Deletion of Ldb1 normalizes gene expression in Lmo2-tg DN thymocytes. (A) Principal component (PC1/2) analysis of RNA-seq gene expression data from B6 (wt, 4 replicates), Lmo2-tg (5 replicates), and Rag1-Cre;Ldb1^fl/fl;Lmo2-tg (5 replicates) DN2/3 and DN4 thymocytes. (B) Venn diagrams depicting number of differentially expressed (DE) genes in the DN2/3 population comparisons. Deletion of Ldb1 lowers the number of Lmo2-induced DE genes from 3369 to 809. (C) Gene set enrichment analysis of RNA-seq data. Genes associated with immune system processes, T-cell differentiation, activation, signaling, or cell proliferation and survival are downregulated in Lmo2-tg DN2/3 thymocytes, whereas genes associated with negative regulation of cell proliferation and growth are upregulated in Lmo2-tg DN2/3 thymocytes. Expression of these genes is normalized in Rag1-Cre;Ldb1^fl/fl;Lmo2-tg DN2/3 thymocytes. (D) Genes previously reported to be up- or downregulated in Lmo2-tg DN thymocytes relative to wt (non–Lmo2-tg; B6) thymocytes are substantially normalized by deletion of Ldb1. (E) Genes previously shown to be positively regulated by Ldb1/Lmo2 complexes in hematopoietic progenitor cells¹⁷  are upregulated in Lmo2-tg DN2/3 thymocytes, and their expression is substantially normalized by deletion of Ldb1. For each of the genes shown in panels C-E, normalization of gene expression in Rag1-Cre;Ldb1^fl/fl;Lmo2-tg thymocytes (ie, trending toward that of wt [B6] compared with Lmo2-tg) was statistically significant (P < .05). FDR, false discovery rate.