Heterochromatin protein 2 (HP2), a partner of HP1 in Drosophila heterochromatin

Abstract

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.

Fig 1.

Interacting domain of HP1. Subdomains of the HP1 coding region fused to the LexA DNA-binding domain were tested for interaction with the polypeptide expressed by clone 2–90. The rate of growth of yeast carrying each construct on medium lacking leucine is indicated (Leu column) [no growth (−); strong growth (+++)], as is the specific activity of the induced β-gal reporter gene (β-gal column). Numbers above the lines indicate chromo and shadow domain boundaries, whereas those below the lines indicate the boundaries of subdomains used in the fusion constructs, with the exception of construct 4, which starts at amino acid 41, and construct 5, which starts at amino acid 93.

Fig 2.

The structure of the gene encoding HP2. A portion of genomic P1 clone DS00857 is shown (DNA), with the location of the 2–90 clone distal to the insertion site for P element l(2)07214. Various cDNAs and RT-PCR products sequenced to confirm the intron–exon boundaries are indicated. mRNA shows the predicted structures of the two transcripts produced by this locus (L, long; S, short). The nine exons are diagrammed below; solid triangles mark the start and stop of translation, and two solid rectangles mark the AT hooks. The locations of the genetic lesions in Su(var)2-HP2²³⁰, Su(var)2-HP2²⁸⁸, and Su(var)2-HP2⁶⁹² also are indicated.

Fig 3.

Immunolocalization of HP1 and HP2. (Upper) Polytene chromosomes stained with anti-HP1 (red, Left) and anti-HP2-L (Ab P-6; green, Right). (Lower) Close-up view of the chromocenter. Single antibody signals are shown in black and white; the color merge of HP1 signal (red) and HP2-L signal (green) is shown in the center. Note the staining of the chromocenter, in a banded pattern along the fourth chromosome (double arrowhead) and of 5–6 bands in 31B (arrow). The arrowhead identifies a euchromatic band positive for HP1 but not HP2.

Fig 4.

β-gal (Upper) and HP2 (Lower) immunofluorescent staining of polytene chromosomes from heat shock-treated flies containing a transgene expressing a chimeric β-gal-HP1-Polycomb fusion protein. The staining pattern in the absence of heat shock shows only minor amounts of euchromatic staining by antibodies against HP2 (similar to Fig. 3), whereas the staining pattern after heat shock shows extensive relocalization of HP2 (Lower) to euchromatic regions marked by the presence of the fusion protein (Upper).

Fig 5.

Genetic analysis of Su(var)2-HP2. (Upper) The chromosomal region around the gene for HP2 is shown. Known and predicted genes are indicated by solid arrows (locations of putative primary transcripts). The location of the P element l(2)07214 is indicated by the green triangle. The boundaries of the two deletions used are shown. In addition to Su(var)2-HP2, Df(2R)B11 deletes tra2 and RpI1, three putative genes, CG12868, CG12869, and CG10127, and the last five exons of ttv, and ends 94 bases upstream of the start of transcription of LamC [map based on Adams et al. (37) as interpreted by Flybase (http://flybase.bio.indiana.edu/) (38)]. (Lower) Eye phenotypes of the three Su(var)2-HP2 alleles in males carrying a w^m4h X chromosome. In each picture, the left fly carries the indicated mutation, whereas the right fly is a sibling carrying a WT copy of Su(var)2-HP2. Both Su(var)2-HP2²³⁰ and Su(var)2-HP2²⁸⁸ suppress variegated w^m4h expression, whereas Su(var)2-HP2⁶⁹² does not.