The Elgin Lab

      We are interested in the role that chromatin structure plays in gene regulation, considering both effects from packaging large domains and local effects of the nucleosome array. We work with Drosophila, combining biochemical, genetic and cytological approaches. 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 can be observed when the P-element is inserted into pericentric heterochromatin, telomeres, or the small fourth chromosome. Further investigations to examine the mechanism(s) of gene silencing are in progress; changes in the local nucleosome array, as well as spatial organization in the nucleus, appear critical (see Heterochromatin Abstracts). More recent work has focused on the role of RNAi in targeting heterochromatin formation to appropriate domains.

      Earlier work in the lab identified Heterochromatin Protein 1 (HP1) as a protein preferentially associated with the pericentric heterochromatin, and in a banded pattern with the small fourth chromatin (see figure below). Subsequent analysis showed that HP1 is encoded by Su(var)2-5; both mutations that would be expected to reduce the level of HP1, and a point mutation (shown by others to disrupt the interaction of HP1 with H3-mK9), result in suppression of Position Effect Variegation, suggesting that HP1 plays a key role in establishing heterochromatic structure. Using a yeast two-hybrid screen, we have identified a protein that interacts with HP1. This protein, HP2, shows a similar distribution pattern on polytene chromosomes. We have recovered 17 alleles of HP2; some are strong suppressors of PEV, while others are not (see HP1 and HP2 Abstracts).

      Transgene inserts showing a variegating phenotype have been recovered not only in the pericentric heterochromatin and telomere of the fourth chromosome, but within the banded region of that chromosome. In contrast, we have identified several P-element insertion sites on the fourth chromosome that allow full gene expression, suggesting that this region has closely interspersed heterochromatic and euchromatic domains. More detailed examination has shown that proximity to repetitious elements, specifically the 1360 transposon, is critical for heterochromatin formation on the fourth chromosome. As RNAi appears to play a role in heterochromatin formation in Drosophila, we suspect that the DNA transposons are targeted for silencing by this system (see Fourth Chromosome Abstracts).

      The above studies require a detailed knowledge of the test gene, hsp26. Previous work from our lab and others has shown that correct assembly of the hsp26 regulatory region in an activatable form requires two (CT)_n sites, which bind GAGA factor (see hsp26 Abstracts). In a collaboration with D. Gilmour, Penn State, we have found that the presence of an immediately adjacent TFIID binding site is critical. The well-characterized hsp26 gene has also proven useful for studies of silencing by the Polycomb system, carried out in collaboration with V. Pirrotta, Rutgers.

Pericentric heterochromatin and the fourth chromosome are asociated with HP1 Immunofluorescent staining of the polytene chromosomes of Drosophila larvae show heterochromatin protein 1 (HP1) to be concentrated at the chromocenter and in a banded pattern along the fourth chromosome (left hand picture). Minor sites are seen along the other chromosome arms and at the telomeres. In contrast such staining shows GAGA factor, present at sites in the euchromatic arms, absent from the heterochromatic chromocenter (right hand picture). Two major sites of GAGA factor localization are seen on the fourth chromosome (arrow).