Extended Data Fig. 6: ThPOK−/− impacts global splicing and cellular process networks.
From: ThPOK is a critical multifaceted regulator of myeloid lineage development
(a, b) UMAP plot of single-cell Fluidigm RNA-Seq analysis of prior profiled (a) wild-type hematopoietic progenitors and (b) Ly6c-ThPOK-/- GMPs aligned to cKit+ CITE-Seq. Consistent alignment of cell populations in the UMAP space indicates a lack of apparent batch effects. c) Gene Ontology enrichment analysis of ThPOK-/- dependent alternative splicing events in proNeu1 cells, for splicing events with the opposite pattern of exon inclusion in proNeu1 versus MultiLin (discordant = more immature splicing) or (d) with the same pattern (concordant = promoting neutrophil specification associated splicing). (e-f) Validation of deregulated gene expression in ThPOK-/- versus WT progenitors for important transcripts as observed in CITE seq data: e) qRTPCR analyses of indicated flow-sorted progenitors (pooled from 4 mice/genotype) for monocyte/DC genes (Irf8, Zeb2, Runx1), granulocyte lineage genes (Cebpe, Pde4d, Cd63 and Nkg7) and RNA binding protein genes (Ddx3x and Srsf5). The experiment was repeated 3 times independently with similar results. f) Expression ratio of Hmga1 alternate transcript versus reference transcript in indicated progenitor populations (pooled from 4 mice/genotype). g) Representative confocal micrographs of EZH2 protein (top), DAPI (middle), and H3K27Me3 (bottom) staining in indicated subsets. Each experiment was reproduced twice and significant differences between ThPOK-/- and WT mice were determined by two-sided unpaired T test with Welch’s correction, and indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001). h) Heat maps showing relative mRNA expression of indicated genes in WT progenitors (left panel), or in ThPOK-/- versus WT progenitor subsets (right panel) for genes that are relatively up-regulated in any ThPOK-/- progenitor population and that are known Ezh2 targets in WT immune cells by ChIP-seq (GSE181873). Note that in WT mice most of these genes show marked stage-specific regulation.
