Signal- and Development-Dependent Alternative Splicing of LEF1 in T Cells Is Controlled by CELF2

Abstract

The HMG-box transcription factor LEF1 controls many developmentally regulated genes, including genes that activate expression of the T-cell antigen receptor alpha chain (TCR-alpha) in developing thymocytes. At least two distinct isoforms of LEF1 are expressed, resulting from variable inclusion of LEF1 exon 6; however, the expression pattern of these isoforms and mechanism of splicing regulation have not been explored. Here we demonstrate that inclusion of LEF1 exon 6 is increased during thymic development and in response to signaling in a cultured T-cell line in a manner which temporally correlates with increased expression of TCR-alpha. We further find that inclusion of exon 6 is dependent on the signal-induced increase in expression and binding of the splicing factor CELF2 to two intronic sequences flanking the regulated exon. Importantly, loss of exon 6 inclusion, through knockdown of CELF2 or direct block of the exon 6 splice site, results in reduced expression of TCR-alpha mRNA. Together, these data establish the mechanistic basis of LEF1 splicing regulation and demonstrate that LEF1 alternative splicing is a contributing determinant in the optimal expression of the TCR-alpha chain.

Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes

“…At one extreme, CELF2 is highly expressed in thymus/immune tissues where the RBFOX proteins show minimal expression (Mallory et al 2011and this study), while at the other end of the spectrum, heart and skeletal muscle down-regulate CELF protein expression during development but express abundant levels of RBFOX proteins (Kalsotra et al 2008;Singh et al 2014). Notably, both CELF2 and RBFOX2 are highly expressed and nuclear-localized in the brain (Otsuka et al 2009;Lee et al 2016).…”

“…Recently, we have shown that CELF2 expression increases during thymic development and upon activation of mature T cells (Mallory et al 2011. We have also identified CELF2-dependent alternative splicing of ∼100 genes in immature, mature, and cultured T cells and have mapped the transcriptome-wide association of CELF2 with pre-mRNA in cultured Jurkat T cells Martinez et al 2015;Ajith et al 2016).…”

“…Altered expression of CELF2 in neuronal and muscle cells has also been shown to correlate with splicing changes (Charlet et al 2002;Dasgupta and Ladd 2011;Wang et al 2015). Although these studies have not demonstrated a causal role of CELF2 in the observed splicing changes, CELF2 expression does increase in developing thymocytes and activated T cells and regulates splicing to promote T cell receptor expression and signaling (Mallory et al 2011;Martinez et al 2015). Similarly, proper expression of RBFOX2 in brain and muscle of several species is required for appropriate splicing and tissue function (Kuroyanagi et al 2007;Gallagher et al 2011;Singh et al 2014).…”

Alternative splicing networks regulated by signaling in human T cells

“…Among these signal-induced changes in alternative splicing, 90 (51%) of the exons displayed increased inclusion, and 88 (49%) of the exons displayed increased skipping upon PMA stimulation. Notably, this list of ''top candidates'' captures previously defined and biologically important signal-responsive exons, including all three of the immune-regulated alternative exons in the CD45 gene, and also exons in the FYN, CUGBP2, and MAP4K2 genes (Lynch and Weiss 2000;Rothrock et al 2003;Ip et al 2007;Mallory et al 2011). We next used low-cycle RT-PCR to validate predicted changes in the levels of inclusion of 46 additional exons among the list of 178.…”

Recent insights into the transcriptional control of theTcra/Tcrdlocus by distant enhancers during the development of T-lymphocytes

“…68 In support of this hypothesis, rearranged Tcra transcription responds positively to treatments with phorbol esters. [69][70][71][72][73] It is interesting to note that in contrast to the positive effect of pre-TCR-transduced signaling in DN3a thymocytes, TCRab-transduced signaling in lDP thymocytes has a negative effect on the function of Ea. In support of the involvement of different signaling pathways in Tcra germline transcription by Ea activation during b-selection compared with the transcription of the rearranged Tcra during later stages of development, treatments with ionomycin synergistically activate the former in DN3a thymocytes but inhibit the latter in postselected lDP thymocytes.…”