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Impact of the Chromatin Remodeller SMARCAD1 on Murine Intestinal Intraepithelial Lymphocyte and White Adipose Tissue Biology

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Impact of the Chromatin Remodeller SMARCAD1 on Murine Intestinal Intraepithelial Lymphocyte and White Adipose Tissue Biology Impact of the chromatin remodeller SMARCAD1 on murine intestinal intraepithelial lymphocyte and white adipose tissue biology. Keith Michael Porter The Babraham Institute Homerton College September 2016 This dissertation is submitted for the degree of Doctor of Philosophy Declaration This research the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface or specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface or specified in the text. It does not exceed the prescribed word limit for the Biological Sciences Degree Committee. Keith Michael Porter Keith Michael Porter 1 Impact of the chromatin remodeller SMARCAD1 on murine intestinal intraepithelial lymphocyte and white adipose tissue biology. Chromatin remodelling factors use the energy of ATP hydrolysis to drive the movement of and/or affect molecular changes to the nucleosome. One such factor, SMARCAD1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A containing DEAD/H box 1), has been previously shown to restore heterochromatin at the replication fork in vitro. This project aimed to assess the impact of SMARCAD1 on mammalian biology, utilising an animal model in which the catalytic ATPase domain of murine SMARCAD1 had been deleted using Cre/lox technology. Preliminary results had implicated SMARCAD1 in adaptive-immunity and white adipose tissue biology, and SMARCAD1 expression in these tissues/cells was confirmed by tissue-panel western blot. This project therefore aimed to build on these results to understand better the impact of SMARCAD1 on adaptive immune development and white adipose tissue biology. In addition, fewer than expected viable Smarcad1-/- homozygous offspring were produced during Smarcad1+/- x +/- matings, which both confirmed the observation from a previous knockout model of Smarcad1, and limited the number of knockout animals available for this study. Investigation of systemic B- and T-cells in the bone marrow, thymus and spleen had previously suggested there was no significant defect in adaptive immune development in Smarcad1-/- mice, however a tissue-specific and age-related loss of intra-epithelial (IEL) T-lymphocytes was found in the small intestine by flow cytometry. Analysis by qPCR of duodenal RNA suggested that differentiation rather than inflammation may underpin any loss-of-IEL phenotype, although further examination of cell-proliferation and crypt/villus anatomy by EdU incorporation and immunofluorescence revealed no overt cell-anatomical or proliferative difference in the knockout mice. The requirement for large numbers of aged mice made further investigation of the intestinal IEL phenotype logistically prohibitive. The reduction of epididymal white adipose tissue (eWAT) size had also been observed in male Smarcad1-/- mice, and serum from these mice showed elevated triglyceride (TG) and free fatty acids (FFA) levels. Transcriptomic analysis by RNA-seq of whole-WAT revealed an elevation in macrophage-related markers in knockout mice, which was confirmed by flow cytometry. As a number of reports have implicated SMARCAD1 in stem cell biology, putative adipose stem cells were isolated from +/+ and -/- mice by FACS and used for adipogenic differentiation assays ex-vivo In parallel, mouse embryonic fibroblasts from +/- and -/- mice were also assayed for adipogenic differentiation. While no significant differences in adipogenesis were observed, Smarcad1-/- mice challenged with a (60%) high fat diet did show increased weight gain over +/+ mice, and measurements of adipocyte size and cell cycle/cell proliferation analysis suggested hyperplasia rather than defects in adipogenesis may drive any WAT- related pathology in these mice. Keith Michael Porter 2 Table of acknowledgement of assistance received during course of thesis. 1) Initial training in techniques and laboratory practice and subsequent mentoring: Louise Matheson, Rachel Walker and Marc Veldhoen – FACS and IEL preparation for FACS Animal handling and dissection – Marc Veldhoen and Dan Bolland. 2) Data obtained from a technical service provider (e.g. DNA sequencing, illustrations, simple bioinformatics information etc) Kristina Tabbada – Illumina platform RNA-sequencing Simon Andrews – RNA-seq results genomic alignment Rachel Walker/Arthur Davis/Lynzi Waugh/Rebecca Roberts - Cell Sorting Keith Burling (Core Biochemistry Assay Laboratory Addenbrookes hospital) – serum assays Anne Segonds-Pichon – statistical methods. 3) Data produced jointly (e.g. where it was necessary or desirable to have two pairs of hands) Irem Kiris (visiting student) – Tissue Panel Western Blots and culture of mouse embryonic fibroblasts. 4) Data/materials provided by someone else (e.g. one-off analysis, bioinformatics analysis, where parallel data or technical provision in a very different area is needed to provide a connected account in the thesis) Keith Burling (Core Biochemistry Assay Laboratory Addenbrookes hospital) – serum assays Keith Michael Porter 3 Acknowledgements. I would like to thank Dr. Patrick Varga-Weisz not just for the opportunity to pursue a PhD, but for allowing me through the looking glass of research savoir-faire. The down-to-earth perspective of Professor Anne Ferguson-Smith FRS was invaluable and gave exactly the taste of scientific cut-and-thrust this student desired, while never sparing of support when needed. Similarly the endless patience of Dr Anne Corcoran allowed me to mentally explore without reproach or suffocation, and whose leadership and success provide inspiration for many. No pivotal moment of my life would be right without mention of my late friend, Richard Dhamesin. Moments of struggle are always grounded by his memory, and I’m certain that he’s somewhere chortling that I managed to pull this one off I’ll be raising a glass to you, but keeping the PhD to myself thank you very much ;) The biggest dedications have to go to family and friends. My parents have never pressured me in whatever I do, but instead always have an ear and supportive words to encourage me along the way. My partner Carol Edwards thought it would be a good idea to apply to the Babraham Institute, and has always offered scientific and non-scientific advice – but most importantly has always been there. We also shared possibly the most expensive and life-changing bottle of Rosé wine in Carpentras in 2013, for which we will forever be picking up the tab –x- There aren’t the words to describe the gratitude I have for the entry of Alicia Erin Porter into my life. So rather than try and fail, I’ll just say that moments after she was born she opened her eyes and looked me up and down, which made it clear we were equals from the get-go; nothing more, nothing less. More than any seminar, the reality of biology unfolding before my eyes is the greatest influence and celebration of the subject I try and articulate over the next couple of hundred pages –x- Keith Michael Porter 4 Contents Chapter 1 - General Introduction ................................................................................ 1 1.1 Chromatin, nucleosomes and DNA compaction ............................................. 2 1.2 Histone modification and the histone code. .................................................... 2 1.3 Chromatin remodelling and chromatin remodeller families. .......................... 7 1.3.1. Chromatin remodelling families. ................................................................. 8 1.3.2. Impact of chromatin remodellers on development. ..................................... 9 1.3.3. SWI/SNF Chromatin remodellers. ............................................................. 15 1.3.4. The SWI/SNF family chromatin remodeller SMARCAD1. ...................... 17 1.3.10 SMARCAD1 in human disease. ............................................................... 29 1.4 Genomic imprinting and the influence of imprinted genes on metabolism. 35 1.4.1. Imprinted genes and metabolism. .............................................................. 35 1.4.2. SWI/SNF chromatin remodellers and the KCNQ1 imprinted locus. ......... 38 1.4.3. Chromatin remodelling controls Dlk1 expression during adipogenesis. ... 38 1.5 Adipocyte lineage differentiation and chromatin. ......................................... 39 1.5.1. Chromatin remodellers and adipocyte differentiation. .............................. 39 1.5.2. Major pathways involved in adipogenesis. ................................................ 42 1.5.3. Approaches to the study of white-adipose tissue. ...................................... 43 1.5.4 Putative white adipocyte stem cells for studying adipogenesis. ................. 44 1.5.5. Mouse Embryonic Fibroblasts (MEFs). ..................................................... 47 1.6 Intestinal microbiota and metabolism. .......................................................... 49 1.7 Intestinal anatomy and development. ............................................................ 55 1.7.1.
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