Analysis of the transcriptional profiles of developing thymocytes has shown that T lineage commitment is associated with loss of stem cell and early progenitor gene signatures and the acquisition of T cell gene signatures. Less well understood are the epigenetic alterations that accompany or enable these transcriptional changes. Here, we show that the histone demethylase Lsd1 (Kdm1a) performs a key role in extinguishing stem/progenitor transcriptional programs in addition to key repressive gene programs during thymocyte maturation. Deletion of Lsd1 caused a block in late T cell development and resulted in overexpression of interferon response genes as well as genes regulated by the Gfi1, Bcl6, and, most prominently, Bcl11b transcriptional repressors in CD4+CD8+ thymocytes. Transcriptional overexpression in Lsd1-deficient thymocytes was not always associated with increased H3K4 trimethylation at gene promoters, indicating that Lsd1 indirectly affects the expression of many genes. Together, these results identify a critical function for Lsd1 in the epigenetic regulation of multiple repressive gene signatures during T cell development.

Prolonged or enhanced expression of the proto-oncogene Lmo2 is associated with a severe form of T-cell acute lymphoblastic leukemia (T-ALL), designated early T-cell precursor ALL, which is characterized by the aberrant self-renewal and subsequent oncogenic transformation of immature thymocytes. It has been suggested that Lmo2 exerts these effects by functioning as component of a multi-subunit transcription complex that includes the ubiquitous adapter Ldb1 along with b-HLH and/or GATA family transcription factors; however, direct experimental evidence for this mechanism is lacking. In this study, we investigated the importance of Ldb1 for Lmo2-induced T-ALL by conditional deletion of Ldb1 in thymocytes in an Lmo2 transgenic mouse model of T-ALL. Our results identify a critical requirement for Ldb1 in Lmo2-induced thymocyte self-renewal and thymocyte radiation resistance and for the transition of preleukemic thymocytes to overt T-ALL. Moreover, Ldb1 was also required for acquisition of the aberrant preleukemic ETP gene expression signature in immature Lmo2 transgenic thymocytes. Co-binding of Ldb1 and Lmo2 was detected at the promoters of key upregulated T-ALL driver genes (Hhex, Lyl1, and Nfe2) in preleukemic Lmo2 transgenic thymocytes, and binding of both Ldb1 and Lmo2 at these sites was reduced following Cre-mediated deletion of Ldb1. Together, these results identify a key role for Ldb1, a nonproto-oncogene, in T-ALL and support a model in which Lmo2-induced T-ALL results from failure to downregulate Ldb1/Lmo2-nucleated transcription complexes which normally function to enforce self-renewal in bone marrow hematopoietic progenitors.

The T-lineage restricted protein THEMIS has been shown to play a critical role in T cell development. THEMIS, via its distinctive CABIT domains, inhibits the catalytic activity of the tyrosine phosphatase SHP1 (PTPN6). SHP1 and THEMIS bind to the ubiquitous cytosolic adapter GRB2, and the purported formation of a tri-molecular THEMIS–GRB2–SHP1 complex facilitates inactivation of SHP1 by THEMIS. The importance of this function of GRB2 among its numerous documented activities is unclear as GRB2 binds to multiple proteins and participates in several signaling responses in thymocytes. Here, we show that similar to Themis−/− thymocytes, the primary molecular defect in GRB2-deficient thymocytes is increased catalytically active SHP1 and the developmental block in GRB2-deficient thymocytes is alleviated by deletion or inhibition of SHP1 and is exacerbated by SHP1 overexpression. Thus, the principal role of GRB2 during T cell development is to promote THEMIS-mediated inactivation of SHP1 thereby enhancing the sensitivity of TCR signaling in CD4+CD8+ thymocytes to low affinity positively selecting self-ligands.

The T cell lineage–restricted protein THEMIS plays a critical role in T cell development at the positive selection stage. In the SHP1 activation model, THEMIS is proposed to enhance the activity of the tyrosine phosphatase SHP1 (encoded by Ptpn6 ), thereby dampening T cell antigen receptor (TCR) signaling and preventing the inappropriate negative selection of CD4 + CD8 + thymocytes by positively selecting ligands. In contrast, in the SHP1 inhibition model, THEMIS is proposed to suppress SHP1 activity, rendering CD4 + CD8 + thymocytes more sensitive to TCR signaling initiated by low-affinity ligands to promote positive selection. We sought to resolve the controversy regarding the molecular function of THEMIS. We found that the defect in positive selection in Themis −/− thymocytes was ameliorated by pharmacologic inhibition of SHP1 or by deletion of Ptpn6 and was exacerbated by SHP1 overexpression. Moreover, overexpression of SHP1 phenocopied the Themis −/− developmental defect, whereas deletion of Ptpn6 , Ptpn11 (encoding SHP2), or both did not result in a phenotype resembling that of Themis deficiency. Last, we found that thymocyte negative selection was not enhanced but was instead impaired in the absence of THEMIS. Together, these results provide evidence favoring the SHP1 inhibition model, supporting a mechanism whereby THEMIS functions to enhance the sensitivity of CD4 + CD8 + thymocytes to TCR signaling, enabling positive selection by low-affinity, self-ligand–TCR interactions.