The GAGA transcription factor of Drosophila melanogaster is ubiquitous and plays multiple roles. Characterization of cDNA clones and detection by domain- specific antibodies has revealed that the 70-90 kDa major GAGA species are encoded by two open reading frames producing GAGA factor proteins of 519 amino acids (GAGA-519) and 581 amino acids (GAGA-581), which share a common N-terminal region that is linked to two different glutamine-rich C-termini. Purified recombinant GAGA-519 and GAGA-581 proteins can form homomeric complexes that bind specifically to a single GAGA sequence in vitro. The two GAGA isoforms also function similarly in transient transactivation assays in tissue culture cells and in chromatin remodeling experiments in vitro . Only GAGA-519 protein accumulates during the first 6 h of embryogenesis. Thereafter, both GAGA proteins are present in nearly equal amounts throughout development; in larval salivary gland nuclei they colocalize completely to specific regions along the euchromatic arms of the polytene chromosomes. Coimmunoprecipitation of GAGA-519 and GAGA-581 from crude nuclear extracts and from mixtures of purified recombinant proteins, indicates direct interactions. We suggest that homomeric complexes of GAGA-519 may function during early embryogenesis; both homomeric and heteromeric complexes of GAGA-519 and GAGA-581 may function later.

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 heatshock-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.

Heterochromatin protein 1 (HP1), first discovered in Drosophila melanogaster, is a highly conserved chromosomal protein implicated in both heterochromatin formation and gene silencing. We report here characterization of an HP1-interacting protein, heterochromatin protein 2 (HP2), which codistributes with HP1 in the pericentric heterochromatin. HP2 is a large protein with two major isoforms of approximately 356 and 176 kDa. The smaller isoform is produced from an alternative splicing pattern in which two exons are skipped. Both isoforms contain the domain that interacts with HP1; the larger isoform contains two AT-hook motifs. Mutations recovered in HP2 act as dominant suppressors of position effect variegation, confirming a role in heterochromatin spreading and gene silencing.

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