Single cell ATAC-seq (scATAC) yields sparse data that makes application of conventional analysis approaches challenging. We developed chromVAR, an R package for analyzing sparse chromatin accessibility data by estimating gain or loss of accessibility within peaks sharing the same motif or annotation while controlling for technical biases. chromVAR enables accurate clustering of scATAC-seq profiles and enables characterization of known and de novo sequence motifs associated with variation in chromatin accessibility.

Human hematopoiesis involves cellular differentiation of multipotent cells into progressively more lineage-restricted states. While the chromatin accessibility landscape of this process has been explored in defined populations, single-cell regulatory variation has been hidden by ensemble averaging. We collected single-cell chromatin accessibility profiles across 10 populations of immunophenotypically defined human hematopoietic cell types and constructed a chromatin accessibility landscape of human hematopoiesis to characterize differentiation trajectories. We find variation consistent with lineage bias toward different developmental branches in multipotent cell types. We observe heterogeneity within common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) and develop a strategy to partition GMPs along their differentiation trajectory. Furthermore, we integrated single-cell RNA sequencing (scRNA-seq) data to associate transcription factors to chromatin accessibility changes and regulatory elements to target genes through correlations of expression and regulatory element accessibility. Overall, this work provides a framework for integrative exploration of complex regulatory dynamics in a primary human tissue at single-cell resolution.

SUMMARY Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread of this lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression.

Transcription factors canonically bind nucleosome-free DNA, making the positioning of nucleosomes within regulatory regions crucial to the regulation of gene expression. Using the assay of transposase accessible chromatin (ATAC-seq), we observe a highly structured pattern of DNA fragment lengths and positions around nucleosomes in Saccharomyces cerevisiae, and use this distinctive two-dimensional nucleosomal "fingerprint" as the basis for a new nucleosome-positioning algorithm called NucleoATAC. We show that NucleoATAC can identify the rotational and translational positions of nucleosomes with up to base-pair resolution and provide quantitative measures of nucleosome occupancy in S. cerevisiae, Schizosaccharomyces pombe, and human cells. We demonstrate the application of NucleoATAC to a number of outstanding problems in chromatin biology, including analysis of sequence features underlying nucleosome positioning, promoter chromatin architecture across species, identification of transient changes in nucleosome occupancy and positioning during a dynamic cellular response, and integrated analysis of nucleosome occupancy and transcription factor binding. [Supplemental material is available for this article.]Chromatin accessibility modulates the ability of transcription factors (TFs) and transcriptional machinery to interact with DNA. Within regions of increased accessibility, nucleosomes and TFs compete for access to regulatory DNA (Lickwar et al. 2012). While sequence content has been shown to influence nucleosome positioning, the specific locations of nucleosomes in vivo are also dynamically modulated by chromatin remodelers, transcription factors, and transcriptional machinery (Kaplan et al. 2009;Zhang et al. 2009;Valouev et al. 2011). Therefore, methods for producing base-pair resolved nucleosome maps with quantitative occupancy information within regulatory DNA promise to provide insight into the interplay between chromatin organization and transcriptional regulators-a crucial step toward a comprehensive and predictive understanding of how regulatory elements control gene expression.Methods for inferring nucleosome positions through MNase digestion of chromatin followed by high-throughput sequencing of protected fragments have provided a window into the relationship between TF binding and nucleosome positioning, revealing that certain TFs are consistently flanked by well-positioned nucleosomes, while others show considerable heterogeneity in the positioning of proximal nucleosomes (Valouev et al. 2011;Gaffney et al. 2012;Kundaje et al. 2012). However, MNase-based methods are limited in their ability to infer high-resolution nucleosome positions and provide quantitative measures of nucleosome occupancy by the enzyme's processive nature of DNA digestion and intrinsic digestion sequence bias (Chung et al. 2010;Fan et al. 2010). Alternatively, chemical mapping approaches in both Saccharomyces cerevisiae and Schizosaccharomyces pombe have provided base-pair resolved maps of nucleosome positions (Brogaard et al. 2012;...