ATAC-Me Captures Prolonged DNA Methylation of Dynamic Chromatin Accessibility Loci during Cell Fate Transitions.

DNA methylation of enhancers is dynamic, cell-type specific, and vital for cell fate progression. However, current models inadequately define its role within the hierarchy of gene regulation. Analysis of independent datasets shows an unanticipated overlap between DNA methylation and chromatin accessibility at enhancers of steady-state stem cells, suggesting that these two opposing features might exist concurrently. To define their temporal relationship, we developed ATAC-Me, which probes accessibility and methylation from single DNA library preparations. We identified waves of accessibility occurring rapidly across thousands of myeloid enhancers in a monocyte-to-macrophage cell fate model. Prolonged methylation states were observed at a majority of these sites, while transcription of nearby genes tracked closely with accessibility. ATAC-Me uncovers a significant disconnect between chromatin accessibility, DNA methylation status, and gene activity. This unexpected observation highlights the value of ATAC-Me in constructing precise molecular timelines for understanding the role of DNA methylation in gene regulation.

Cross-tissue patterns of DNA hypomethylation reveal genetically distinct histories of cell development.

BACKGROUND: Establishment of DNA methylation (DNAme) patterns is essential for balanced multi-lineage cellular differentiation, but exactly how these patterns drive cellular phenotypes is unclear. While > 80% of CpG sites are stably methylated, tens of thousands of discrete CpG loci form hypomethylated regions (HMRs). Because they lack DNAme, HMRs are considered transcriptionally permissive, but not all HMRs actively regulate genes. Unlike promoter HMRs, a subset of non-coding HMRs is cell type-specific and enriched for tissue-specific gene regulatory functions. Our data further argues not only that HMR establishment is an important step in enforcing cell identity, but also that cross-cell type and spatial HMR patterns are functionally informative of gene regulation. RESULTS: To understand the significance of non-coding HMRs, we systematically dissected HMR patterns across diverse human cell types and developmental timepoints, including embryonic, fetal, and adult tissues. Unsupervised clustering of 126,104 distinct HMRs revealed that levels of HMR specificity reflects a developmental hierarchy supported by enrichment of stage-specific transcription factors and gene ontologies. Using a pseudo-time course of development from embryonic stem cells to adult stem and mature hematopoietic cells, we find that most HMRs observed in differentiated cells (~ 60%) are established at early developmental stages and accumulate as development progresses. HMRs that arise during differentiation frequently (~ 35%) establish near existing HMRs (≤ 6 kb away), leading to the formation of HMR clusters associated with stronger enhancer activity. Using SNP-based partitioned heritability from GWAS summary statistics across diverse traits and clinical lab values, we discovered that genetic contribution to trait heritability is enriched within HMRs. Moreover, the contribution of heritability to cell-relevant traits increases with both increasing HMR specificity and HMR clustering, supporting the role of distinct HMR subsets in regulating normal cell function. CONCLUSIONS: Our results demonstrate that the entire HMR repertoire within a cell-type, rather than just the cell type-specific HMRs, stores information that is key to understanding and predicting cellular phenotypes. Ultimately, these data provide novel insights into how DNA hypo-methylation provides genetically distinct historical records of a cell's journey through development, highlighting HMRs as functionally distinct from other epigenomic annotations.

Adipose-Derived Stromal Vascular Fraction and Cultured Stromal Cells as Trophic Mediators for Tendon Healing.

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous population of cells that yields a homogeneous population of plastic-adherent adipose tissue-derived stromal cells (ASC) when culture-expanded. SVF and ASC have been used clinically to improve tendon healing, yet their mechanism of action is not fully elucidated. The objective of this study was to investigate the potential for ASC to act as trophic mediators for tendon healing. Flexor digitorum superficialis tendons and adipose tissue were harvested from adult horses to obtain SVF, ASC, and tenocytes. Growth factor gene expression was quantified in SVF and ASC in serial passages and growth factors were quantified in ASC-conditioned medium (CM). Microchemotaxis assays were performed using ASC-CM. Tenocytes were grown in co-culture with autologous ASC or allogeneic SVF. Gene expression for insulin-like growth factor 1 (IGF-1), stromal cell-derived factor-1α (SDF-1α), transforming growth factor-ß1 (TGF-ß1) and TGF-ß3 was significantly higher in SVF compared to ASC. Concentrations were significantly increased in ASC-CM compared to controls for IGF-1 (4-fold) and SDF-1α (6-fold). Medium conditioned by ASC induced significant cell migration in a dose-dependent manner. Gene expression for collagen types I and III, decorin, and cartilage oligomeric matrix protein was modestly, but significantly increased following co-culture of tenocytes with autologous ASC. Our findings support the ability of SVF and ASC to act as trophic mediators in tendon healing, particularly through chemotaxis, which stands to critically impact the intrinsic healing response. In vivo studies to further delineate the potential for SVF and/or ASC to improve tendon healing are warranted. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1429-1439, 2019.