Transcriptional regulation of Msx2 in the AERs of developing limbs is dependent on multiple closely spaced regulatory elements.
In developing limb buds, Msx2 transcripts are expressed in the apical ectodermal ridge (AER) and in various regions of the limb mesenchyme. To identify DNA sequences responsible for Msx2 expression in the AER, we characterized the expression of LacZ reporter constructs driven by chicken Msx2 regulatory sequences in transgenic mice. We have identified a 55-bp enhancer that can direct AER-specific reporter gene expression. This 55-bp enhancer contains three elements that are evolutionary conserved among five vertebrate Msx2 genomic sequences. AER expression of reporter constructs in transgenic mice is lost or reduced when mutations are introduced into each of these three regions. Moreover, changing the relative orientation by reverse complementing one of the three elements also results in loss of expression, suggesting that the relative orientations of transcription factor binding is important. To identify the transcription factor(s) binding to these elements, we conducted one-hybrid screening and identified Dlx5 and Sox11. Both Dlx5 and Sox11 are expressed in the AER, and the proteins encoded by these genes bind to separate conserved elements, supporting their possible roles in regulating Msx2 expression.
Cheng
Hsu-Chen
HC
Department of BioStructure and Function, School of Dental Medicine, University of Connecticut Health Center, Farmington, CT 06030-3705, USA.
Wang
Chi-Kuang Leo
CK
Upholt
William B
WB
D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004475:Ectoderm / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005121:Extremities / Q000196:embryology*; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter / Q000235:genetics; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D017403:In Situ Hybridization; D008156:Luciferases; D051379:Mice / Q000196:embryology* / Q000378:metabolism; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D055751:SOXC Transcription Factors; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins; C501120:Dlx5 protein, mouse; D006609:High Mobility Group Proteins; D018398:Homeodomain Proteins; C084119:MSX2 protein; D055751:SOXC Transcription Factors; C490793:Sox11 protein, mouse; D014157:Transcription Factors; D008156:Luciferases