The Elgin Lab

Mol Cell Biol. 2004 Sep;24(18):8210-20.

cis-Acting determinants of heterochromatin formation on Drosophila melanogaster chromosome four

Sun FL, Haynes K, Simpson CL, Lee SD, Collins L, Wuller J, Eissenberg JC, Elgin SC.
Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

The heterochromatic domains of Drosophila melanogaster (pericentric heterochromatin, telomeres, and the fourth chromosome) are characterized by histone hypoacetylation, high levels of histone H3 methylated on lysine 9 (H3-mK9), and association with heterochromatin protein 1 (HP1). While the specific interaction of HP1 with both H3-mK9 and histone methyltransferases suggests a mechanism for the maintenance of heterochromatin, it leaves open the question of how heterochromatin formation is targeted to specific domains. Expression characteristics of reporter transgenes inserted at different sites in the fourth chromosome define a minimum of three euchromatic and three heterochromatic domains, interspersed. Here we searched for cis-acting DNA sequence determinants that specify heterochromatic domains. Genetic screens for a switch in phenotype demonstrate that local deletions or duplications of 5 to 80 kb of DNA flanking a transposon reporter can lead to the loss or acquisition of variegation, pointing to short-range cis-acting determinants for silencing. This silencing is dependent on HP1. A switch in transgene expression correlates with a switch in chromatin structure, judged by nuclease accessibility. Mapping data implicate the 1360 transposon as a target for heterochromatin formation. We propose that heterochromatin formation is initiated at dispersed repetitive elements along the fourth chromosome and spreads for approximately 10 kb or until encountering competition from a euchromatic determinant.

Model: repetitious element 1360 as an initiator of heterochromatin. Local deletions and duplications, occuring on re-mobilitzation of the reporter P element, indicated that proximity to 1360 determines whether or not the regions will be assembled into heterochromatin.

Proc Natl Acad Sci USA. 2000 May 9;97(10):5340-5.

The fourth chromosome of Drosophila melanogaster: interspersed euchromatic and heterochromatic domains

Sun FL, Cuaycong MH, Craig CA, Wallrath LL, Locke J, Elgin SC.
Department of Biology, Washington University, St. Louis, MO 63130, USA.

The small fourth chromosome of Drosophila melanogaster (3.5% of the genome) presents a puzzle. Cytological analysis suggests that the bulk of the fourth, including the portion that appears banded in the polytene chromosomes, is heterochromatic; the banded region includes blocks of middle repetitious DNA associated with heterochromatin protein 1 (HP1). However, genetic screens indicate 50-75 genes in this region, a density similar to that in other euchromatic portions of the genome. Using a P element containing an hsp70-white gene and a copy of hsp26 (marked with a fragment of plant DNA designated pt), we have identified domains that allow for full expression of the white marker (R domains), and others that induce a variegating phenotype (V domains). In the former case, the hsp26-pt gene shows an accessibility and heat-shock-inducible activity similar to that seen in euchromatin, whereas in the latter case, accessibility and inducible expression are reduced to levels typical of heterochromatin. Mapping by in situ hybridization and by hybridization of flanking DNA sequences to a collection of cosmid and bacterial artificial chromosome clones shows that the R domains (euchromatin-like) and V domains (heterochromatin-like) are interspersed. Examination of the effect of genetic modifiers on the variegating transgenes shows some differences among these domains. The results suggest that heterochromatic and euchromatic domains are interspersed and closely associated within this 1.2-megabase region of the genome.

"Heterochromatic" and "euchromatic" domains are interspersed on the fourth chromosome. Following mobilization of our test P-element, we have recovered 15 lines with P-element inserts at new sites on the fourth chromosome, selected for high levels of gene expression (full red yee). Work is in progress to further characterize these lines and others recovered in a similar screen; the results of in situ hybridization show that of the lines examined to date, all have insertion sites within the banded region of the fourth chromosome. These findings indicate that a standard test gene can be used to identify different functional domains along the fourth chromosome. The results indicate that in this instance, domains imposing a variegating phenotype (heterochromatin) are closely interspersed with domains allowing full expression (euchromatin).