Knockout of Rcor1 results in the upregulation of myeloid genes. (A) Volcano plot from RNA-Seq showing all expressed genes. The y-axis shows statistical significance. A false discovery rate (FDR) –adjusted P value of .05 is 1.3 on this scale. The x-axis shows the magnitude of change (mutant/control). Red dots, all upregulated genes with fold change ≥2 (P < .05) and an adjusted tag number of >50 (average mutant reads minus average control reads; for details, see “Materials and methods”). Green dots, all downregulated genes with fold change ≤2 (P < .05) and an adjusted tag number of >50 (average control reads minus average mutant reads). Blue dots, all other genes. (B) Confirmation of representative RNA-Seq results by qPCR. (C) Correlation between qPCR and RNA-Seq data based on data points from (B). (D) GSEA showing that genes that are upregulated in mutant Rcor1^−/− cells correlate positively with genes that are enriched in monocytes. Heat map: genes are ranked according to their relative expression levels in monocytes to “other” hematopoietic cell types (LT-HSC, NK cells, monocytes, granulocytes, erythrocytes, naive CD4 cells, naive CD8 cells, active CD4 cells, active CD8 cells, B cells). Red, highly enriched in monocytes. Black vertical lines represent single genes from the upregulated Rcor1^−/− gene set, which are positioned with respect to the ranked list of the reference cell type expression data set. The green line is the running sum for calculating the enrichment score (ES). Positive and negative ES indicate enrichment at the top and bottom of the ranked reference list, respectively. ES is normalized (NES) on the basis of differences in gene set size and correlations between gene sets and is used to compare GSEA results across experiments. (E) GSEA showing that genes upregulated in Rcor1^−/− cells are correlated positively with genes that are highly expressed in HSCs. Heat map: genes are ranked according to their relative expression levels in HSCs relative to CMPs, GMPs, and MEPs.²¹  Colors and NES as described in (D). (F) Examples of de-repressed genes from Rcor1^−/− cells. Red colored genes have been reported to block erythropoiesis when de-repressed in RBC progenitors.