An evolutionarily conserved enhancer regulates Bmp4 expression in developing incisor and limb bud.
To elucidate the transcriptional regulation of Bmp4 expression during organogenesis, we used phylogenetic footprinting and transgenic reporter analyses to identify Bmp4 cis-regulatory modules (CRMs). These analyses identified a regulatory region located ∼46 kb upstream of the mouse Bmp4 transcription start site that had previously been shown to direct expression in lateral plate mesoderm. We refined this regulatory region to a 396-bp minimal enhancer, and show that it recapitulates features of endogenous Bmp4 expression in developing mandibular arch ectoderm and incisor epithelium during the initiation-stage of tooth development. In addition, this enhancer directs expression in the apical ectodermal ridge (AER) of the developing limb and in anterior and posterior limb mesenchyme. Transcript profiling of E11.5 mouse incisor dental lamina, together with protein binding microarray (PBM) analyses, allowed identification of a conserved DNA binding motif in the Bmp4 enhancer for Pitx homeoproteins, which are also expressed in the developing mandibular and incisor epithelium. In vitro electrophoretic mobility shift assays (EMSA) and in vivo transgenic reporter mutational analyses revealed that this site supports Pitx binding and that the site is necessary to recapitulate aspects of endogenous Bmp4 expression in developing craniofacial and limb tissues. Finally, Pitx2 chromatin immunoprecipitation (ChIP) demonstrated direct binding of Pitx2 to this Bmp4 enhancer site in a dental epithelial cell line. These results establish a direct molecular regulatory link between Pitx family members and Bmp4 gene expression in developing incisor epithelium.
Division of Genetics, Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston Massachusetts, United States of America.
Dolrudee
D
Jumlongras
Salil A
SA
Lachke
Daniel J
DJ
O'Connell
Anton
A
Aboukhalil
Xiao
X
Li
Sung E
SE
Choe
Joshua W K
JW
Ho
Annick
A
Turbe-Doan
Erin A
EA
Robertson
Bjorn R
BR
Olsen
Martha L
ML
Bulyk
Brad A
BA
Amendt
Richard L
RL
Maas
D000818:Animals; D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000235:genetics / Q000502:physiology*; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000502:physiology*; D018398:Homeodomain Proteins / Q000378:metabolism; D007180:Incisor / Q000254:growth & development* / Q000378:metabolism; D061346:Laser Capture Microdissection; D018878:Limb Buds / Q000254:growth & development* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D016296:Mutagenesis; D040081:Protein Array Analysis; D013045:Species Specificity; D014157:Transcription Factors / Q000378:metabolism; D001616:beta-Galactosidase; D000097577:Homeobox Protein PITX2
D055415:Bone Morphogenetic Protein 4; D017931:DNA Primers; D018398:Homeodomain Proteins; D014157:Transcription Factors; D001616:beta-Galactosidase; D000097577:Homeobox Protein PITX2; C525810:Bmp4 protein, mouse