Journal: The Journal of Experimental Medicine

Article Title: Subset- and tissue-defined STAT5 thresholds control homeostasis and function of innate lymphoid cells

doi: 10.1084/jem.20150907

Figure Lengend Snippet: Genome-wide distribution of STAT5 in NK cells. (A–F) STAT5 ChIP-seq was performed in ex vivo and IL-15–treated NK cells. (A) Venn diagram denotes the total number of unique and overlapping peaks. Percentages denote the fraction of peaks shared between the two conditions. (B) Line graphs show the fraction of peaks and amplitude (mean tags per peak) for STAT5 peaks at varying distances from the nearest transcriptional start sites (TSS). (C) Histogram shows the relative amplitude (mean tags per base pair) for peaks shared between ex vivo and IL-15–treated NK cells. Bar graph shows percentage and absolute number of genes exclusively associated with peaks found in ex vivo or IL-15–treated NK cells. Denominator is all genes assigned to the 3,332 or 9,410 peaks described in A. (D–F) Genome browser tracks show STAT5 peaks at selected loci exhibiting overlapping (D), “IL-15 only” (E), or “ex vivo only” (F) distributions. (G and H) Weblogos depict the top enriched STAT (G) and non-STAT (H) transcription factor–binding motifs under STAT5 peaks from ex vivo and/or IL-15–treated NK cells. Shown are corrected p-values and rank of each motif among all database hits. (A–H) Data are presented from one of two biological replicates per condition.

Article Snippet: 10 7 NK cells were purified from spleens of WT mice by magnetic bead separation (90% purity; negative selection kit by Miltenyi Biotec) and fixed with 1% formaldehyde either directly ex vivo or after treatment with mouse IL-15 (2 h, 20 ng/ml).

Techniques: Genome Wide, ChIP-sequencing, Ex Vivo, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Subset- and tissue-defined STAT5 thresholds control homeostasis and function of innate lymphoid cells

doi: 10.1084/jem.20150907

Figure Lengend Snippet: Transcriptional activity and cooperativity of STAT5 downstream of IL-15. Transcriptomes were compared between ex vivo and IL-15–treated NK cells. Differentially expressed genes were then segregated on the basis of whether or not STAT5-binding sites were detected near transcriptional start sites in IL-15–treated NK cells. (A) Left donut plot reports the total number of positively or negatively regulated genes from among all STAT5-bound genes. Right donut plot reports the total number of STAT5-bound, positively or negatively regulated genes from among all IL-15–regulated genes. (B) GSEA was applied to STAT5-bound and STAT5-unbound, IL-15–regulated genes. Bar graphs show hallmark pathway genes sets that were reduced or enhanced by IL-15. FDR, false discovery rate; NES, normalized enrichment score. (C) Left heat map shows the fold change in RPKM (log 2 of IL-15–treated ÷ untreated) for all IL-15/STAT5 signature genes across three biological replicates. Right heat maps shows the RPKM fold change for a selection of signature genes and peak amplitude for the highest associated STAT5 peaks in untreated or IL-15–treated cells. (D) Left pie chart shows the number of IL-15/STAT5 signature genes among NK cell signature genes. Right pie chart shows the number of genes with nearby STAT5 peaks among NK cell signature genes that are not included in the STAT5 signature. (E) Transcriptomes were measured in IL-15–treated WT and T-BET–deficient NK cells and GSEA performed using STAT5 signature or IL-15–regulated, STAT5-unbound gene sets. Circle plot relays whether gene sets were enhanced or reduced in T-BET–deficient NK cells (y axis), along with gene set count (element size; number shown) and false discovery rate (y axis). Two biological replicates were included per genotype. (F) Genome browser tracks show STAT5 and T-BET distributions at selected loci. (G) Histogram shows the position and amplitude of T-BET-binding sites relative to STAT5-binding sites in ex vivo NK cells.

Article Snippet: 10 7 NK cells were purified from spleens of WT mice by magnetic bead separation (90% purity; negative selection kit by Miltenyi Biotec) and fixed with 1% formaldehyde either directly ex vivo or after treatment with mouse IL-15 (2 h, 20 ng/ml).

Techniques: Activity Assay, Ex Vivo, Binding Assay, Selection