title: Molecular dissection of cis-regulatory modules at the Drosophila bithorax complex reveals critical transcription factor signature motifs.
abstract: At the Drosophila melanogaster bithorax complex (BX-C) over 330kb of intergenic DNA is responsible for directing the transcription of just three homeotic (Hox) genes during embryonic development. A number of distinct enhancer cis-regulatory modules (CRMs) are responsible for controlling the specific expression patterns of the Hox genes in the BX-C. While it has proven possible to identify orthologs of known BX-C CRMs in different Drosophila species using overall sequence conservation, this approach has not proven sufficiently effective for identifying novel CRMs or defining the key functional sequences within enhancer CRMs. Here we demonstrate that the specific spatial clustering of transcription factor (TF) binding sites is important for BX-C enhancer activity. A bioinformatic search for combinations of putative TF binding sites in the BX-C suggests that simple clustering of binding sites is frequently not indicative of enhancer activity. However, through molecular dissection and evolutionary comparison across the Drosophila genus we discovered that specific TF binding site clustering patterns are an important feature of three known BX-C enhancers. Sub-regions of the defined IAB5 and IAB7b enhancers were both found to contain an evolutionarily conserved signature motif of clustered TF binding sites which is critical for the functional activity of the enhancers. Together, these results indicate that the spatial organization of specific activator and repressor binding sites within BX-C enhancers is of greater importance than overall sequence conservation and is indicative of enhancer functional activity.
authors.affiliation: Biology Department, Harvey Mudd College, Claremont, CA 91711, USA.
authors.forename: Michael O
authors.initials: MO
authors.lastname: Starr
authors.forename: Margaret C W
authors.initials: MC
authors.lastname: Ho
authors.forename: Eric J M
authors.initials: EJ
authors.lastname: Gunther
authors.forename: Yen-Kuei
authors.initials: YK
authors.lastname: Tu
authors.forename: Andrey S
authors.initials: AS
authors.lastname: Shur
authors.forename: Sara E
authors.initials: SE
authors.lastname: Goetz
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Borok
authors.forename: Victoria
authors.initials: V
authors.lastname: Kang
authors.forename: Robert A
authors.initials: RA
authors.lastname: Drewell
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004330:Drosophila / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics*; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000196:embryology / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D019143:Evolution, Molecular; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000235:genetics*; D017403:In Situ Hybridization; D008957:Models, Genetic; D008969:Molecular Sequence Data; D005810:Multigene Family; D009687:Nuclear Proteins / Q000235:genetics; D059372:Nucleotide Motifs / Q000235:genetics; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D013045:Species Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: C062393:Abd-B proteins, Drosophila; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; C066177:Ubx protein, Drosophila; C066591:abd-A protein, Drosophila