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Human Monocyte-To-Macrophage Dekkers et al. Epigenetics & Chromatin (2019) 12:34 https://doi.org/10.1186/s13072-019-0279-4 Epigenetics & Chromatin RESEARCH Open Access Human monocyte-to-macrophage diferentiation involves highly localized gain and loss of DNA methylation at transcription factor binding sites Koen F. Dekkers1†, Annette E. Neele2†, J. Wouter Jukema3, Bastiaan T. Heijmans1*‡ and Menno P. J. de Winther2,4*‡ Abstract Background: Macrophages and their precursors monocytes play a key role in infammation and chronic infamma- tory disorders. Monocyte-to-macrophage diferentiation and activation programs are accompanied by signifcant epigenetic remodeling where DNA methylation associates with cell identity. Here we show that DNA methylation changes characteristic for monocyte-to-macrophage diferentiation occur at transcription factor binding sites, and, in contrast to what was previously described, are generally highly localized and encompass both losses and gains of DNA methylation. Results: We compared genome-wide DNA methylation across 440,292 CpG sites between human monocytes, naïve macrophages and macrophages further activated toward a pro-infammatory state (using LPS/IFNγ), an anti-infam- matory state (IL-4) or foam cells (oxLDL and acLDL). Moreover, we integrated these data with public whole-genome sequencing data on monocytes and macrophages to demarcate diferentially methylated regions. Our analysis showed that diferential DNA methylation was most pronounced during monocyte-to-macrophage diferentiation, was typically restricted to single CpGs or very short regions, and co-localized with lineage-specifc enhancers irrespec- tive of whether it concerns gain or loss of methylation. Furthermore, diferentially methylated CpGs were located at sites characterized by increased binding of transcription factors known to be involved in monocyte-to-macrophage diferentiation including C/EBP and ETS for gain and AP-1 for loss of methylation. Conclusion: Our study highlights the involvement of subtle, yet highly localized remodeling of DNA methylation at regulatory regions in cell diferentiation. Keywords: DNA methylation, Diferentiation, Epigenomics, Macrophage, Monocyte, Transcription factor *Correspondence: [email protected]; [email protected] †Koen F. Dekkers and Annette E. Neele contributed equally to this work ‡Bastiaan T. Heijmans and Menno P. J. de Winther contributed equally to this work 1 Molecular Epidemiology, Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden, The Netherlands 2 Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, Meibergdreef 9, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dekkers et al. Epigenetics & Chromatin (2019) 12:34 Page 2 of 13 Background macrophage diferentiation. Of interest, such DNA meth- Infammation is characterized by the recruitment of ylation changes are highly localized, often afecting sin- monocytes upon migration from the blood diferenti- gle CpGs only, and primarily located in enhancer regions ated into macrophages [1]. Tese cells participate in defned by specifc transcription factor binding sites. many aspects of infammation such as host defense, tis- Interestingly, we found that gain of methylation, a previ- sue remodeling and wound healing [2]. Due to their ously ignored phenomenon in monocyte–macrophage broad range of functional capacities, macrophages are diferentiation, was associated with increased binding of important regulators of disease outcome. Local environ- lineage determining TFs. mental triggers can induce diferent activation states of macrophages. In vitro, cells treated with lipopolysaccha- Results ride (LPS) plus interferon gamma (IFNγ) or interleukin-4 Marked DNA methylation changes occur (IL-4) are at the two extreme ends of the macrophage during monocyte‑to‑macrophage diferentiation activation spectrum, with the frst having pro-infam- but not upon subsequent macrophage activation matory characteristics and the latter being considered Monocytes were isolated from four healthy donors and anti-infammatory [3]. Foam cells (i.e., macrophage that diferentiated to macrophages in vitro. Tey were sub- have engulfed modifed lipids in the arterial wall) play an sequently activated with LPS/IFNγ, IL-4, oxidized low- important role in the development of atherosclerosis, the density lipoprotein (oxLDL) or acetylated LDL (acLDL) primary cause of cardiovascular disease, which can result to obtain diferent activation states. An overview of the in a myocardial infarction or stroke [4]. Understanding study design and a summary of the primary characteriza- the molecular mechanisms controlling macrophage dif- tion of the cells are shown in Fig. 1 and Additional fle 1: ferentiation and activation will aid in understanding their Figure S1. functioning in health and disease. We evaluated DNA methylation during diferentia- Diferentiation and activation processes of mac- tion and further activation at 440,292 CpGs. Samples rophages are accompanied by pronounced epige- clustered primarily on sex (principal component 1, netic remodeling to accommodate the changes in their 37% variance explained), donor (principal component transcriptional repertoire [5]. DNA methylation is an 2 and 3, 17% and 16% variance explained, respectively), essential component of the epigenome and defnes cell in line with the genetic component of DNA methyla- identity. It occurs predominantly at CpG sites and previ- tion [8, 13] and also on monocyte versus macrophage ous studies identifed loss of methylation as being domi- diferentiation (principal component 4, 6% variance nant during monocyte-to-macrophage diferentiation explained) (Additional fle 1: Figure S2). A paired analy- [6, 7]. While loss of methylation at promoter regions is sis testing for diferences within donors identifed 5870 traditionally thought to increase gene expression, recent data challenge this view indicating that both gain and loss of methylation can be associated with increased tran- scriptional activity in particular at non-promoter regions monocytemacrophage [8, 9]. What is known is that regions of dynamic DNA 6-days methylation between diferent cell types co-localize with enhancers and transcription factor binding sites [10], M-CSF in line with a role of transcription factors in the meth- ylation process. Recent studies showed that changes in 24h DNA methylation can be both a cause [9] and conse- quence [11] of transcription factor binding and may also be involved in stabilizing regulatory states [12]. So while the exact molecular role of DNA methylation appears to be context-specifc, mapping dynamic DNA methylation may be instrumental in identifying the regulatory regions in the genome that control macrophage diferentiation and activation. Here, we investigated both genome-wide and whole- M(LPS/IFNy) M(IL-4) M(oxLDL) M(acLDL) genome DNA methylation changes in monocyte-to-mac- Fig. 1 Study design. Monocytes were isolated from four healthy rophage diferentiation and subsequent activation (LPS/ donors and diferentiated to macrophages in the presence of MCSF. IFNγ, IL-4, modifed lipids). We found that not only loss Macrophages were subsequently activated to obtain diferent but also gain of DNA methylation is common during macrophage subsets Dekkers et al. Epigenetics & Chromatin (2019) 12:34 Page 3 of 13 Genes linked to DMCs are enriched for processes diferentially methylated CpGs (DMCs) (PFDR < 0.05, mean squares > 0.025) between the six cell types (Table 1 involved in monocyte‑to‑macrophage diferentiation and Additional fle 2: Table S2). Te large majority of and macrophage activation for both gain and loss DMCs could be attributed to monocyte-to-macrophage of methylation diferentiation (98%, 5780 DMCs, Table 1). In contrast To identify potential pathways afected by DNA meth- to previous data (6), DMCs included both gain of DNA ylation changes, we subsequently mapped the DMCs methylation (gain-DMCs, n = 4283) and loss of DNA to their nearest genes (Additional fle 2: Table S2). We methylation (loss-DMCs, n = 1497), with the frst being observed that these genes included hallmark examples most common. DMCs had a mean methylation level that of genes involved in monocyte-to-macrophage difer- was uniformly distributed from ~ 0 to ~ 100%, and like- entiation for both gain-DMCs (e.g., IRF8, CEBPB) and wise the diference between monocytes and macrophages loss-DMCs (e.g., PPARG) [14, 15] (Fig. 2). Additionally, was similar across methylation levels (mean diference in we also found genes for LPS/IFNγ macrophage-specifc methylation: 13%, Additional fle 1: Figure S3). However, activation (e.g., CCL5). some DMCs switched from hypomethylated to hyper- Pathway analysis showed strong enrichment for pro- methylated and vice versa during diferentiation (Addi- cesses involved in monocyte-to-macrophage diferen- tional fle 1: Figure S3).
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