Joint single-cell DNA accessibility and protein epitope profiling reveals environmental regulation of epigenomic heterogeneity.

Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. Pi-ATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1α protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells

Transcript-indexed ATAC-seq for precision immune profiling.

T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4+ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy

Genome-Wide Transcriptional Response to Varying RpoS Levels in Escherichia coli K-12

The alternative sigma factor RpoS is a central regulator of many stress responses in Escherichia coli. The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We found that 23% of genes in the E. coli genome are regulated by RpoS, and we identified many RpoS-transcribed genes and promoters. We observed three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS (“sensitive” genes), and genes whose expression changes very little with the production of a little RpoS (“insensitive”). We show that sequences outside the core promoter region determine whether an RpoS-regulated gene is sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase and may also contribute to the diversity of stress responses directed by RpoS

Clustering of transcription factor expression patterns in <i>C. elegans</i>.

<p>(a) The expression patterns of members of each of the seven clusters determined by Mfuzz. The gray vertical line indicates the onset of gastrulation. (b) The relative representation of different TF families in each cluster. Blue stars signify statistically significant over-representation of a TF family in a cluster; red stars signify statistically significant under-representation of a TF family in a cluster.</p

TF and TF family expression throughout embryogenesis based on microarray or RNA-seq data.

<p>The gray vertical line in each plot indicates the onset of gastrulation. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066826#pone.0066826.s005" target="_blank">Table S2</a> shows the numbers of TFs and members of TF families included in each dataset used for this figure.</p

Comparison of TF expression clusters in <i>Xenopus tropicalis</i> (XT) and <i>Danio rerio</i> (DR).

<p>(a) The expression profiles of the cluster pairs. The gray vertical line indicates the onset of gastrulation. The plot in the third column shows the values of the centers of the clusters at each of the common time points. Orange is <i>Xenopus tropicalis</i> and blue is <i>Danio rerio</i>. (b) Dendrogram showing hierarchical clustering of the clusters. (c) TF family representation in each cluster. The dark gray represents <i>Xenopus tropicalis</i> and the light gray <i>Danio rerio</i>. Blue stars signify statistically significant over-representation of a TF family in a cluster pair; red stars signify statistically significant under-representation of a TF family in a cluster pair.</p

TF and TF family expression through embryogenesis based on <i>in situ</i> hybridization data.

<p>The gray vertical line in each plot indicates the onset of gastrulation. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066826#pone.0066826.s004" target="_blank">Table S1</a> shows the numbers of TFs and members of TF families included in each dataset used for this figure.</p

Similarity of TF transcriptomes between species.

<p>Heatmaps showing the similarity of the TF transcriptome at all time points for (a) <i>Xenopus tropicalis</i> and <i>Danio rerio</i> and (b) <i>Drosophila melanogaster</i> and <i>Anopheles gambiae</i>. Darker blue/violet shading indicates that the TF trancriptomes for the two species at the given times during development are more similar. The profiles on the right shows the minimum distance between (a) each time piont of <i>Xenopus tropicalis</i> and any time point in <i>Danio rerio</i> and (b) each time point of <i>Drosophila melanogaster</i> and any time point in <i>Anopheles gambiae</i>. In (b), the bracketed time periods for <i>Drosopila</i> are those included in the study by Kalinka et al. (2010) that showed that the time period between 8–10 hours is when the transcriptomes of different <i>Drosophila</i> species are most similar. That time period (indicated by a *) is a local maximum for TF transcriptome similarity between the two insects considered in this study; the 16–18 hour time period for <i>Drosophila melanogaster</i> (indicated by a **) is the time period with the greatest TF transcriptome similarity to <i>Anopheles gambiae</i>.</p

chromVAR: Inferring transcription factor-associated accessibility from single-cell epigenomic data

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

Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions

Objectives: This study sought to answer two questions: (1) what are the characteristics of young Kenyans aged 18-24 who use contraception obtained at pharmacies, and (2) why are pharmacies appealing sources of contraception? Design and setting: This was a mixed-methods study in one peri-urban part of Kwale County, Kenya. Methods included cross-sectional survey (n=740), six focus group discussions, 18 in-depth interviews and 25 key-informant interviews. Quantitative data analysis identified factors pushing young people to pharmacies for modern contraception versus other sources. Qualitative data analysis identified reasons pharmacies were perceived to be appealing to young clients. Participants: Participants were (1) young people aged 18-24 from the study area, including a subset who had recently purchased contraception from a pharmacy; or (2) pharmacy personnel and pharmacy stakeholders. Results: Among surveyed participants who had ever had sexual intercourse and had used modern contraception at last sexual intercourse, 59% obtained it from a pharmacy. In multivariable analysis, participants who used a condom or emergency contraception as well as those living alone were significantly more likely to get contraception from pharmacies. Pharmacies were valued for their convenience, privacy, non-judgmental and personable staff, service speed, as well as predictable and affordable prices. Conclusions: Our findings indicate a high percentage of young people in Coastal Kenya use pharmacies for contraception. Our inclusion of emergency contraception users partially explains this. Pharmacies were perceived to be everything that health facilities are not: fast, private and non-limiting. Policy-makers should recognise the role of pharmacies as contraception providers and look for opportunities to link pharmacies to the public health system. This would create a network of accessible and appealing contraception services for young people. Keywords: community child health; public health; qualitative research; reproductive medicine