Phosphorylation of BRN2 Modulates Its Interaction with the Pax3 Promoter To Control Melanocyte Migration and Proliferation

Abstract

MITF-M and PAX3 are proteins central to the establishment and transformation of the melanocyte lineage. They control various cellular mechanisms, including migration and proliferation. BRN2 is a POU domain transcription factor expressed in melanoma cell lines and is involved in proliferation and invasion, at least in part by regulating the expression of MITF-M and PAX3. The T361 and S362 residues of BRN2, both in the POU domain, are conserved throughout the POU protein family and are targets for phosphorylation, but their roles in vivo remain unknown. To examine the role of this phosphorylation, we generated mutant BRN2 in which these two residues were replaced with alanines (BRN2TS→BRN2AA). When expressed in melanocytes in vitro or in the melanocyte lineage in transgenic mice, BRN2TS induced proliferation and repressed migration, whereas BRN2AA repressed both proliferation and migration. BRN2TS and BRN2AA bound and repressed the MITF-M promoter, whereas PAX3 transcription was induced by BRN2TS but repressed by BRN2AA. Expression of the BRN2AA transgene in a Mitf heterozygous background and in a Pax3 mutant background enhanced the coat color phenotype. Our findings show that melanocyte migration and proliferation are controlled both through the regulation of PAX3 by nonphosphorylated BRN2 and through the regulation of MITF-M by the overall BRN2 level.

ACKNOWLEDGMENTS

We are grateful to F. Relaix (Pax3^IRESnLacZ mice), I. Jackson (Dct::LacZ mice), and C. Lobe (Z/EG mice) for providing mouse strains, to D. Bennett (melan-a cells) for providing melanocyte lines, and to R. Ballotti (MITF-M, DCT, and TYRP1 promoters) and J. Epstein (PAX3 promoter) for providing constructs. We thank the teams caring for the animal colony and imaging facilities of the Institut Curie, especially Y. Bourgeois, F. Cordelières, and H. Harmange.

I.B. and I.P. were supported by fellowships from the Institut Curie and the ARC. L.D. was supported by a fellowship from the Ligue Contre le Cancer (Comité de l'Oise). This work was supported by the Ligue Nationale Contre le Cancer (Equipe labelisée), INCa, and Cancéropole IdF.