The pioneer transcription factors Foxa1 and Foxa2 regulate alternative RNA splicing during thymocyte positive selection.

Ching-In Lau,Jasmine Rowell,Masahiro Ono,Diana C Yanez,Tessa Crompton,Anisha Solanki,Susan Ross

doi:10.1242/dev.199754

Abstract

ABSTRACTDuring positive selection at the transition from CD4+CD8+ double-positive (DP) to single-positive (SP) thymocyte, TCR signalling results in appropriate MHC restriction and signals for survival and progression. We show that the pioneer transcription factors Foxa1 and Foxa2 are required to regulate RNA splicing during positive selection of mouse T cells and that Foxa1 and Foxa2 have overlapping/compensatory roles. Conditional deletion of both Foxa1 and Foxa2 from DP thymocytes reduced positive selection and development of CD4SP, CD8SP and peripheral naïve CD4+ T cells. Foxa1 and Foxa2 regulated the expression of many genes encoding splicing factors and regulators, including Mbnl1, H1f0, Sf3b1, Hnrnpa1, Rnpc3, Prpf4b, Prpf40b and Snrpd3. Within the positively selecting CD69+DP cells, alternative RNA splicing was dysregulated in the double Foxa1/Foxa2 conditional knockout, leading to >850 differentially used exons. Many genes important for this stage of T-cell development (Ikzf1-3, Ptprc, Stat5a, Stat5b, Cd28, Tcf7) and splicing factors (Hnrnpab, Hnrnpa2b1, Hnrnpu, Hnrnpul1, Prpf8) showed multiple differentially used exons. Thus, Foxa1 and Foxa2 are required during positive selection to regulate alternative splicing of genes essential for T-cell development, and, by also regulating splicing of splicing factors, they exert widespread control of alternative splicing.

Highlights

The production of αβ T cells in the thymus involves multiple stages of development during which haematopoietic precursors give rise to mature T cells that can differentiate into functional effector T cells
CD4SP and CD8SP development are impaired in the Foxa1/2 conditional knockout thymus We examined Foxa1 and Foxa2 expression by qRT-PCR in FACS-sorted developing thymocytes from the DN3 stage onwards (Fig. 1A)
Foxa1 and Foxa2 were effectively deleted in Foxa1/2cKO CD4SP and CD8SP thymocytes, as expression of Foxa1 and Foxa2 were below detection by qRT-PCR, but detected in the control (Foxa1fl/flFoxa2fl/flCD4cre−) CD4SP and CD8SP populations (Fig. 1B)

Summary

Introduction

The production of αβ T cells in the thymus involves multiple stages of development during which haematopoietic precursors give rise to mature T cells that can differentiate into functional effector T cells. Maturation from DP to SP follows successful rearrangement of the Tcra locus, and requires TCR signalling: positive selection results in appropriate MHC restriction of SP cells, J.R., 0000-0001-7040-8528; T.C., 0000-0002-8973-4021. For DP thymocytes undergoing positive selection, TCR signal strength and duration influence CD4 and CD8 lineage choice Those cells receiving stronger and longer TCR signals tend towards the CD4SP fate, whereas weaker/more transient signals favour the CD8SP fate, and fate decisions are influenced by the relative timing of cytokine and TCR signalling that a developing cell receives (Littman, 2016; Klein et al, 2014; Bosselut, 2004). The CD4/CD8 lineage decision is influenced by factors from the stroma, such as Notch and Hedgehog (Hh) signalling (Laky and Fowlkes, 2008; Solanki et al, 2018; Furmanski et al, 2012; Rowbotham et al, 2007)

Methods

Results

Conclusion