiation, as seen by the lack of milk domes and the milk protein β casein. Bulk RNA sequencing identified potential KAP1-controlled genes and pathways mediating knockout phenotypes in HC11 cells. This included the downregulation of cholesterol biosynthesis in differentiated knockout cells. Single-cell RNA sequencing identified the downregulation of luminal and alveolar progenitor genes and pathways controlling growth and morphogenesis in knockout pregnancy day 10 glands. Clustering identified progenitors with intermediate gene expression profiles between common luminal and alveolar progenitors. Knockout alveolar progenitors lacked casein 2, which encodes β casein, confirming knockout phenotypes observed in HC11 cells. Knockouts showed significant reductions in genes controlling cholesterol uptake and lipid droplet formation. Total cholesterol levels were decreased in both knockout models. The link between cholesterol biosynthesis and mammary lactogenesis was strengthened in statin-treated HC11 cells where defects in proliferation, dome formation, and casein expression were observed, like those seen with KAP1 loss. While the knockout proliferative defect was rescued by adding the cholesterol pathway metabolite mevalonate, the lactogenic defect was partially rescued. I have identified a novel link between cholesterol metabolism and mammary lactogenesis, potentially mediated by KAP1. This could inform the decision of women with familial hypercholesteremia using statins during pregnancy, due to possible effects on maternal lactation capacity.
dc.identifier.citation	Govindrajan, N. (2024). Investigating the role of KAP1 in mammary gland development (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.
dc.identifier.uri	https://hdl.handle.net/1880/119720
dc.language.iso	en
dc.publisher.faculty	Graduate Studies
dc.publisher.institution	University of Calgary
dc.rights	University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.
dc.subject	mammary gland development
dc.subject	cell fate specification
dc.subject	novel mouse knockout models
dc.subject	gene expression profiling
dc.subject	pregnancy
dc.subject	lactogenic differentiation
dc.subject	cholesterol biosynthesis
dc.subject.classification	Biology--Cell
dc.title	Investigating the Role of KAP1 in Mammary Gland Development
dc.type	doctoral thesis
thesis.degree.discipline	Biological Sciences
thesis.degree.grantor	University of Calgary
thesis.degree.name	Doctor of Philosophy (PhD)
ucalgary.thesis.accesssetbystudent	I do not require a thesis withhold – my thesis will have open access and can be viewed and downloaded publicly as soon as possible.