ChIP-seq of plasma cell-free nucleosomes identifies gene expression programs of the cells of origin

Abstract

Blood contains cfDNA fragments derived from dying cells 1 . cfDNA has a half-life of ~15 min 2 and, therefore, represents events that occurred close to sampling time. cfDNA analysis is used for assessment of fetus chromosomal aberrations, graft rejection, monitoring tumor dynamics and targeted treatment [3][4][5][6][7] . These applications rely on genetic differences between the host and the tissue of interest. Analysis of CpG methylation in cfDNA is emerging as an alternative independent of genetic alteration 5,[8][9][10][11] . CpG methylation profiles are determined during differentiation and are stable afterwards and, thus, are highly informative about cell identity (for example, liver or lung). However, genetic and methylation-based approaches do not report on recent transcriptional events, as mutations and methylation changes occur over developmental time scales.The basic repeating unit of chromatin is the nucleosome, which is a histone-DNA complex encompassing ~150 base pairs (bp) of DNA 12 . Histone proteins are subject to multiple covalent modifications, which are involved in nearly all aspects of messenger RNA (mRNA) biogenesis [13][14][15][16] . Histone modification patterns reflect recent events related to chromatin regulation and activity of RNA polymerase 13,15 , and different combinations of such modifications mark the location and activity of non-coding regions, enhancers, promoters and gene bodies [17][18][19][20][21][22] . Chromatin immunoprecipitation and sequencing (ChIP-seq) enables genome-wide mapping of histone modifications and provides detailed understanding of the regulatory activity within cells [17][18][19][23][24][25][26][27] .Upon cell death, the genome is fragmented, and chromatin, mostly in the form of nucleosomes, is released into the circulation as cell-free nucleosomes (cf-nucleosomes) 28-30 that retain some histone modifications [31][32][33] . We reasoned that capturing and DNA sequencing of modified nucleosomes from plasma might inform on DNA-related activities, including transcription, within the cells of origin (Fig. 1a). This currently inaccessible epigenetic information extends beyond cfDNA modalities examined to date [4][5][6][7][8][9][10][11][34][35][36][37][38][39][40][41][42][43] .In this study, we performed chromatin immunoprecipitation and sequencing of cell-free nucleosomes directly from human plasma (cfChIP-seq). We show that cfChIP-seq recapitulates the original genomic distribution of modifications associated with transcriptionally active promoters, enhancers and gene bodies, demonstrating that plasma nucleosomes retain the epigenetic information of their

Cell Types of Origin in the Cell Free Transcriptome in Human Health and Disease

“…S5C). The predominant cell types and their respective proportions we observe are generally consistent with recently published estimates for serum cfRNA 1 and plasma cfDNA 16 . We also observed small fractional contributions from endothelial cells, pancreatic cells, intestinal enterocytes, kidney epithelia, club cells, goblet cells, pancreatic acinar cells, and other pancreatic cells (Fig.…”

Epigenetics of Hepatic Insulin Resistance

“…Such regions could offer biomarkers which may inform appropriate lifestyle interventions to reduce risk of IR-related disease. More recently, Sadeh et al developed a method to identify liver-specific histone modification signatures in plasma cell-free nucleosomes using ChIP-seq (249). Despite the relatively small sample size and patient heterogeneity, the authors were able to detect liverdisease associated changes in histone modification enrichment at specific loci (249).…”

“…More recently, Sadeh et al developed a method to identify liver-specific histone modification signatures in plasma cell-free nucleosomes using ChIP-seq (249). Despite the relatively small sample size and patient heterogeneity, the authors were able to detect liverdisease associated changes in histone modification enrichment at specific loci (249). This methodology is expected to be taken up in the future to facilitate the high-throughput interrogation of disease signatures in patient blood samples.…”

“…Due to higher associated costs and technical challenges, the profiling of histone modifications has not really matched the pace of methylome-wide studies. Nevertheless, exciting research has recently demonstrated the potential to once more harness the correlation between liver and blood epigenomic profiles, this time by performing ChIP-seq on circulating cell-free DNA (249). Future studies are expected to leverage on this new technology to identify additional biomarkers of hepatic IR, assisting in the prevention of NAFLD, T2D and other cardiometabolic diseases, which together have an enormous health, societal and economic impact.…”

Detection of Cell Types Contributing to Cancer From Circulating, Cell-Free Methylated DNA

“…However, signal abundance can be increased by leveraging signals from all cfDNA molecules rather than the smaller subset of fragments containing specific tumorrelated mutations (Figures 2A,C). This can be accomplished by targeting tumor-specific epigenetic changes that occur early on during carcinogenesis and thus are found at higher abundance in early stage cancers than tumor-related mutations (Snyder et al, 2016;Ulz et al, 2016;Wong et al, 2016;Leygo et al, 2017;Jiang et al, 2018Jiang et al, , 2019Cristiano et al, 2019;Gai and Sun, 2019;Ivanov et al, 2019;Panagopoulou et al, 2019;Sun et al, 2019;Van der pol and Mouliere, 2019;Sadeh et al, 2021). Further, combining tumor-cell derived signals with those from the surrounding host microenvironment can increase signal abundance (Hoadley et al, 2018;Liu et al, 2018;Haigis et al, 2019;Lam et al, 2019).…”