The Essential Function for Serum Response Factor in T-Cell Development Reflects Its Specific Coupling to Extracellular Signal-Regulated Kinase Signaling

Abstract

Serum response factor (SRF) recruits members of two families of signal-regulated coactivators, the extracellular signal-regulated kinase (ERK)-regulated ternary complex factors (TCFs) and the actin-regulated myocardin-related transcription factors (MRTFs), to its target genes through its DNA-binding domain. Whether coactivator association is required for SRF function in vivo and whether particular SRF functions reflect specific coupling to one or the other signal pathway have remained largely unexplored. We show that SRF is essential for thymocyte positive selection and thymic T_reg and NK T-cell development but dispensable for early thymocyte development and negative selection. Expression of wild-type SRF, or mutants lacking the N-terminal phosphorylation sites or C-terminal transcriptional activation domain, restores positive selection in SRF null thymocytes. In contrast, SRF.V194E, which cannot recruit TCF or MRTF family members, is inactive, although it is recruited to target genes. Fusion of a TCF C-terminal activation domain to SRF.V194E effectively restores ERK-dependent single-positive (SP) thymocyte development. The resulting SP thymocytes exhibit normal surface marker expression and proliferation following T-cell receptor cross-linking. Thus, ERK signaling through the TCF pathway to SRF is necessary and sufficient for SRF function in thymocyte positive selection.

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ACKNOWLEDGMENTS

We thank Facundo Batista, Cyril Esnault, and Caetano Reis e Sousa for helpful discussions, advice, and communication of data prior to publication; Derek Davies and the staff of the LRI Flow Cytometry facility for FACS support; and the LRI Biological Resources Unit for animal husbandry.

Research at LRI is supported by Cancer Research UK through institutional core funding. A.M. was supported by an EMBO long-term fellowship.

A.M. and P.C. devised and executed thymus analysis and retroviral transduction experiments and wrote the paper. R.N. and M.S. devised and performed DNA-binding studies and gene expression and chromatin immunoprecipitation experiments. D.M. performed the analysis of DP-APC conjugation and calcium signaling. D.T. and D.D. provided the Srf^f/f animals. R.T. conceived of the project, devised experiments, and wrote the paper.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01058-10.