Date Published: February 3, 2017
Publisher: Public Library of Science
Author(s): Başak Kandemir, Ugur Dag, Burcu Bakir Gungor, İlknur Melis Durasi, Burcu Erdogan, Eray Sahin, Ugur Sezerman, Isil Aksan Kurnaz, Jinsong Zhang.
Pea3 transcription factor belongs to the PEA3 subfamily within the ETS domain transcription factor superfamily, and has been largely studied in relation to its role in breast cancer metastasis. Nonetheless, Pea3 plays a role not only in breast tumor, but also in other tissues with branching morphogenesis, including kidneys, blood vasculature, bronchi and the developing nervous system. Identification of Pea3 target promoters in these systems are important for a thorough understanding of how Pea3 functions. Present study particularly focuses on the identification of novel neuronal targets of Pea3 in a combinatorial approach, through curation, computational analysis and microarray studies in a neuronal model system, SH-SY5Y neuroblastoma cells. We not only show that quite a number of genes in cancer, immune system and cell cycle pathways, among many others, are either up- or down-regulated by Pea3, but also identify novel targets including ephrins and ephrin receptors, semaphorins, cell adhesion molecules, as well as metalloproteases such as kallikreins, to be among potential target promoters in neuronal systems. Our overall results indicate that rather than early stages of neurite extension and axonal guidance, Pea3 is more involved in target identification and synaptic maturation.
ETS domain transcription factors are characterized by an evolutionarily-conserved ETS domain of about 85 amino acids that facilitates binding to DNA sequences with a central GGAA/T core consensus and flanking nucleotides . Around 30 members of the ETS proteins have been identified in mammals and are categorized within several subfamilies. Among them, PEA3 subfamily members, most notably Pea3/ETV4, Erm/ETV5 and Er81/ETV1, also bind to the DNA core sequence GGAA/T , and contain an acidic activation domain in the N-terminus as well as a C-terminal activation domain . Pea3 family members are involved in several processes, including breast cancer, prostate cancer , motor neuron connectivity and dendritic arborization  as well as neuronal differentiation [6,7].
The aim of this combinatorial study was to identify novel transcriptional targets for Pea3 with respect to its neuron-specific functions. To that end, our first approach was an in silico analysis through manual curation of predicted target promoters for Pea3/ETV4 (Fig 1a). 404 human genes related to neuronal migration and 47 human genes related to axonal guidance were manually curated, and promoter sequences for 428 of these were found through nucleotide databases (Fig 1). Out of these, 123 candidate promoters crossed the threshold (5% dissimilarity rate) for Pea3/ETV4 binding (Fig 1b).
ETS transcription factors were shown to be regulated in a temporally regulated manner at later stages of nervous system development, in particular for normal sensory neuron differentiation and during branching . Pea3 family of proteins are expressed from E9.5 till birth, and in some cases after birth, starting with brain regions followed by expression in lung, thymus, cartilage and mammary tissue of mouse . Pea3 and Er81 appear to be particularly important at later stages of neural development, whereas Erm seems to be involved in early differentiation of neural crest stem cells .