DNA Methylation Dynamics of Human Hematopoietic Stem Cell Differentiation

摘要

class="head no_bottom_margin" id="sec1title">IntroductionAll blood cells originate from hematopoietic stem cells (HSCs), which represent the apex of a differentiation cascade of progenitor cell types that gives rise to billions of new cells every day. HSC differentiation is believed to progress through stepwise restriction of lineage potential, a concept that is summarized by the classical tree model of murine hematopoiesis (, ).HSC differentiation in human is less well understood than in mouse. Despite recent progress (reviewed in , , ), several aspects of human hematopoiesis have remained controversial (, , , , , , ).We sought to use DNA methylation for in vivo dissection of human hematopoiesis. DNA methylation is well suited for studying cellular differentiation because its patterns are cell-type-specific and retain an epigenetic memory of a cell’s developmental history. For example, cell-of-origin-specific DNA methylation patterns are detectable among induced pluripotent stem cells (, ), and such patterns of epigenetic tissue memory predict primary tumor location in metastatic cancers (, href="#bib35" rid="bib35" class=" bibr popnode">Moran et al., 2016).Previous studies have established a close connection between stem cell differentiation and widespread epigenome remodeling. DNA methylation has been studied in early mammalian development (href="#bib44" rid="bib44" class=" bibr popnode">Smallwood et al., 2011, href="#bib45" rid="bib45" class=" bibr popnode">Smith et al., 2012), mouse HSC differentiation (href="#bib5" rid="bib5" class=" bibr popnode">Bock et al., 2012, href="#bib9" rid="bib9" class=" bibr popnode">Cabezas-Wallscheid et al., 2014, href="#bib21" rid="bib21" class=" bibr popnode">Ji et al., 2010), neural differentiation (href="#bib31" rid="bib31" class=" bibr popnode">Lister et al., 2013), pluripotent stem cells (href="#bib4" rid="bib4" class=" bibr popnode">Bock et al., 2011, href="#bib20" rid="bib20" class=" bibr popnode">Habibi et al., 2013), and a broad collection of human tissue samples (href="#bib28" rid="bib28" class=" bibr popnode">Kundaje et al., 2015, href="#bib57" rid="bib57" class=" bibr popnode">Ziller et al., 2013). Chromatin accessibility has been mapped using the assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) in multiple cell types of the human blood lineage (href="#bib11" rid="bib11" class=" bibr popnode">Corces et al., 2016), and three recent studies used chromatin immunoprecipitation sequencing (ChIP-seq) to map histone modifications in the developing mouse embryo (href="#bib12" rid="bib12" class=" bibr popnode">Dahl et al., 2016, href="#bib32" rid="bib32" class=" bibr popnode">Liu et al., 2016, href="#bib56" rid="bib56" class=" bibr popnode">Zhang et al., 2016).To establish a basis for epigenome-wide analysis and data-driven modeling of the human hematopoietic lineage, we applied our protocol for low-input and single-cell whole genome bisulfite sequencing (href="#bib15" rid="bib15" class=" bibr popnode">Farlik et al., 2015) to 17 hematopoietic cell types (href="/pmc/articles/PMC5145815/figure/fig1/" target="figure" class="fig-table-link figpopup" rid-figpopup="fig1" rid-ob="ob-fig1" co-legend-rid="lgnd_fig1">Figure 1A). HSCs and multipotent progenitors (MPPs) were sorted from fetal liver, cord blood, bone marrow, and peripheral blood. Eight additional progenitor cell types and six differentiated cell types were sorted from peripheral blood, and megakaryocytes were sorted from bone marrow. For each stem and progenitor cell type, we sequenced an average of 32 low-input methylomes from three individuals, and we bioinformatically integrated them into meta-epigenomic profiles (href="#bib53" rid="bib53" class=" bibr popnode">Wijetunga et al., 2014). Additionally, we sequenced an average of 26 single-cell methylomes for seven cell types (HSC, MPP, common lymphoid progenitor [CLP], common myeloid progenitor [CMP], immature multi-lymphoid progenitor [MLP0], granulocyte macrophage progenitor [GMP], and megakaryocytes) to assess cell-to-cell heterogeneity.href="/pmc/articles/PMC5145815/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">class="inline_block ts_canvas" href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=5145815_gr1.jpg" target="tileshopwindow">target="object" href="/pmc/articles/PMC5145815/figure/fig1/?report=objectonly">Open in a separate windowclass="figpopup" href="/pmc/articles/PMC5145815/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">Figure 1Charting the DNA Methylation Landscape of Human Hematopoietic Differentiation(A) Conceptual outline of human hematopoietic differentiation, highlighting the 17 hematopoietic cell types whose genome-wide DNA methylation patterns were profiled in this study. Arrows denote established differentiation trajectories, dashed arrows indicate uncertainty about the in vivo differentiation potential of lymphoid progenitors, and the inset illustrates the sorting of four subsets of immature multi-lymphoid progenitors.(B) Fluorescence-activated cell sorting panel used to purify 10 stem and progenitor cell types from peripheral blood.(C) Violin plots and boxplots showing the distribution of DNA methylation levels in 5-kb tiling regions for hematopoietic cell types sorted from peripheral blood.(D) Distribution of DNA methylation levels across cell types for different sets of genomic regions. Gene and promoter annotations are based on GENCODE, CpG islands are from the UCSC Table Browser, enhancer elements are from Ensembl, and tiling regions were calculated with a custom script.(E) Distribution of average DNA methylation levels across cell types for putative regulatory regions annotated by the Ensembl BLUEPRINT Regulatory Build.(F) DNA methylation at putative regulatory regions for illustrative gene loci. Black bars denote the position of regions annotated by the BLUEPRINT Regulatory Build, and dashed horizontal black lines indicate sample medians for the respective regions. Colored vertical bars connect the highest and lowest DNA methylation levels that have been measured in any sample of the indicated cell type.dis, distal element; prox, proximal element; TSS, transcriptional start site. See also href="#mmc1" rid="mmc1" class=" supplementary-material">Figure S1 and href="http://blueprint-methylomes.computational-epigenetics.org" data-ga-action="click_feat_suppl" ref="reftype=extlink&article-id=5145815&issue-id=280608&journal-id=445&FROM=Article%7CBody&TO=External%7CLink%7CURI" target="_blank">http://blueprint-methylomes.computational-epigenetics.org.