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We are interested in the role that chromatin structure plays in gene
regulation, both effects from packaging large domains and local effects of
the nucleosome array. Working with Drosophila melanogaster, we have used a
transposable P element containing a copy of the white gene, a visible marker
for gene silencing, and a copy of hsp26, a well-characterized inducible
gene, to examine the effect of insertion into different chromosomal domains.
While these genes are fully active in euchromatic domains, silencing
(similar to Position Effect Variegation) is observed on insertion into
pericentric heterochromatin, telomeres, and sites within the small fourth
chromosome. Both changes in the local nucleosome array, and the spatial
organization of the nucleus, appear critical in determining gene silencing.
While many P element insertion sites on the fourth chromosome induce
silencing, others allow full expression; these sites are interspersed,
indicating closely interspersed heterochromatic and euchromatic domains. We
are characterizing these domains and their impact on gene expression.
Mapping experiments indicate that heterochromatin formation is targeted by
the presence of a repetitious element, 1360, and perhaps by other similar
elements. Genetic analysis has shown that heterochromatic silencing is
dependent on the RNAi machinery. Work is ongoing to determine the mechanism
of heterochromatin targeting, and to analyze the role of critical
heterochromatin-associated proteins, including HP1 and HP2.
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Our P-element construct (based on vector A412 from V. Pirrotta) contains a visible marker for variegation, hsp70-white, and a marked copy of hsp26 for subsequent studies of chromatin structure. Several fly lines have been recovered showing a PEV phenotype (A); all have P element inserts in the pericentric heterochromatin (as shown for this case by in situ hybridization of the polytene chromosomes with the entire P element; see B), telomeres and the small fourth chromosome.
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