A critical regulator of Bcl2 revealed by systematic transcript discovery of lncRNAs associated with T-cell differentiation
Résumé
Normal T-cell differentiation requires a complex regulatory network which supports a series of maturation steps, including lineage commitment, T-cell receptor (TCR) gene rearrangement, and thymic positive and negative selection. However, the underlying molecular mechanisms are difficult to assess due to limited T-cell models. Here we explore the use of the pro-T-cell line P5424 to study early T-cell differentiation. Stimulation of P5424 cells by the calcium ionophore ionomycin together with PMA resulted in gene regulation of T-cell differentiation and activation markers, partially mimicking the CD4-CD8-double negative (DN) to double positive (DP) transition and some aspects of subsequent T-cell maturation and activation. Global analysis of gene expression, along with kinetic experiments, revealed a significant association between the dynamic expression of coding genes and neighbor lncRNAs including many newly-discovered transcripts, thus suggesting potential co-regulation. CRISPR/Cas9-mediated genetic deletion of Robnr, an inducible lncRNA located downstream of the anti-apoptotic gene Bcl2, demonstrated a critical role of the Robnr locus in the induction of Bcl2. Thus, the pro-T-cell line P5424 is a powerful model system to characterize regulatory networks involved in early T-cell differentiation and maturation. T lymphocytes are one of the main players of the adaptive immunity. T-cell development in the thymus requires temporally regulated rearrangements of the T-cell receptor (Tcr) genes and a series of selection events, whereby newly assembled TCR complexes signal for cell survival, proliferation and differentiation processes 1,2. The Tcrb locus rearranges in the most immature thymocytes, known as CD4 − CD8 − double-negative (DN) thymocytes. Thymocytes that have successfully rearranged a Tcrb allele differentiate into CD4 + CD8 + double-positive (DP) thymocytes in a process known as β-selection. This process is driven by signaling through the pre-TCR, which is composed of TCRβ and the invariant pTα protein, and through cooperation with the Notch signaling pathway 1,3. The β-selection process triggers the activation of Tcra rearrangements and transcription along with complex intracellular pathways resulting in wide changes in the transcriptional and epigenetic programs of the immature T cells 4-6. The expression of a functionally rearranged Tcra gene leads to the formation of a variable TCRαβ heterodimer and, ultimately, to the selection of TCRαβ expressing cells which will terminally differentiate into CD4 + or CD8 + single positive (SP) T cells. Disruptions of these genetic and epigenetic processes might result in oncogenic transformation of T-cell precursors (i.e. leukemia and lymphoma 7,8) or immune-related pathologies 9. Long non-coding RNAs (lncRNAs) are a heterogeneous group of non-coding genes transcribed by RNA pol-ymerase II from intergenic or intragenic regions and varying in length from 200 nt to over 100 kb 10. Many studies have demonstrated that lncRNAs are key components of the repertoire of regulatory elements that control normal
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