ABSTRACT
Pax3 plays a role in regulating Hes1 and Neurog2 activity and thereby stem cell maintenance and neurogenesis. A mechanism for Pax3 regulation of these two opposing events, during caudal neural tube development, is examined in this study. Pax3 acetylation on C-terminal lysine residues K437 and K475 may be critical for proper regulation of Hes1 and Neurog2. Removal of these lysine residues increased Hes1 but decreased Neurog2 promoter activity. SIRT1 deacetylase may be a key component in regulating Pax3 acetylation. Chromatin immunoprecipitation assays showed that SIRT1 is associated with Hes1 and Neurog2 promoters during murine embryonic caudal neural tube development at E9.5, but not at E12.5. Overexpression of SIRT1 decreased Pax3 acetylation, Neurog2 and Brn3a positive staining. Conversely, siRNA-mediated silencing of SIRT1 increased these factors. These studies suggest that Pax3 acetylation results in decreased Hes1 and increased Neurog2 activity, thereby promoting sensory neuron differentiation.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Homeodomain Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurogenesis/genetics , Paired Box Transcription Factors/metabolism , Sensory Receptor Cells/metabolism , Sirtuin 1/genetics , Sirtuin 1/metabolism , Acetylation , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Line, Tumor , Chromatin Immunoprecipitation/methods , Homeodomain Proteins/genetics , Humans , Mice , Nerve Tissue Proteins/genetics , Neural Tube/metabolism , PAX3 Transcription Factor , Paired Box Transcription Factors/genetics , Promoter Regions, Genetic/genetics , Protein Processing, Post-Translational/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Transcription Factor HES-1ABSTRACT
Pax3 is expressed early during embryonic development in spatially restricted domains including limb muscle, neural crest, and neural tube. Pax3 functions at the nodal point in melanocyte stem cell differentiation, cardiogenesis and neurogenesis. Additionally Pax3 has been implicated in migration and differentiation of precursor cell populations. Currently there are questions about how Pax3 regulates these diverse functions. In this study we found that in the absence of functional Pax3, as in Splotch embryos, the neural crest cells undergo premature neurogenesis, as evidenced by increased Brn3a positive staining in neural tube explants, in comparison with wild-type. Premature neurogenesis in the absence of functional Pax3 may be due to a change in the regulation of basic helix-loop-helix transcription factors implicated in proliferation and differentiation. Using promoter-luciferase activity measurements in transient co-transfection experiments and electro-mobility shift assays, we show that Pax3 regulates Hairy and enhancer of split homolog-1 (Hes1) and Neurogenin2 (Ngn2) by directly binding to their promoters. Chromatin immunoprecipitation assays confirmed that Pax3 bound to cis-regulatory elements within Hes1 and Ngn2 promoters. These observations suggest that Pax3 regulates Hes1 and Ngn2 and imply that it may couple migration with neural stem cell maintenance and neurogenesis.
