The genomics of human development Understanding the trajectory of a developing human requires an understanding of how genes are regulated and expressed. Two papers now present a pooled approach using three levels of combinatorial indexing to examine the single-cell gene expression and chromatin landscapes from 15 organs in fetal samples. Cao et al. focus on measurements of RNA in broadly distributed cell types and provide insights into organ specificity. Domcke et al. examined the chromatin accessibility of cells from these organs and identify the regulatory elements that regulate gene expression. Together, these analyses generate comprehensive atlases of early human development. Science, this issue p. eaba7721, p. eaba7612 Chromatin accessibility is examined at the single-cell level during early human development. INTRODUCTION In recent years, the single-cell genomics field has made incredible progress toward disentangling the cellular heterogeneity of human tissues. However, the overwhelming majority of effort has been focused on single-cell gene expression rather than the chromatin landscape that shapes and is shaped by gene expression. Toward advancing our understanding of the regulatory programs that underlie human cell types, we set out to generate single-cell atlases of both chromatin accessibility (this study) and gene expression (Cao et al., this issue) from a broad range of human fetal tissues. RATIONALE Regions of accessible chromatin in our genome, such as enhancers, play key roles in the determination and maintenance of cell fates. Accessible regions are also markedly enriched for genetic variation that contributes to common disease heritability. The vast majority of chromatin accessibility data collected to date lacks single-cell resolution, limiting our ability to infer patterns such as which cell types are most relevant to each common disease. We previously demonstrated single-cell profiling of chromatin accessibility using combinatorial indexing, based on two rounds of in situ molecular barcoding. Here, we describe an improved assay that uses three levels of combinatorial indexing and does not rely on custom reagents. The method, sci-ATAC-seq3, reduces costs and opens the door to the scales necessary for generating a human cell atlas of chromatin accessibility. RESULTS We applied sci-ATAC-seq3 to 59 human fetal samples ranging from 89 to 125 days in estimated postconceptual age and representing 15 organs, altogether obtaining high-quality chromatin accessibility profiles from ~800,000 single cells. Gene expression data collected on an overlapping set of tissues were leveraged to annotate cell types. We asked which transcription factor (TF) motifs found in the accessible sites of each cell best explain its cell type affiliation, revealing both known and potentially previously unknown regulators of cell fate specification and/or maintenance. Many TFs could be putatively assigned as activators or repressors depending on whether their expression and the accessibility of their cognate motif were positively or negatively correlated across cell types. Comparing chromatin accessibility from cell types that appear in multiple tissues revealed that whereas blood cell types are highly similar across organs, endothelial cells exhibit organ-specific chromatin accessibility, which appears to be controlled combinatorially by several TFs with overlapping expression patterns. We leveraged our master set of 1.05 million accessible sites, spanning 532 Mb or 17% of the reference human genome, to score cell type–specific links between candidate enhancers and genes based on coaccessibility, to detect cell type–specific enrichment of heritability for specific common human diseases, and to identify genetic variants affecting chromatin accessibility in cis. Comparisons with chromatin accessibility in corresponding adult tissues allowed us to identify fetal-specific cell subtypes and nominate POU2F1 as a potential regulator of excitatory neuron development. CONCLUSION Sci-ATAC-seq3 adds to a growing repertoire of single-cell methods that use combinatorial indexing, a technical paradigm whose advantages include exponential scaling and greater range to profile diverse aspects of single-cell biology. We anticipate that the intersection of single-cell chromatin accessibility and gene expression will critically accelerate the field’s long-term goal of establishing a deep, predictive understanding of gene regulation. An interactive website facilitates the exploration of these freely available data by tissue, cell type, locus, or motif (descartes.brotmanbaty.org). A human cell atlas of fetal chromatin accessibility enables the exploration of in vivo gene regulation across diverse cell types. We devised a three-level combinatorial indexing assay (sci-ATAC-seq3) and profiled chromatin accessibility in ~800,000 single cells from 15 fetal organs. This rich resource enables, for example, identification of cell type–specific regulatory elements and TFs, classification of TFs into activators and repressors, and quantification of cell type–specific enrichments of complex trait heritability, as well as chromatin accessibility dynamics. The chromatin landscape underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of chromatin accessibility and gene expression in fetal tissues. For chromatin accessibility, we devised a three-level combinatorial indexing assay and applied it to 53 samples representing 15 organs, profiling ~800,000 single cells. We leveraged cell types defined by gene expression to annotate these data and cataloged hundreds of thousands of candidate regulatory elements that exhibit cell type–specific chromatin accessibility. We investigated the properties of lineage-specific transcription factors (such as POU2F1 in neurons), organ-specific specializations of broadly distributed cell types (such as blood and endothelial), and cell type–specific enrichments of complex trait heritability. These data represent a rich resource for the exploration of in vivo human gene regulation in diverse tissues and cell types.

中文翻译：

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胎儿染色质可及性的人类细胞图谱

人类发展的基因组学 了解人类发展的轨迹需要了解基因的调控和表达方式。现在有两篇论文提出了一种合并方法，使用三个级别的组合索引来检查胎儿样本中 15 个器官的单细胞基因表达和染色质图谱。曹等人。专注于测量广泛分布的细胞类型中的 RNA，并提供对器官特异性的见解。多姆克等人。检查了来自这些器官的细胞的染色质可及性，并确定了调节基因表达的调节元件。这些分析共同生成了人类早期发展的综合图谱。科学，这个问题 p。eaba7721，第。eaba7612 在人类早期发育过程中在单细胞水平上检查染色质可及性。简介 近年来，单细胞基因组学领域在解开人体组织的细胞异质性方面取得了令人难以置信的进展。然而，绝大多数的努力都集中在单细胞基因表达上，而不是塑造基因表达并由基因表达塑造的染色质景观。为了加深我们对人类细胞类型基础的调控程序的理解，我们着手从广泛的人类胎儿中生成染色质可及性（本研究）和基因表达（Cao 等人，本期）的单细胞图谱。组织。基本原理 我们基因组中可接近的染色质区域，例如增强子，在决定和维持细胞命运方面起着关键作用。可及区域也显着丰富了有助于常见疾病遗传的遗传变异。迄今为止收集的绝大多数染色质可及性数据缺乏单细胞分辨率，这限制了我们推断模式的能力，例如哪种细胞类型与每种常见疾病最相关。我们之前基于两轮原位分子条形码，使用组合索引展示了染色质可及性的单细胞分析。在这里，我们描述了一种改进的检测方法，它使用三个级别的组合索引并且不依赖于自定义试剂。该方法 sci-ATAC-seq3 降低了成本，并为生成染色质可及性人类细胞图谱所需的尺度打开了大门。结果我们将 sci-ATAC-seq3 应用于 59 个人类胎儿样本，估计受孕后年龄从 89 天到 125 天不等，代表 15 个器官，总共获得了约 800 个高质量的染色质可及性图谱，000 个单元格。利用在一组重叠组织上收集的基因表达数据来注释细胞类型。我们询问在每个细胞的可接近位点中发现的哪些转录因子 (TF) 基序最能解释其细胞类型从属关系，揭示已知和可能以前未知的细胞命运规范和/或维持的调节器。许多 TF 可以被假定为激活剂或阻遏物，这取决于它们的表达和它们的同源基序的可及性在细胞类型之间是正相关还是负相关。比较出现在多个组织中的细胞类型的染色质可及性表明，虽然血细胞类型在器官间高度相似，但内皮细胞表现出器官特异性染色质可及性，这似乎是由几个具有重叠表达模式的 TF 组合控制的。我们利用我们的 105 万个可访问位点的主集，跨越 532 Mb 或参考人类基因组的 17%，基于可访问性对候选增强子和基因之间的细胞类型特异性联系进行评分，以检测特定细胞类型遗传性的特定富集常见的人类疾病，并确定影响顺式染色质可及性的遗传变异。与相应成人组织中染色质可及性的比较使我们能够识别胎儿特异性细胞亚型并指定 POU2F1 作为兴奋性神经元发育的潜在调节剂。结论 Sci-ATAC-seq3 增加了越来越多的使用组合索引的单细胞方法，一种技术范式，其优势包括指数缩放和更大范围来描绘单细胞生物学的各个方面。我们预计，单细胞染色质可及性和基因表达的交叉将极大地加速该领域建立对基因调控的深入、预测性理解的长期目标。一个交互式网站有助于按组织、细胞类型、位点或基序 (descartes.brotmanbaty.org) 探索这些免费可用的数据。胎儿染色质可及性的人类细胞图谱使探索不同细胞类型的体内基因调控成为可能。我们设计了一个三级组合索引分析 (sci-ATAC-seq3) 并分析了来自 15 个胎儿器官的约 800,000 个单细胞的染色质可及性。例如，这种丰富的资源使 识别细胞类型特异性调控元件和 TF，将 TF 分类为激活子和抑制子，量化复杂性状遗传性的细胞类型特异性富集，以及染色质可及性动态。人类细胞类型规范背后的染色质景观具有根本的意义。我们在胎儿组织中生成了染色质可及性和基因表达的人类细胞图谱。对于染色质可及性，我们设计了一个三级组合索引分析，并将其应用于代表 15 个器官的 53 个样本，分析了约 800,000 个单细胞。我们利用由基因表达定义的细胞类型来注释这些数据，并对数十万个表现出细胞类型特异性染色质可及性的候选调控元件进行编目。我们研究了谱系特异性转录因子（如神经元中的 POU2F1）的特性、广泛分布的细胞类型（如血液和内皮细胞）的器官特异性特化，以及复杂性状遗传性的细胞类型特异性富集。这些数据代表了在不同组织和细胞类型中探索体内人类基因调控的丰富资源。