The Elgin Lab

Contacts:

Overview:

   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 (see figure below). 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.
   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. 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, result in suppression of Position Effect Variegation, suggesting that HP1 plays a role in establishing heterochromatic structure. All of the known mutant alleles of Su(var)2-5 result in greater expression from our test transgenes inserted in pericentric heterochromatin and the fourth chromosome, but have no impact on expression of the same transgenes in the telomeres of the second or third chromosome, indicating a difference in these heterochromatic domains. Work is ongoing to identify additional heterochromatin-associated proteins, screening for interactions with HP1. A candidate has been identified using a yeast two-hybrid screen; this protein, HP2, shows a similar distribution pattern on polytene chromosomes.
   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. Recently 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. Utilizing such lines, we plan to create a functional map, allowing characterization of such domains and their boundaries. Results to date indicate consistent differences in the chromatin structure of heterochromatic and euchromatic domains.
   We are also looking at the effect of a homeotic regulatory signal, PRE, on hsp26 structure and function (collaboration with V. Pirrotta, Geneva); such signals may maintain silencing by altering chromatin structure. Thus, shifts in the nucleosome array, and concommitant shifts in higher order structure, might be a key distinction between activatable and silent genes programmed during development.
   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. We are examining the effect of various mutations in the hsp26 promoter on GAGA factor function and chromatin assembly, in particular asking whether the presence of an immediately adjacent TFIID binding site is critical (collaboration with D. Gilmour, Penn State).

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 to the polytene chromosomes with the entire P element; see B), telomeres, or the fourth chromosome. (Note that the P element also hybridizes to the sites of the endogenous white, hsp70, and hsp26 genes.) See Elgin et al, 1993, and Wallrath and Elgin, 1995; full citations and abstracts below.