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Effects of Glucocorticoids in Macrophages

Alasdair Jubb

Presented for the degree Doctor of Philosophy
University of Edinburgh
2015
PhD – The University of Edinburgh – 2015
PhD – The University of Edinburgh – 2015
Declaration

Unless explicitly stated otherwise the work presented here is my own. The work has
not been submitted for any other degree or qualification.

……………………………………….. Date …………………
Alasdair Jubb

PhD – The University of Edinburgh – 2015

Contents

Acknowledgements ..................................................................................................... 9!
Abstract ..................................................................................................................... 11!
Figures ....................................................................................................................... 13!
Tables ......................................................................................................................... 15!
Abbreviations .............................................................................................................. ii!
Published, presented and submitted work from this thesis ..................................... i!
Chapter 1: Introduction ............................................................................................. 1!
1.1! Glucocorticoids ................................................................................................ 1!
1.1.1! Discovery, use and problems .................................................................... 1!
1.1.2! Towards a mechanism for glucocorticoid action ...................................... 6!
1.1.3! The role of GR dimerization and the ‘dissociated’ glucocorticoid ........ 10!
1.2! Chromatin and the control of gene expression .............................................. 11!
1.2.1! Chromatin ............................................................................................... 11!
1.2.2! Long range control of gene regulation ................................................... 12!
1.2.3! GC effects on chromatin ......................................................................... 25!
1.3! Macrophages .................................................................................................. 26!
1.3.1! Overview and relevance ......................................................................... 26!
1.3.2! Macrophage response to GC .................................................................. 27!
1.4! Models, conservation and divergence ........................................................... 29!
1.5! Summary ........................................................................................................ 31!
1.6! Thesis aims .................................................................................................... 31!
Chapter 2: Methods .................................................................................................. 33!
2.1! Laboratory procedures ................................................................................... 33!
2.1.1! Ethics ...................................................................................................... 33!
2.1.2! Cell culture ............................................................................................. 33!
2.1.3! RNA extraction and processing .............................................................. 34!
2.1.4! Chromatin Immunoprecipitation ............................................................ 34!
2.1.5! Single cell RT-qPCR .............................................................................. 37!
2.1.6! 3D DNA FISH ........................................................................................ 37!
2.2! Data Analysis ................................................................................................. 40!
2.2.1! Expression data ....................................................................................... 41!
2.2.2! Single cell PCR ....................................................................................... 41!
2.2.3! Promoter Analysis .................................................................................. 41!
2.2.4! Comparison to GWAS results and inflammatory genes ......................... 42!
2.2.5! Functional Annotation ............................................................................ 42!
2.2.6! ChIP-sequencing ..................................................................................... 42!
2.2.7! Interspecies and evolutionary analysis ................................................... 43!
2.2.8! Analysis of 3D FISH data ....................................................................... 44!
Chapter 3: The transcriptional response of macrophages to glucocorticoids .... 45!
3.1! Introduction ................................................................................................... 45!
3.2! The response of mouse bone marrow derived macrophages to
dexamethasone ....................................................................................................... 46!
3.3! The response of human monocyte derived macrophages to dexamethasone 51!
3.4! The expression response is different and is not linked to promoter variation
54!
3.5! Targets of glucocorticoids in human macrophages enrich for risk variants for
inflammatory disease .............................................................................................. 61!
3.6! Discussion ...................................................................................................... 63!
3.7! Summary ........................................................................................................ 65!
Chapter 4: Glucocorticoid receptor binding in macrophages .............................. 67!
4.1! Introduction ................................................................................................... 67!
4.2! GR binding in Mouse Bone Marrow Derived Macrophages ......................... 68!
4.3! GR binding in Human Monocyte Derived Macrophages .............................. 76!
4.4! Glucocorticoid receptor binding is associated with induced, not repressed
genes ....................................................................................................................... 80!
4.5! Inter-species differences of glucocorticoid receptor binding are associated
with sequence changes that lead to motif loss ........................................................ 82!
4.6! Discussion ...................................................................................................... 91!
4.7! Summary ........................................................................................................ 95!
Chapter 5: Dynamic regulation of chromatin structure in macrophages by
glucocorticoids .......................................................................................................... 97!
5.1! Introduction ................................................................................................... 97!
5.2! GR binding is associated with rapid and prolonged chromatin decompaction
98!
5.3! Rapid chromatin decompaction of Fkbp5 locus does not depend on
transcription .......................................................................................................... 105!
5.4! GR binds rapidly at enhancers in the Fkbp5 locus ...................................... 108!
5.5! Rapid decompaction may be a feature of GR bound loci ............................ 112!
5.6! Discussion .................................................................................................... 115!
5.7! Summary ...................................................................................................... 117!
Chapter 6: Conclusions and future work ............................................................. 119!
6.1! Conclusions ................................................................................................. 119!
6.1.1! The genome wide response to GC in macrophages .............................. 119!
6.1.2! GC effects on chromatin organisation in macrophages ........................ 119!
6.2! Future work ................................................................................................. 120!
6.2.1! Enhancer turnover and variability ........................................................ 120!
6.2.2! Kinetics of the macrophage response to GC and mechanisms of
repression .......................................................................................................... 123!
6.2.3! GC driven chromatin dynamics ............................................................ 123!
6.3! Summary ...................................................................................................... 127!
References ............................................................................................................... 129!
Appendix 1 .............................................................................................................. 149!
Appendix 2 .............................................................................................................. 155!

Acknowledgements
Thanks must first go to the directors of the Edinburgh Clinical Academic Track
Brian Walker and John Iredale and the Wellcome Trust/NES for giving me the post
in the first place. The flexibility of the programme and advice they have given me,
more recently also Andrew Jackson, has enabled me to identify and take on a much
more ambitious project than I could have done otherwise.

Thanks to my supervisors David Hume and Wendy Bickmore for taking me on
without any track record to speak of and helping me put together a worthwhile
project. Both were and continue to be incredibly supportive of my attempts to scale
what remains at times a pretty precipitous learning curve.

There is no way I could have done any of this without the teaching and support
provided by the Bickmore and Hume and wider Maclab group members. Thankfully
there are plenty of them to spread the load, some are named below but thanks are due
to all. From the Bickmoreans Rob Illingworth has to get first mention due to the
sheer amount of time I spent bending his ear about ChIP. Rob Young added welcome
and necessary computational and enhancer expertise and Shelagh Boyle enabled all
things FISH related. Thanks are due to all : Hazel, Pradeep ‘should be fine’ MM, Liz,
Gillian (GD), Sehrish, Iain, Nezha, Pierre, Celine, Leisha, Jess and Andrew as well
as the wider E3 community for the unfailingly entertaining environment. Paul and
Matt for helping me out in imaging at HGU. At Roslin thanks must go to the core of
the lab Clare, Kristen, Lindsay, Anna, and Gemma; to those who helped me get
going Kyle Upton and Ronan Kapetanovic; to Tom Freeman for introducing me to
the world of expression analysis and his lab for providing good chat and gender
balance; to Malcom Fisher and latterly Sara Clohisey and Gemma Davies for blood
days Maggie for the fish, Bob in imaging. Specific mention to my ECAT forerunner
at Roslin, Kenny Baillie, for help, advice, and being consistently distractible by the
promise of a haggis roll.

And finally, of course, thanks to Francesca and Saoirse.