www.nature.com/scientificreports OPEN Neuron-specifc analysis of histone modifcations with post-mortem brains Kagari Koshi-Mano1, Tatsuo Mano1, Maho Morishima2, Shigeo Murayama2, Akira Tamaoka3, Shoji Tsuji1, Tatsushi Toda1 & Atsushi Iwata1* Histone modifcations govern chromatin structures and regulate gene expression to orchestrate cellular functions in the central nervous system, where neuronal cells are postmitotic and developmentally inactive, the functional and age-dependent changes also accumulate in the epigenetic states. Because the brain is composed of several types of cells, such as the neurons, glial cells, and vascular cells, the analysis of histone modifcations using bulk brain tissue might obscure alterations specifc to neuronal cells. Furthermore, among the various epigenetic traits, analysis of the genome-wide distribution of DNA methylation in the bulk brain is predominantly a refection of DNA methylation of the non- neuronal cells, which may be a potential caveat of previous studies on neurodegenerative diseases using bulk brains. In this study, we established a method of neuron-specifc ChIP-seq assay, which allows for the analysis of genome-wide distribution of histone modifcations specifcally in the neuronal cells derived from post-mortem brains. We successfully enriched neuronal information with high reproducibility and high signal-to-noise ratio. Our method will further facilitate the understanding of neurodegeneration. Histone modifcation is a part of the epigenome that includes covalent post-translational methylation, acetyla- tion, or ubiquitylation of histone proteins. Tese modifcations co-operate with other epigenetic factors1 such as DNA methylation or non-coding RNA, to alter chromatin structures, orchestrate gene expression, and regulate cellular functions, including cell division, growth, and diferentiation during the developmental process2,3. In the central nervous system, where mature neuronal cells are postmitotic and developmentally inactive, histone mod- ifcations play a key role in memory formation and learning process contributing to neuronal plasticity4,5. Aging is also associated with chromatin remodeling, and a better understanding of the phenomenon could be leveraged to induce a variety of responses to restore youthful functionalities in old tissues6–8. Furthermore, in neurodegen- erative conditions, such as Alzheimer’s and Parkinson’s disease, profound efects on histone modifcations are thought to refect the pathogenic neurodegenerative processes9,10. As a result, histone modifcations that exist in mature neuronal cells have a complex structure, post hoc modifcations corresponding to physiological and/or pathological process, layered on a priori modifcation specifc to neuronal cells. Tus, genome-wide profles of histone modifcations specifc to neuronal cells can facilitate the elucidation of physiological mechanisms of the brain related to learning and memory, and pathomechanisms, where various life-long factors converge to cause neurodegeneration. When analyzing histone modifcations in brain samples, we must consider the fact that the brain is composed of several types of cells, including neuronal cells that directly contribute to learning and memory, glial cells that support neuronal activities or provoke infammation, and vascular cells that deliver oxygen and nutrition to the brain. Each type of cell has its own specifc histone modifcation corresponding to its developmental process, and subsequently acquires alterations in the modifcations based on its physiological and pathological condi- tion. Terefore, histone modifcation of the bulk brain derived from the cerebral cortex is a mixture of that of neuronal and non-neuronal origins. Considering that neurons comprise approximately 40%11–13 of all the cells in the cortex, bulk brain analysis is not representative of the neuronal epigenome. Tus, we hypothesized that the genome-wide profles of histone modifcation in neuronal cells cannot be estimated by using bulk brain tissue, 1Department of Neurology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan. 2Department of Neuropathology, Tokyo Metropolitan Geriatric Hospital, 35-2 Sakaecho, Itabashi, Tokyo, 173-0015, Japan. 3Department of Neurology, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305- 8575, Japan. *email: [email protected] SCIENTIFIC REPORTS | (2020) 10:3767 | https://doi.org/10.1038/s41598-020-60775-z 1 www.nature.com/scientificreports/ www.nature.com/scientificreports and this motivated us to develop a method for understanding the genome-wide profles of histone modifcations specifc to neuronal cells. Chromatin immunoprecipitation sequencing (ChIP-seq) is a method used to identify genome-wide profiles of histone modifications, where the genomic DNA that is wrapped around histone proteins is co-immunoprecipitated using a modifcation-specifc anti-histone antibody to prepare libraries for next gen- eration sequencing. For neuron-specifc analysis, we applied fuorescence activated cell sorting (FACS)-based isolation of neuronal nuclei. Formerly, large number of cells was required for robust and reproducible ChIP-seq analysis and this used to be a major challenge for FACS isolation of neuronal nuclei where the number of the nuclei that could be isolated was limited. Especially, for studying neurodegenerative conditions where post-mortem brain samples are used and the amount of sample available for the assay is limited, the number of the nuclei required for the assay should ideally be low. Te condition of the sample used in the assays is also critical for reproducibility because post-mortem brain samples are inevitably afected by the post-mortem time to autopsy and subsequent freeze-thaw processes. To overcome these issues, we optimized each step of the FACS and ChIP-seq that enabled multiple genome-wide histone modifcation analyses. Here, we demonstrate that neuron-specifc histone modifcations are completely diferent from non-neuron-specifc, and bulk brain histone modifcations, emphasizing the importance of neuronal isolation for post-mortem brain epigenome analysis. Results Optimization of crosslinking methods. Te frst step in the ChIP assay is the crosslinking of the nucle- osome, which is composed of genomic DNA wrapped around histone proteins, and uniform reaction across the tissue is essential for reproducibility14. Generally, fxation in the early steps ensures optimum crosslinking. However, when using tissue sample, fxing brain tissue en block has a serious disadvantage in that the surface of the brain may be fxed more than its inside potentially leading to uneven ChIP-seq assay. Given that the sep- aration of neuronal nuclei from non-neuronal ones using FACS is required to achieve specifcity in neuronal ChIP-seq15, optimization of this step is crucial. Terefore, we tested two diferent time points for fxing the nucle- osome complexes; (1) immediately afer homogenization of the frozen brain or (2) afer FACS. All the brains were obtained from the patients without any pathological conditions in the brain. When compared to the yield of genomic DNA extracted before DNA fragmentation, the yield was higher and more reproducible when the nuclei were fxed immediately afer homogenization (Mean ± SD: 26.2 ± 8.4% vs 8.5 ± 10.2%) (Fig. 1a). We speculated that this was because before fxation the bare nuclear membrane can be easily fragmented during FACS. With this method, we obtained 47.4 ± 19.3 neuronal nuclei and 78.8 ± 30.1 non-neuronal nuclei from 100 mg of the brain tissue (Fig. S1a). Separation of neuronal and non-neuronal nuclei confrmed by immunofuorescence staining and western blotting (Figs. 2a and S1b). On the completion of nuclear isolation, the neuronal or non-neuronal nuclei were subjected to sonication to fragment the genomic DNA into lengths of 150–250 bp according to the standard sonication protocol, such that the genomic DNA was fragmented into single nucleosome units (Fig. S2). Optimization of ChIP assay. We then optimized the amount of antibodies used in the immunoprecipi- tation of two representative histone modifcations, H3K4me3 and H3K27ac, that are positively correlated with gene expressions16. In general, the amount of DNA fragments captured by antibodies depends on the amount of antibodies used for the immunoprecipitation, however, excessive amounts of antibodies could potentially result in non-specifc binding and increase undesirable signal in regions without target modifcations. On the other hand, a low yield of DNA fragments requires more PCR cycles resulting in reduced library complexity and skew- ing of the library. Tus, optimization of the amount of antibody is crucial to obtain a good quality library with high sensitivity and specifcity. To validate the yield and specifcity, we performed qPCR with the immunoprecip- itated DNA fragments. To assess the specifcity of our neuronal and non-neuronal ChIP, we chose three genomic regions for qPCR as below, where are supposed to be enriched with the histone modifcations analyzed in this study according to their expressions. ChIP reaction was performed using 2 × 106 nuclei in the reaction volume of 1 mL. We measured the fold enrichment of TSS regions of GAPDH (endogenous positive control), GRIN2B (neuron-specifc marker) and HBB (negative control that is not expressed in the central nervous system)17. Along with the increasing amounts of antibodies, the fold enrichment of GAPDH, which is the universally expressed gene in all tissues, was enhanced