Direct and Indirect Regulation of SIX1+EYA Transcriptional Activity by PA2G4, MCRS1, and SOBP

Abstract Branchio-oto-renal (BOR) syndrome is an autosomal dominant condition characterized by variable craniofacial malformations, hearing loss and in some patients, renal dysfunction. Recently, a clinical study showed that patients with mutations causative of BOR also present with craniosynostosis, indicating that this could be an undiagnosed feature of BOR. Approximately half of all patients presenting with BOR have a variant in either SIX1 or its activating co-factor EYA1 . The genes underlying BOR in the other 50% of patients remain unknown. To date, most studies on the role of SIX1 in the inner ear have focused on its role in the placode-based development of the sensorineural epithelia (cochlear and vestibular organs), as sensorineural hearing loss has long term effects on human health and development. The role of SIX1 in the neural crest cells (NCCs) of the first pharyngeal ( a.k.a. , mandibular) arch, which will give rise to the upper and lower jaws as well as the middle ear ossicles which may be affected in patients with BOR, is less well understood. To that end, we examine herein the role of three proteins that have been identified in Xenopus laevis as SIX1 co-factors to determine whether their function as regulators of SIX1+EYA transcriptional activity is conserved in mouse and begin to elucidate their function in the NCCs of the mandibular arch. Our data reveal that SIX1 is co-expressed with PA2G4, MCRS1, and SOBP within the oral domain of the mandibular arch, although each has a very distinct expression pattern. We further demonstrate that MCRS1 and SOBP are bona fide SIX1 co-factors that can repress SIX1+EYA transcriptional activity. PA2G4, on the other hand, does not bind to SIX1 in the mouse but can indirectly enhance the transcriptional activity of the SIX1+EYA transcriptional complex. Further, we show that both MCRS1 and SOBP are also able to translocate EYA to the nucleus via direct ( i.e. , SOBP) or indirect ( i.e. , MCRS1) mechanisms. We also show that later, during development of the mandible and incisors that SOBP is co-expressed with SIX1 within these anlagen, confirming the biological relevance of these transcriptional complexes in craniofacial bone formation and within the derivatives of the mandibular NCCs. This study, therefore, highlights the complexity of SIX1+EYA transcriptional regulation suggesting that there are multiple, and potentially redundant, mechanisms in place to ensure precise levels of SIX1+EYA transcriptional activity during craniofacial development.