LBC AND ITS ROLE IN DEVELOPMENTAL MODULATION OF INSULATORS IN DROSOPHILA MELANOGASTER

Abstract

Chromatin insulators are architectural elements that determine the 3-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 and Fab-8 insulators from the Drosophila Bithorax complex (BX-C) are required for the parasegment specific expression of the Abd-B gene. The aim of this thesis is to understand the molecular mechanism underlying Fab-7 and Fab-8 boundary activity. Utilizing an endogenous replacement strategy, sequences that are necessary and sufficient for Fab-7 boundary function in BX-C were identified. Fab-7 boundary activity is known to depend on factors that are stage specific. A novel ~700kD complex, the Late Binidng Complex (LBC), that binds to Fab-7 sequences that have insulator function in late embryos and adults is described. Using an electrophoretic mobility shift assay (EMSA), it is shown that the LBC is enriched in nuclear extracts from late but not early embryos and that it contains three insulator proteins, GAF, Mod(mdg4) and E(y)2. Its DNA bindings properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display little sequence similarities. Finally we show that mutations, which abrogate LBC binding in vitro inactivate the Fab-7 boundary in BX-C.

Previous works show that Fab-7 and Fab-8 can replace each other indicating that Fab-8 shares similar trans-acting factors with Fab-7. To address this question, I subdivided the adjacent boundary, Fab-8, into small fragments and used these as probes for EMSA with nuclear extracts from staged embryos. EMSA results reveal that Fab-8 has sub-elements that are recognized by stage specific DNA binding factors. Moreover, there is evidence that at one early (Elba) and one late complex (LBC) are common to Fab-8 and Fab-7. LBC is shown to recognize a 65bp sub-element in Fab-8, which contains 4 distinct sequence motifs that are important for binding. This sub-element is capable of conferring boundary activity in the adult fly. These results further advance our understanding of the developmental based regulation of insulators in the BX-C. More importantly, based on these findings we can postulate new rules that govern the refinement of a fundamental cellular process, transcription.