University of Southampton Human Development and Health Faculty of Medicine CDKN2A: A Predictive Marker for Bone Mineral Density or Direct Role in Osteoblast Function? Eloïse Cook PhD Thesis July 2016 Supervisors: Prof Karen Lillycrop, Prof Nicholas Harvey and Dr Stuart Lanham UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF MEDICINE ACADEMIC UNIT OF HUMAN DEVELOPMENT AND HEALTH Doctor of Philosophy CDKN2A: A Predictive Marker for Bone Mineral Density or Direct Role in Osteoblast Function? By Eloïse Cook As the population is becoming more aged, osteoporosis is becoming more prevalent and the number of fragility fractures that are occurring is increasing. One of the main predictors of developing osteoporosis in later life is a bone mineral density that is greater than 2.5 standard deviations below the young adult sex-matched mean, though studies have only been able to explain 5% of the variance seen in bone mineral densities between individuals. There is now increasing evidence that the development of osteoporosis can begin in utero and that epigenetic processes, such as DNA methylation, may be central to the mechanism by which early development influences bone mineral density and later bone health. Previous work within the group has identified associations of a 300bp differentially methylated region within the CDKN2A locus with bone mineral content, bone area and areal bone mineral density in offspring from the Southampton Women’s Survey (SWS) cohort. As methylation of CDKN2A increases, bone mineral content, bone area and areal bone mineral density all decrease at both 4 and 6 years of age. The two cell cycle regulators p16INK4A and p14ARF are transcribed from this gene as well as the lncRNA ANRIL. Various techniques were used to determine whether this differentially methylated region has a functional role in osteoblasts or whether it is just a potential marker for predicting bone mineral density. It was shown that there was specific binding in the osteoblast-like SaOS-2 cell line to this region and that methylation of these CpGs led to a decrease in transcription factor binding. Treatment of both SaOS-2 cells and primary osteoblasts with siRNAs against ANRIL caused a decrease in cell number, a decrease in cell proliferation, an increase in cells in G0/G1 phase of the cell cycle and, in SaOS-2 cells, caused an increase in the expression of bone differentiation markers RUNX2 and ALP. Knock down of ANRIL expression also caused a change in genes that have roles in osteoblast specific pathways as well as genes that were involved in molecular functions such as nuclear regulation and acetylation. Associations were found between CpG methylation in RXRA and offspring bone outcomes at birth in both the SWS and MAVIDOS cohorts. Daily maternal cholecalciferol supplementation was also shown to decrease the methylation of CpG loci within RXRA in offspring umbilical cord and further investigation showed that this effect of maternal supplementation was only seen in children born during the winter months. Together, these findings suggest that the association between CDKN2A methylation at birth and later bone health could indicate a potential role for CDKN2A in bone development and function and raises the possibility that differential methylation of CDKN2A may allow the identification of individuals at increased risk of osteoporosis in later life. Due to the effect of ANRIL knock down in primary osteoblasts, which could not be explained through normal cell cycle pathways, results suggest that ANRIL has potential trans-regulatory functions in these cells. These findings suggest that ANRIL could potentially play a role in bone differentiation, cell proliferation and metabolism and that dysregulation of the expression of this lncRNA could have adverse effects on bone health, potentially increasing the risk of individuals developing osteoporosis in later life. I Table of Contents Abstract – I Table of contents – II List of figures – VII List of tables – X Declaration of authorship – XI Acknowledgements – XII Abbreviations – XIII Page Number 1.0 Introduction 1 1.1 Osteoporosis 3 1.1.1 Epidemiology of osteoporosis 3 1.1.1.1 Fracture 3 1.1.1.2 Morbidity 4 1.1.1.3 Mortality 5 1.1.1.4 Economic cost 6 1.2 Bone formation 6 1.2.1 Osteoblast differentiation 7 1.2.1.1 Transcription factors 9 1.2.1.2 Signalling pathways 10 1.2.1.3 Systemic regulation 16 1.2.2 Osteoclasts 17 1.2.3 Bone remodelling 18 1.2.4 Genome-wide association studies (GWASs) 19 1.2.5 Pathogenesis of osteoporosis 19 1.3 Developmental origins of health and disease (DOHaD) 21 1.3.1 Human studies 22 1.3.2 Animal studies 24 1.3.3 Evidence for the developmental origins of osteoporosis 25 1.3.3.1 Population studies 25 1.3.1.2 Physiological studies 26 1.3.3.3 Maternal lifestyle 27 1.3.3.4 Childhood growth 27 1.3.4 Vitamin D 28 1.4 Epigenetics 29 1.4.1 Epigenetic mechanisms 30 1.4.1.1 DNA methylation 30 1.4.1.2 Histone modifications 31 1.4.1.3 microRNAs (miRNAs) 31 1.4.1.4 Long non-coding RNAs (lncRNAs) 32 1.4.1.5 Evidence epigenetic processes mediate developmental origins of adult disease using animal models 33 1.4.1.6 Vitamin D and epigenetic mechanisms 33 1.4.2 Epigenetics and early life environment 34 1.4.3 Epigenetics and bone 36 1.5 Identification of potential predictive markers of offspring bone outcomes 38 1.5.1 Epigenetic biomarkers of later health 38 1.5.2 Epigenetic markers of later bone health 39 II 1.5.2.1 RXRα 39 1.5.2.2 Endothelial nitric oxide synthase (eNOS) 40 1.5.2.3 CDKN2A 41 1.6 Aims and hypotheses 43 2.0 Materials and Methods 45 2.1 Materials 47 2.2 Methods 50 2.2.1 Cell culture 50 2.2.1.1 Osteosarcoma SaOS-2 cell line 50 2.2.1.2 Human bone marrow stromal cells (hBMSCs) 50 2.2.2 Transfection of SaOS-2 with luciferase reporter constructs 52 2.2.2.1 ANRIL/p14ARF luciferase reporter constructs 52 2.2.2.2 Inoculation and minipreparation of plasmid DNA 52 2.2.2.3 Preparing plasmid DNA for transfection 53 2.2.2.4 Transfection of plasmid DNA 53 2.2.2.5 Preparation of cells for Dual-Luciferase assay 54 2.2.3 Isolation of nuclear extract 54 2.2.4 Analysis of transcription factor binding by electrophoretic mobility shift assays 54 2.2.4.1 Annealing oligonucleotides 54 2.2.4.2 Electrophoretic mobility shift assay (EMSA) 55 2.2.5 Transfection with siRNA against ANRIL 57 2.2.5.1 SaOS-2 57 2.2.5.2 hBMSCs and primary osteoblasts 59 2.2.6 Isolation of total RNA 59 2.2.6.1 TRI Reagent 59 2.2.6.2 RNeasy kit 60 2.2.7 Illumina Human H12 Gene Expression Array 60 2.2.8 DNase I treatment 60 2.2.9 cDNA synthesis 61 2.2.10 Real time quantitative polymerase chain reaction (RT-qPCR) 61 2.2.10.1 GeNorm assay 61 2.2.10.2 TaqMan assays 61 2.2.11 Fluorescence-activated cell sorting (FACS) 62 2.2.12 Measuring methylation levels of MAVIDOS umbilical cord 63 2.2.12.1 Isolation of genomic DNA from umbilical cord 63 2.2.12.2 Bisulfite conversion of genomic DNA 66 2.2.12.3 Polymerase chain reaction (PCR) 66 2.2.12.4 Pyrosequencing 68 2.2.13 Statistical analysis 68 2.2.13.1 Statistical analysis of siRNA transfected cells 68 2.2.13.2 Statistical analysis of MAVIDOS cohort 68 3.0 Identification of CDKN2A as a predictive marker of bone mineral density 71 3.1 Introduction 73 3.1.1 CDKN2A gene locus 73 3.2 Aims 76 3.3 Results 77 3.3.1 Does the DMR control CDKN2A expression? 77 III 3.3.2 Is there specific binding to the CpGs within the CDKN2A DMR in bone cells? 79 3.3.3 Does the methylation of CpGs -887 and -907 in the CDKN2A DMR alter the binding of transcription factors? 82 3.3.4 What transcription factors bind to CpGs -887 and -907? 85 3.4 Discussion 89 3.5 Summary 93 3.6 Future work 93 4.0 Functional significance of ANRIL in SaOS-2, an osteoblast-like cell line 95 4.1 Introduction 97 4.2 Aims 99 4.3 Results 100 4.3.1 Is ANRIL expressed in bone-related cells? 100 4.3.2 Does ANRIL knock down affect SaOS-2 cell number? 103 4.3.3 Does knock down of ANRIL expression cause cell cycle arrest? 106 4.3.4 Does ANRIL knock down have an effect on osteoblast differentiation? 108 4.3.5 Does ANRIL knock down have an effect on mineralisation? 110 4.3.6 Does ANRIL mediate its affect through altered expression of the CDKN2A/B locus? 111 4.4 Discussion 113 4.5 Summary 118 5.0 Characterisation of ANRIL in human bone marrow stromal cells and primary osteoblasts 121 5.1 Introduction 123 5.2 Aims 123 5.3 Results 124 5.3.1 ANRIL knock down causes a decrease in hBMSC and primary osteoblast cell number 124 5.3.2 ANRIL knock down does not have an effect on the viability of primary cells 129 5.3.3 Knock down of ANRIL expression causes a decrease in the proliferation of primary cells 131 5.3.4 Knock down of ANRIL expression causes cell cycle arrest in hBMSCs and primary osteoblasts 133 5.3.5 Does ANRIL mediate its affect through altered expression of the CDKN2A/B locus in primary cells? 135 5.3.6 hBMSCs and primary osteoblasts treated with siRNAs against ANRIL are not undergoing replicative senescence 138 5.4 Discussion 140 5.5 Summary 142 5.6 Future work 143 6.0 Effects of ANRIL knock down on gene expression in human primary osteoblasts 145 6.1 Introduction 147 6.2 Aims 148 6.3 Results 149 6.3.1 Validation of Illumina Human H12 Gene Expression Array results 149 IV 6.3.2 ANRIL knock down in primary osteoblasts alters osteoblast- specific pathways 154 6.4 Discussion 163 6.5 Summary 166 6.6 Future work 167 7.0 The association between the methylation status of CDKN2A, RXRA and NOS3 with bone outcomes at birth 169 7.1 Introduction 171 7.1.1 DNA methylation as a marker for a later phenotype 171 7.1.2 Maternal vitamin D and offspring bone health