ABSTRACT
The Fab-7 boundary is required to ensure that the iab-6 and iab-7 cis-regulatory domains in the Drosophila Bithorax complex can function autonomously. Though Fab-7 functions as a boundary from early embryogenesis through to the adult stage, this constitutive boundary activity depends on subelements whose activity is developmentally restricted. In the studies reported here, we have identified a factor, called early boundary activity (Elba), that confers Fab-7 boundary activity during early embryogenesis. The Elba factor binds to a recognition sequence within a Fab-7 subelement that has enhancer-blocking activity during early embryogenesis, but not during mid-embryogenesis or in the adult. We found that the Elba factor is present in early embryos but largely disappears during mid-embryogenesis. We show that mutations in the Elba recognition sequence that eliminate Elba binding in nuclear extracts disrupt the early boundary activity of the Fab-7 subelement. Conversely, we find that early boundary activity can be reconstituted by multimerizing the Elba recognition site.
Subject(s)
Drosophila Proteins/physiology , Drosophila melanogaster/embryology , Embryonic Development , Enhancer Elements, Genetic , Nuclear Proteins/physiology , Transcription Factors/physiology , Animals , Binding Sites , Cell Nucleus , Embryo, NonmammalianABSTRACT
In the work reported here we have analyzed the role of the GAGA factor [encoded by the Trithorax-like (Trl) gene] in the enhancer-blocking activity of Frontabdominal-7 (Fab-7), a domain boundary element from the Drosophila melanogaster bithorax complex (BX-C). One of the three nuclease hypersensitive sites in the Fab-7 boundary, HS1, contains multiple consensus-binding sequences for the GAGA factor, a protein known to be involved in the formation and/or maintenance of nucleosome-free regions of chromatin. GAGA protein has been shown to localize to the Fab-7 boundary in vivo, and we show that it recognizes sequences from HS1 in vitro. Using two different transgene assays we demonstrate that GAGA-factor-binding sites are necessary but not sufficient for full Fab-7 enhancer-blocking activity. We show that distinct GAGA sites are required for different enhancer-blocking activities at different stages of development. We also show that the enhancer-blocking activity of the endogenous Fab-7 boundary is sensitive to mutations in the gene encoding the GAGA factor Trithorax-like.
Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/metabolism , Enhancer Elements, Genetic/physiology , Gene Expression Regulation/physiology , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , DNA-Binding Proteins/genetics , Drosophila/genetics , Drosophila Proteins/genetics , Molecular Sequence Data , Mutation , Phenotype , Transcription Factors/geneticsABSTRACT
The Fab-7 boundary functions to ensure the autonomous activity of the iab-6 and iab-7 cis-regulatory domains in the Drosophila Bithorax Complex from early embryogenesis through to the adult stage. Although Fab-7 is required only for the proper development of a single posterior parasegment, it is active in all tissues and stages of development that have been examined. In this respect, Fab-7 resembles conventional constitutive boundaries in flies and other eukaryotes that act through ubiquitous cis-elements and trans-acting factors. Surprisingly, however, we find that the constitutive activity of Fab-7 is generated by combining sub-elements with developmentally restricted boundary function. We provide in vivo evidence that the Fab-7 boundary contains separable regions that function at different stages of development. These findings suggest that the units (domains) of genetic regulation that boundaries delimit can expand or contract by switching insulator function off or on in a temporally regulated fashion.
