bioRxiv preprint doi: https://doi.org/10.1101/274043; this version posted March 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Chromatin accessibility landscape of articular knee cartilage reveals aberrant 2 enhancer regulation in osteoarthritis 3 4 Ye Liu1,2+, Jen-Chien Chang1+, Chung-Chau Hon3, Naoshi Fukui4,5, Nobuho Tanaka4, 5 Zhenya Zhang2, Ming Ta Michael Lee6,7,8*, Aki Minoda1* 6 7 1. Epigenome Technology Exploration Unit, Division of Genomic Technologies, 8 RIKEN Center for Life Science Technologies (CLST), Yokohama, Japan. 9 2. Graduate School of Life and Environmental Sciences, University of Tsukuba, 10 Tsukuba, Japan. 11 3. Genome Information Analysis Team, Division of Genomic Technologies, RIKEN 12 Center for Life Science Technologies (CLST), Yokohama, Japan. 13 4. Clinical Research Center, National Hospital Organization Sagamihara Hospital, 14 Kanagawa, Japan. 15 5. Department of Life Sciences, Graduate School of Arts and Sciences, The University 16 of Tokyo, Tokyo, Japan. 17 6. Genomic Medicine Institute, Geisinger, Danville, PA, USA. 18 7. Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC. 19 8. Laboratory for International Alliance on Genomic Research, RIKEN Center for 20 Integrative Medical Sciences, Yokohama, Japan. 21 22 + Authors contributed equally to this work 23 * Co-corresponding. Address correspondence to Aki Minoda, Epigenome Technology 24 Exploration Unit, Division of Genomic Technologies, RIKEN Center for Life Science 1 bioRxiv preprint doi: https://doi.org/10.1101/274043; this version posted March 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 25 Technologies (CLST), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. 26 E-mail: [email protected]; Tel: 81-45-503-9309. 27 Ming Ta Michael Lee, Genomic Medicine Institute, Geisinger Health System, 100 N. 28 Academy Ave. Danville, PA, 17822, USA. E-mail: [email protected]; Tel: 1-570- 29 214-1005. 30 31 ABSTRACT 32 Background: Osteoarthritis (OA) is a common joint disorder with increasing impact 33 in an aging society; however, there is no cure or effective treatments so far due to lack 34 of sufficient understanding of its pathogenesis. While genome-wide association studies 35 (GWAS) and DNA methylation profiling identified many non-coding loci associated 36 to OA, the interpretation of them remains challenging. 37 Methods: Here, we employed Assay for Transposase-Accessible Chromatin with high 38 throughput sequencing (ATAC-seq) to map the accessible chromatin landscape in 39 articular knee cartilage of OA patients and to identify the chromatin signatures relevant 40 to OA. 41 Results: We identified 109,215 accessible chromatin regions in cartilage and 71% of 42 these regions were annotated as enhancers. We found these accessible chromatin 43 regions are enriched for OA GWAS single nucleotide polymorphisms (SNPs) and OA 44 differentially methylated loci, implying their relevance to OA. By linking these 45 enhancers to their potential target genes, we have identified a list of candidate enhancers 46 that may be relevant to OA. Through integration of ATAC-seq data with RNA-seq data, 47 we identified genes that are altered both at epigenomic and transcriptomic levels. These 48 genes are enriched in pathways regulating ossification and mesenchymal stem cell 49 (MSC) differentiation. Consistently, the differentially accessible regions in OA are 2 bioRxiv preprint doi: https://doi.org/10.1101/274043; this version posted March 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 50 enriched for mesenchymal stem cell-specific enhancers and motifs of transcription 51 factor families involved in osteoblast differentiation (e.g. bZIP and ETS). 52 Conclusions: This study marks the first investigation of accessible chromatin 53 landscape on clinically relevant hard tissues and demonstrates how accessible 54 chromatin profiling can provide comprehensive epigenetic information of a disease. 55 Our analyses provide supportive evidence towards the model of endochondral 56 ossification-like cartilage-to-bone conversion in OA knee cartilage, which is consistent 57 with the OA characteristic of thicker subchondral bone. The identified OA-relevant 58 genes and their enhancers may have a translational potential for diagnosis or drug 59 targets. 60 61 Keywords 62 osteoarthritis, epigenomics, enhancer, ATAC-seq, endochondral ossification. 63 64 Background 65 Osteoarthritis (OA) is a degenerative joint disease [1,2] that is one of the most common 66 causes of chronic disability in the world [3,4], of which the knee OA is the most 67 common. Main features of OA include cartilage degradation, subchondral bone 68 thickening, joint space narrowing and osteophytes formation [5], resulting in stiffness, 69 swelling, and pain in the joint. Currently available treatments are either pain relief or 70 joint function improvement by strengthening the supporting muscles. However, OA 71 progression ultimately leads to costly total joint replacement surgery, making it a 72 growing global health burden. 73 Although the causes of OA are not well understood, risk factors such as age, 74 weight, gender, and genetic factors have been identified [4]. Several models for OA 75 initiation, such as mechanical injury, inflammatory mediators from synovium, defects 3 bioRxiv preprint doi: https://doi.org/10.1101/274043; this version posted March 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 76 in metabolism and endochondral ossification, have been proposed to explain 77 pathogenesis of this disease [6–12]. To date, GWAS have identified more than 20 loci 78 to be associated with the risk of developing OA [13]. While next generation sequencing 79 data is being generated to discover rare variants with larger effect size, the identified 80 variants are often located in the non-coding regions of the genome [14], complicating 81 the identification of the causal genes. Transcriptomic analyses of cartilage in diseased 82 joints of OA patients (taken from the replacement surgeries) provided an opportunity 83 to pinpoint transcriptionally dysregulated genes and pathways relevant to OA [15,16]. 84 However, such studies have yet to fully reveal the underlying molecular mechanism of 85 how the transcription of these genes are dysregulated. 86 Recently, epigenetic tools have been applied to gain further insight into the 87 pathogenesis of OA. There have been several reports of DNA methylation status [17– 88 19] in cartilage of diseased joints. However, relatively few DNA methylation 89 alterations were found in OA degenerated tissues, despite many genes being 90 differentially expressed. Consistent with these findings, it was also found that change 91 of gene expression is rarely associated with DNA methylation alterations at promoters 92 [16,19,20]. Many of the identified differentially methylated sites in fact fall in enhancer 93 regions, which are non-coding regulatory elements, disruption of which may lead to 94 dysregulated transcription, and many are cell type-specific [21,22]. Recent large-scale 95 studies, such as FANTOM5 [23], Roadmap Epigenomics Project [24] and GTEx [25] 96 have enabled the prediction of regulatory networks between enhancers and their 97 potential target genes (e.g. JEME [26]), which could be applied in a clinical context to 98 explore the roles of enhancers in disease pathogenesis. 99 Here, we set out to investigate alterations of enhancers associated with OA by 100 applying ATAC-seq [27] on the knee joint cartilages from OA patients, using an 4 bioRxiv preprint doi: https://doi.org/10.1101/274043; this version posted March 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 101 optimized protocol for cartilage sample preparation. ATAC-seq maps the accessible 102 chromatin regions, which are often regulatory regions such as promoters and enhancers 103 that play roles in regulation of gene expression. By integrating our ATAC-seq data with 104 the publicly available genetic, transcriptomic and epigenomic data, we identified 105 dysregulated enhancers and their potential target genes. Our data highlights a number 106 of OA risk loci and differentially methylated loci that potentially play roles in cartilage 107 degradation during OA development. 108 109 Methods 110 Knee joint tissues collection 111 Human knee joint tibial plates were collected from patients who undertook joint 112 replacement surgery due to severe primary OA at the National Hospital Organization 113 Sagamihara Hospital (Kanagawa, Japan). Demographic information for patients is 114 listed in Additional file 1: Table S1. Diagnosis of OA was based on the criteria of the 115 American College of Rheumatology [28], and all the knees were medially involved in 116 the disease. Tissues were stored in 4°C cultured medium after removal. Informed 117 consent was obtained from each patient enrolled in this study. This study was approved 118 by the institutional review board of all the participating institutions (Sagamihara 119 National Hospital and RIKEN). 120 121 Processing of cartilage samples 122 Fresh cartilage was separated from the subchondral bone by a scalpel immediately after 123 surgery and stored in 4°C Dulbecco's Modified Eagle Medium until they were 124 processed. 100 mg cartilage from both outer region of lateral tibial plateau (oLT) and 125 inner region of medial tibial plateau (iMT) (Fig. 1a and Additional file 2: Figure S1a) 5 bioRxiv preprint doi: https://doi.org/10.1101/274043; this version posted March 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.