Molecular characterization of SF-1, a novel chromatin boundary from the Drosophila Antennapedia complex
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My work has focused on understanding the biological function and the molecularmechanism of SF-1, a novel chromatin boundary from the Scr-ftz region in the DrosophilaAntennapedia complex. Chromatin boundaries regulate gene activity by modulating enhancer-promoter interactions (insulator activity) and protecting genes from the influences of neighboringchromatin (barrier activity). Previous work showed that SF-1 contains both of these activities butthey reside in separate DNA fragments: the insulator activity is associated with SF-1/b, whereasthe barrier activity lies mainly in SF-1/c. Our transgenic studies show that SF-1/b but not SF-1/cis capable of blocking various enhancers. In particular, SF-1/b blocks the ftz distal enhancer, anelement that does not rely on promoter competition for the selection of appropriate target. On theother hand, SF-1/c but not SF-1/b prevents the spread of silent chromatin initiated at the Scr PREas revealed by the mini-white reporter assay. Based on these results we propose a model for thedual function of SF-1 in the region: a) it protects the Scr promoter from inappropriate activationby nearby ftz enhancers and b) it protects a non-homeotic gene ftz from the effects of Scr-proximal PRE. The molecular mechanism of insulator and barrier activities appears to bedistinct, i.e. SF-1/b critically depends on the GAGA factor, whereas SF-1/c does not. To gainfurther insights into the insulator mechanism, we identified the minimal sequence, SF-1/b3,necessary for enhancer-blocking. This sequence is highly conserved among four Drosophilaspecies separated by approximately 5 million years, suggesting an important biological function.SF-1/b3 was used as a bait in a yeast one-hybrid screen of the Drosophila cDNA library. Fivecandidate trans-factors were isolated in this screen. To test biological relevance of these proteinswe devised a novel cell culture-based insulator assay, utilizing the inducible expression of GFPdriven by the metallothionin enhancer. The transcripts of the candidate genes were targeted byRNAi. The knock-down of e(bx) and GAGA factor were found to attenuate the enhancer-blocking activity of SF-1/b in this assay. We demonstrate the utility of this cell culture systemfor the genome-wide RNAi-mediated search for the protein components of insulators.