Single-Cell Chromatin Modification Profiling Reveals Increased Epigenetic Variations with Aging

“We identified differentially expressed genes using the DESeq2 algorithm on the read count matrix (Love et al., 2014).”

“Next, we used L-1 regularized logistic regression models (Friedman et al., 2010) to classify immune cells based on chromatin marks alone utilizing single-cell data from bio rep 1 as the training dataset.”

“We combined the means from both panels (separately for standard and T cell focused panels) and integrated multiple batches by applying ComBat normalization (Johnson et al., 2007).”

“Together with the discoveries of higher euchromatin-associated H3K27me1 and H3K36me1 (Barski et al., 2007; Wagner and Carpenter, 2012) and lower heterochromatin-associated H3K27me3, H4K20me3, and H3K9me2 (Trojer and Reinberg, 2007) in memory T cells, our results suggest that chromatin in memory T…”

“Chromatin marks known to be present at low levels in unperturbed cells, such as DNA double-strand breakinduced g-H2AX (Rogakou et al., 1998), and citrullination marks (Cuthbert et al.”

“Pivotal roles of PRCs in transcription silencing and chromatin compaction have been extensively described (Margueron and Reinberg, 2011).”

“While recent technologic breakthroughs have allowed investigations of chromatin dynamics using a small number of cells or even single cells (Buenrostro et al., 2015; Corces et al., 2016; Gomez et al., 2013; Rotem et al., 2015; Schwartzman and Tanay, 2015), a high-throughput method to measure the overall cellular level…”

“We leveraged the highlymultiplexed capacity and single-cell resolution of mass cytometry (Bendall et al., 2011) to develop EpiTOF for simultaneous measurement of chromatin marks in single immune cells.”

“…dataset (GEO: GSE18927) from the Roadmap Epigenomics Mapping Consortium, where hallmarks of gene silencing and active transcription and H3K27me3 and H3 tri-methylation at lysine 4 (H3K4me3), respectively, were profiled in sorted peripheral CD3+ T cells and CD14+ monocytes (Bernstein et al., 2010).”

“…that showed a statistically significant increase in CV in older subjects (FDR < 5%), more than 50% (17 of 29) involved H3K27me3, H3K27me2, and H2AK119ub, all of which are catalyzed by polycomb-repressive complex 1 or 2 (PRC1 or PRC2) (Di Croce and Helin, 2013; Wang et al., 2004a) (Figure 5B).”

“These chemical moieties alter the architecture of chromatin (Kouzarides, 2007) or serve as docking sites for ‘‘reader’’ proteins that dictate higher-order chromatin compaction or recruit protein complexes with DNA-related biological functions (Taverna et al., 2007).”

“The CD56dim subset, which accounts for approximately 90% of total NK cells, has stronger cytotoxic killer cell functions than the low-abundance CD56bright population (Lanier et al., 1986), whereas the latter is the major source of secreted cytokines (Cooper et al., 2001).”

“Quantification of cell-to-cell variation in chromatin levels (Figure 5) Wemeasured cell-to-cell variation in chromatin levels by using the coefficient of variation (CV) (Martinez-Jimenez et al., 2017; Raser and O’Shea, 2004).”

“The results suggest that this group of genes is differentially regulated, at the levels of both H3K27me3 enrichment and transcription, during the lineage commitment of CLPs and CMPs. Gene ontology (GO) enrichment analysis revealed that a significant number of these genes are involved in the differentiation of megakary…”

“We analyzed the data in conjunction with a ChIP-seq dataset mapping H3K27me3 and H3K4me3 occupancies at genes with vital immune functions and critical roles in cell differentiation in naive CD4+ T cells purified from the same C57BL/6J substrain (Wei et al., 2009).”

“Using publicly available RNA sequencing (RNA-seq) data from sorted immune cells (GEO: GSE74246) (Corces et al., 2016), we found that the expression of EZH1 and EZH2, the two lysine methyltransferases catalyzing H3K27me3, is significantly higher in common lymphoid progenitors (CLPs) and in peripheral T cells than in co…”

“The CD56dim subset, which accounts for approximately 90% of total NK cells, has stronger cytotoxic killer cell functions than the low-abundance CD56bright population (Lanier et al., 1986), whereas the latter is the major source of secreted cytokines (Cooper et al., 2001).”

“…Modification Profiling Reveals Increased Epigenetic Variations with Aging, Cell (2018), https://doi.org/10.1016/j.cell.2018.03.079 influences (Brodin et al., 2015) led us to postulate that environmental and/or other non-heritable factors are the primary driving force for variations in…”

“…that showed a statistically significant increase in CV in older subjects (FDR < 5%), more than 50% (17 of 29) involved H3K27me3, H3K27me2, and H2AK119ub, all of which are catalyzed by polycomb-repressive complex 1 or 2 (PRC1 or PRC2) (Di Croce and Helin, 2013; Wang et al., 2004a) (Figure 5B).”

“Together with the discoveries of higher euchromatin-associated H3K27me1 and H3K36me1 (Barski et al., 2007; Wagner and Carpenter, 2012) and lower heterochromatin-associated H3K27me3, H4K20me3, and H3K9me2 (Trojer and Reinberg, 2007) in memory T cells, our results suggest that chromatin in memory T…”

“…of megakaryocyte-erythroid progenitors (MEPs) and granulocyte-monocyte progenitors (GMPs), such as GATA1, PU.1, and C/EBPa (Pevny et al., 1991; Scott et al., 4 Cell 173, 1–13, May 31, 2018 1994; Zhang et al., 1997), in addition to myeloid cell-specific markers, such as CD33 and PADI4.”

“…that showed a statistically significant increase in CV in older subjects (FDR < 5%), more than 50% (17 of 29) involved H3K27me3, H3K27me2, and H2AK119ub, all of which are catalyzed by polycomb-repressive complex 1 or 2 (PRC1 or PRC2) (Di Croce and Helin, 2013; Wang et al., 2004a) (Figure 5B).”

“…of chromatin dynamics using a small number of cells or even single cells (Buenrostro et al., 2015; Corces et al., 2016; Gomez et al., 2013; Rotem et al., 2015; Schwartzman and Tanay, 2015), a high-throughput method to measure the overall cellular levels of chromatin marks in individual…”

“, 1998), and citrullination marks (Cuthbert et al., 2004; Wang et al., 2004b) had very low signals in EpiTOF.”

“This group of genes contains key regulators of lymphocyte differentiation, such as BLIMP1, FOXP1, and IL7R (Hu et al., 2006; Mazzucchelli and Durum, 2007; Turner et al., 1994).”

“We analyzed data utilizing the classical ACE model, which calculates the fractions of variance attributed to additive genetics (A), common environment (C) or unique environment (E) (Rijsdijk and Sham, 2002).”

“Recent publications have described increased variability in the immune systems of older versus younger human beings (Brodin and Davis, 2017; Kaczorowski et al., 2017).”

“It has been proposed thatMZ twins start with identical epigenomes in the zygotes and, therefore, remain more similar to each other than DZ twins, who develop from independent zygotes with distinct epigenomes (Petronis, 2010).”

“Our findings are in agreement with a comprehensive study showing increases in both H3K4me3 and H3K27me3 marks in murine HSCs from old mice (Sun et al., 2014).”

“…H3K27me1 and H3K36me1 (Barski et al., 2007; Wagner and Carpenter, 2012) and lower heterochromatin-associated H3K27me3, H4K20me3, and H3K9me2 (Trojer and Reinberg, 2007) in memory T cells, our results suggest that chromatin in memory T cells is maintained in a more accessible state than in…”

“Closer concordance in MZ twins than in DZ twins in DNA methylation suggests that heritable influences are not trivial (Kaminsky et al., 2009).”

“Unsupervised clustering based on chromatin marks revealed that T cells expressing ab T cell receptors (TCRs) are characterized by a distinct chromatin modification profile when compared with T cells expressing gd TCRs, which have specialized functions in immune responses (Chien et al., 2014) (Figures 3A and S3A).”

“This group of genes contains key regulators of lymphocyte differentiation, such as BLIMP1, FOXP1, and IL7R (Hu et al., 2006; Mazzucchelli and Durum, 2007; Turner et al., 1994).”

“In memory T cells, phosphorylation of H3 at serine 10 (H3S10ph), which is detected at the transcription start sites of actively transcribed genes (Agalioti et al., 2002), is highly elevated.”

“We found that in addition to the upregulation of EZH1 and EZH2 in Tregs, the expression of KDM6A/UTX and KDM6B/JMJD3, which catalyze H3K27 demethylation (Shi, 2007), is also elevated (Figure S3C).”

“The process of aging is known to involve multiple genes and pathways and, importantly, epigenetic regulation (Benayoun et al., 2015; Dorshkind et al., 2009).”

“Elevated single-cell transcriptional variability with age has been reported in several studies (Bahar et al., 2006; Enge et al., 2017; Martinez-Jimenez et al., 2017); however, the molecular mechanisms underlying the transcriptional variability remain unclear.”

“Elevated single-cell transcriptional variability with age has been reported in several studies (Bahar et al., 2006; Enge et al., 2017; Martinez-Jimenez et al., 2017); however, the molecular mechanisms underlying the transcriptional variability remain unclear.”

“Aging-associated histone loss has been reported in several species, and reduced nucleosome occupancy with age has been implicated in changes in chromatin dynamics and transcription activities (Booth and Brunet, 2016).”

“The process of aging is known to involve multiple genes and pathways and, importantly, epigenetic regulation (Benayoun et al., 2015; Dorshkind et al., 2009).”

“Our findings are highly similar to results from twin analyses on DNA methylation, where environmental influences explain approximately 80% of the variance in DNA methylation (McRae et al., 2014; van Dongen et al., 2016).”

“Cell-cycle-independent incorporation of histone variants further diversifies chromatin states and provides an additional layer of regulation to DNA-templated processes (Maze et al., 2014).”

“Hallmarks of aging include declining hematopoietic output, defective immune cell functions, and an elevated proportion of CD45RO+ memory T cell subsets as we observed in our cohort (Figure S4A) (Wertheimer et al., 2014).”

“Nucleosomes constituted with histone variants change the structure and stability of chromatin and, through unique protein motifs or variant-specific PTMs, alter interactions with chromatin binding proteins (Talbert and Henikoff, 2017).”

“Quantification of cell-to-cell variation in chromatin levels (Figure 5) Wemeasured cell-to-cell variation in chromatin levels by using the coefficient of variation (CV) (Martinez-Jimenez et al., 2017; Raser and O’Shea, 2004).”

“The essential roles of EZH2 in the functions of Tregs have been studied extensively (Arvey et al., 2014; DuPage et al., 2015).”

“This group of genes contains key regulators of lymphocyte differentiation, such as BLIMP1, FOXP1, and IL7R (Hu et al., 2006; Mazzucchelli and Durum, 2007; Turner et al., 1994).”

“While recent technologic breakthroughs have allowed investigations of chromatin dynamics using a small number of cells or even single cells (Buenrostro et al., 2015; Corces et al., 2016; Gomez et al., 2013; Rotem et al., 2015; Schwartzman and Tanay, 2015), a high-throughput method to measure the overall cellular level…”

“Our findings are highly similar to results from twin analyses on DNA methylation, where environmental influences explain approximately 80% of the variance in DNA methylation (McRae et al., 2014; van Dongen et al., 2016).”

“…have allowed investigations of chromatin dynamics using a small number of cells or even single cells (Buenrostro et al., 2015; Corces et al., 2016; Gomez et al., 2013; Rotem et al., 2015; Schwartzman and Tanay, 2015), a high-throughput method to measure the overall cellular levels of chromatin…”

“Recent reports have found substantial chromatin changes as a result of aging (Moskowitz et al., 2017; Ucar et al., 2017).”

“Recent reports have found substantial chromatin changes as a result of aging (Moskowitz et al., 2017; Ucar et al., 2017).”

“While the CD56bright subset is historically considered the precursor of CD56dim NK cells, recent evidence suggests that the two subsets differentiate from independent hematopoietic lineages (Wu et al., 2014).”

“We found that in addition to the upregulation of EZH1 and EZH2 in Tregs, the expression of KDM6A/UTX and KDM6B/JMJD3, which catalyze H3K27 demethylation (Shi, 2007), is also elevated (Figure S3C).”

“Recent publications have described increased variability in the immune systems of older versus younger human beings (Brodin and Davis, 2017; Kaczorowski et al., 2017).”

“This hypothesis helps explain our results on chromatin modifications in twins; however, a unified mechanism for the patterns of chromatin modifications to be reestablished during mitotic cell division remains unclear (Zhu and Reinberg, 2011).”

“Similarly, chromatin modification analyses in blood, while clearly indicating immune cell types, also identify an increase in cell-to-cell variability with age, or “epigenomic noise,” with particular increases in both H3K4me3 and H3K27me3 [177].”

“…suggested including DNA damage, increased epigenetic variations, release of mitochondrial DNA, age-related leakage of intestinal microbiota, persistent viral infections (e.g., with cytomegalovirus), or cellular immunosenescence (Cheung et al., 2018; Cunha et al., 2020; Franceschi et al., 2017).”