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

Abstract

f 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. POU family transcription factors are expressed in a wide variety of cell types. They are involved in diverse functions, such as cell type determination, proliferation, renewal, invasion, and migration. The members of the POU domain family of transcription factors share the POU DNA-binding domain (DBD) called the POU domain. The POU domain consists of two DNA-binding units (POUs for POU specific and POUh for POU homeodomain) connected by a flexible linker (3). This molecular structure allows POU proteins to recognize a large set of DNA targets and also to bind different transactivator proteins, depending on the spacing and the positioning adopted by the two subdomains of the POU DBD (50).POU transcription factor function can be modulated by posttranslational modifications, including sumoylation, oxidation, ubiquitinylation, glycosylation, and particularly phosphorylation (5,20,32). Two residues in the DBD domain, a threonine and a serine, are conserved in all mammalian POU domains (see Table S1 at http://umr3347.curie.fr/fr/quipes-de -recherche/d-veloppement-normal-et-pathologie-des-m-lanocytes /differential-function-non-pho). These serine and/or threonine residues in Oct-1, Pit-1, and BRN2 are phosphorylated by protein kinase A (PKA) (31,46,55).Several lines of evidence suggest that PKA is involved in melanocyte lineage proliferation. Forskolin stimulates the proliferation of human melanocytes (54). Proliferation of human melanocytes is induced in a dose-dependent manner by alphamelanocyte-stimulating hormone (1,57,58). Dibutyryl adenosine cyclic AMP induces the proliferation of epidermal melanocytes in culture (29). PKA phosphorylates claudin-1 and allows its tr...

Profiling and metaanalysis of epidermal keratinocytes responses to epidermal growth factor

“…BRN2, OCT7) appear over-represented in the suppressed genes. This transcription factor has not been noted in keratinocytes before (although it is important in melanocytes and melanoma [37]).…”

In the Wnt-er of life: Wnt signalling in melanoma and ageing

“…For example, in the presence of oncogenic BRAF, Brn2 may drive MITF-mediated proliferation, but in its absence, it may repress MITF and promote invasion. Post-translational modifications such as phosphorylation also regulate the function of Brn2 and its effects on cellular proliferation, as shown by Berlin et al (2012), and MITF is known to function as a ‘rheostat' where, depending on the levels of its expression and activity, MITF signalling can result in both inhibition and promotion of melanoma growth (Goding, 2011). Very low levels of MITF or absence of MITF results in a population of completely arrested, potentially senescent cells (Vachtenheim and Ondrusova, 2015).…”

Octamer-binding transcription factors: genomics and functions

“…Oct7 controls melanocyte migration and proliferation via its regulation of MITF, by the overall Oct7 level and through regulation of Pax3 transcription by Oct7 phosphorylation at T361 and S362 residues (188, 189). Oct7 can counter-regulate NOTCH pathway of melanoma cells with MITF to suppress the differentiated melanocytic phenotype and enhance tumor metastasis (189).…”