Megakaryocytic STAT targets are enriched in granulocytes from ET JAK2-mutant patients but not ET CALR-mutant patients. (A) Schematic of megakaryocyte (MK) cells derived from cord blood (CB) mononuclear cells (MNCs). After an 11-day culture in stem cell growth media (SCGM, Cell Genix) supplemented with 100 ng/mL TPO (Cell Genix) and 10 ng/mL interleukin-1β (IL-1β, Miltenyi), mature MK cells were treated with 50 ng/mL TPO for 30 minutes and used for ChIP with pSTAT3 and pSTAT5 antibodies (Cell Signaling Technologies). (B) Example of a density plot transformed from raw ChIP-seq data reads, displayed in the UCSC Genome Browser. CISH gene structure is shown above the tracks. Venn diagrams depict peak and gene overlaps from ChIP-seq data. Genomic coordinates of STAT-bound peaks were converted to gene lists using UCSC as the gene source. (For a complete list of STAT-bound peaks and genes, see supplementary Tables 1 and 2). (C) ET patient granulocytes were genotyped and expression-profiled by Rampal et al⁶ ; samples that were independently positive for JAK2V617F and CALR were processed in GSEA⁷  with pSTAT3 and pSTAT5 ChIP-Seq data sets from TPO-treated MK cells. (D) GSEA results showing activated STAT signatures enriched in ET patients with JAK2V617F (JAK2) mutation relative to both normal and ET patients positive for CALR mutation (CALR). All 3 GSEA comparisons (JAK2 vs normal, CALR vs normal and JAK2 vs CALR) were evaluated with leading-edge analysis (see supplementary Table 3) to determine which genes contributed to the normalized enrichment score (NES).⁷  Leading-edge genes are core-enriched genes that are ordered in a ranked gene list (as depicted as black and white bars below a GSEA profile) and appear at, or before, an automatically generated enrichment score threshold.⁷  Gene ontology analysis was performed for leading-edge genes using Enrichr⁹  and the Functional Annotation tool of the Database for Annotation, Visualization and Integrated Discovery¹⁰ , version 6.7 (david.abcc.ncifcrf.gov). Nonredundant gene ontology biological process terms with P values < .001 are shown below a Venn diagram of leading-edge genes enriched in JAK2-mutant patients compared with controls, and CALR-mutant patients compared with controls (for full gene ontology biological process table of terms, see supplementary Tables 4-6). For GSEA comparing JAK2 against CALR patients, the 76 leading-edge genes shared by both pSTAT ChIP-seq datasets in MK cells are also shown as a heat map indicating relative expression. Gene expression is denoted pink to red, indicating moderate (mod) to high expression. Gene ontology analysis of these 76 shared leading-edge genes was performed and the main Kyoto Encyclopedia of Genes and Genomes pathway is depicted. *Key canonical JAK-STAT target genes. All P values shown were evaluated by the modified Fisher’s exact test. FDR, false discovery rate; GTPase, guanosine triphosphatase. All supplementary tables are freely available at http://hscl.cimr.cam.ac.uk/genomic_supplementary.html.