We study the mechanisms and control of telomere elongation, insulator functions, and enhancer-promoter interactions in Drosophila. Telomeres are structures at the termini of linear chromosomes and are responsible for a number of functions, such as protection of chromosomal ends from fusion and degradation, and compensation for the inability of DNA polymerases to completely replicate the end of a linear chromosome. The telomeric structure in Drosophila melanogaster and other drosophilids is essentially different from that in other eukaryotes; head-to-tail arrays of telomere-specific non-LTR (long terminal repeat) retrotransposons, HeT-A, and TART make up the endmost sequences instead of simple repeats generated by telomerase. Yeast and mammals assemble protein complexes on the telomerase-generated repeats, whereas Drosophila appears to employ a sequence-independent protein capping complex. This terminal capping complex seems to bind to any stable chromosome end, regardless of the presence of HeT-A or TART. Despite the pronounced differences in structure and properties, orthodox telomerase-generated telomeres and Drosophila retrotransposon-based telomeres may have some features in common. For instance, the conserved DNA repair and DNA damage checkpoint proteins are essential to the function of both telomere types. In most organisms the telomere length is maintained by developmental activity and by controlling telomere accessibility to telomerase. In Drosophila the mechanism of telomere length regulation is unknown.
Regardless of the different origin and sequence composition of the endmost telomeric regions, all eukaryotic nuclear chromosomes contain a span of complex DNA repeats (TAS) adjacent to the telomeric regions. In Drosophila melanogaster TAS arrays consist of 15–26 kb blocks, depending on the end of the chromosome. The expression of transgenes inserted in TAS is strongly inhibited, suggesting that repressive chromatin is formed on the TAS regions of the Drosophila telomeres. We proposed a model suggesting that the repressive chromatin bound to TAS regulates the stability of chromatin formed at the telomeres. If the telomere HeT-A/TART array is relatively short, the chromatin formed on TAS would interfere with the assembly of telomeric chromatin, which would result in higher accessibility of the chromosome end and thus in telomere lengthening. In cases where the HeT-A/TART array at the telomere is sufficiently long, chromatin formed on TAS would not influence the stability of telomeric chromatin that protected the chromosome end, would prevent telomere elongation and lead to telomere shortening. To test our hypothesis, we developed genetic systems based on terminal deficiencies generated by I-SceI cutting in P transposons inserted in TAS. The main goal is to identify new genes important for telomere functioning and compare them with mammalian and yeast homologues.
Eukaryotic enhancers and silencers are able to act over long distances, sometimes interacting with promoters hundreds of kilobase pairs away. A fundamental question in transcriptional regulation is how enhancers find and control their target promoters over long distances. Depending on the location, shielding against positive or negative regulatory effects from neighboring chromatin may be required. Indeed, sequences referred to as insulators that prevent the spread of activation or repression to a promoter have been found in various organisms. The insulators were found at the 3′ ends of the yellow and white genes. The white gene is widely used as a marker to select transgenic lines and as a test gene in the enhancer blocking assay. The existence of insulators 3′ to the yellow and white genes suggests that such insulator-like regulatory elements may be widely distributed in the Drosophila genome. Questions arise as to how widespread insulators are and what role these insulators play in gene regulation. To study the role of insulators in transcriptional regulation, we plan to identify new insulators and proteins that are responsible for the enhancer-blocking and boundary activities. To look into the functions of the insulators, we plan to assess the functional interactions between insulators in various combinations at the same genomic site, by using Cre/LOX, Flp/FRT and newly created I-SceI recombination systems, and to test the physical interactions between the insulators, by running ChiP, Dam methyltransferase, and 3C assays.
Frequently, genes and associated cis-acting sequences do not constitute physically distinct domains on the chromosomes. Rather, genes can overlap, and cis-acting sequences can be found tens and hundreds of kilobases away from the target gene, sometimes with unrelated genes in between. It was found recently that chromatin loop formation might result from direct interactions between transcriptional factors bound at an enhancer and a promoter. The looping model raises the question of how potentially promiscuous enhancers in the cis-regulatory regions are able to avoid other promoters and to locate and physically approach their proper target promoter. The existence of the “tethering elements” in the promoter-proximal region was postulated and that they recruit distal enhancers to the core promoter. It is possible that a special class of proteins ensures correct gene expression by supporting proper interactions between enhancers and promoters. We will attempt to establish a simple assay to map tethering elements in enhancers and promoters. The isolation of such regulatory elements will help identify proteins that are responsible for long-distance enhancer-promoter communication.
Last updated August 2009
