Hox/Pbx and Brn binding sites mediate Pax3 expression in vitro and in vivo.
Pax3 is a paired-homeodomain class transcription factor that serves a role in dorsal-ventral and medial-lateral patterning during vertebrate embryogenesis. Its expression is localized to dorsal domains within the developing neural tube and lateral domains within the developing somite. Additionally, modulation of its expression occurs along the rostral-caudal axis. Previous studies [Development 124 (1997) 617] have localized sequence elements required for expression of Pax3 in the neural tube and neural crest to a 1.6 kbp promoter fragment. In the present study, four discrete DNA elements within the 1.6 kbp promoter fragment are shown by electrophoretic mobility shift assays (EMSA) to exhibit sequence specific interactions with proteins present in nuclear extracts from P19 EC cells induced to express Pax3 by treatment with retinoic acid (RA). Proteins interacting at each of these elements are identified based on biochemical purification using DNA affinity chromatography or a candidate approach. These identifications were confirmed by the ability of specific antibodies to super-shift DNA-protein complexes in EMSA. Two of the four DNA sequence elements are shown to interact with the neural specific Pou-domain class III transcription factors Brn1 and Brn2. The remaining sites contain either consensus binding elements for heterodimers of Pbx and an anterior set of Hox family members, from paralogous groups 1-5, or monomeric Meis and are shown to interact with members of the Pbx and Meis families. Ectopic expression of Brn2 plus HoxA1 but not either factor alone, is sufficient to induce efficient expression from the endogenous Pax3 promoter in P19 EC stem cells under conditions where they would not otherwise express Pax3. Finally, in transgenic mice, mutation of either of the Pou-domain protein binding sites results in reduced expression throughout the neural tube while mutation of the Pbx/Hox binding site results in loss of expression in the anterior domain in which Hox family members from paralogous groups 1-5 are expressed. These observations demonstrate that binding elements for both neural and anterior-posterior position specific transcription factors mediate domains of Pax3 expression.
Pruitt
Steven C
SC
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. steven.pruitt@roswellpark.org
Bussman
Amy
A
Maslov
Alexander Y
AY
Natoli
Thomas A
TA
Heinaman
Roy
R
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D017403:In Situ Hybridization; D066298:In Vitro Techniques; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D009419:Nerve Tissue Proteins; D009432:Neural Crest / Q000196:embryology; D009479:Neuropeptides / Q000096:biosynthesis* / Q000737:chemistry; D000072041:PAX3 Transcription Factor; D050809:POU Domain Factors; D051761:Paired Box Transcription Factors; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D015534:Trans-Activators / Q000096:biosynthesis* / Q000737:chemistry; D014157:Transcription Factors / Q000096:biosynthesis* / Q000378:metabolism; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology; D001616:beta-Galactosidase / Q000378:metabolism
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D000072041:PAX3 Transcription Factor; D050809:POU Domain Factors; D051761:Paired Box Transcription Factors; D015534:Trans-Activators; D014157:Transcription Factors; C085076:transcription factor Brn-2; C068686:Pax3 protein, mouse; C074065:Pou3f3 protein, mouse; D014212:Tretinoin; D004247:DNA; D001616:beta-Galactosidase