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

Abstract

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.

FIG. 1.

A map of the P[hsp26-pt, hsp70-w] construct drawn to scale (40). The hsp26 sequences from −1917 to +490 were fused to a 740-bp fragment of barley cDNA (plant probe), followed by hsp70 transcription termination sequences (dotted box). This fusion gene was cloned into the P element vector A412 possessing an hsp70-white gene. Transcription start sites for hsp26-pt and hsp70-w are marked by bent arrows. The 3′P end is on the left side (horizontally striped box), and the 5′P end is on the right side (vertically striped box). Restriction sites used in the analysis are the following: S, SalI; Sp, SpeI; X, XbaI; B, BglII; D, DpnII; Hp, HpaII; H, HhaI. Primers used for inverse or direct PCR to identify flanking DNA (small half-arrows below the diagram) are the following: 1, 5′-AACTCGAGGCCTCGAGGT-3′; 2, 5′-GACGAAATGACCCACTCGG-3′; 3, 5′-GCTTCGGCTATCGACGGGACCACC-3′.

FIG. 2.

Heterochromatin is not limited to tandem repetitious sequences. The phenotype of P[hsp26-pt, hsp70-w] transgenes indicates that while domains of euchromatin are present, the fourth chromosome is largely heterochromatic. The map of the fourth chromosome (top) (regions 101F to 102F) shows the positions of known and predicted genes and of transposable elements (upper row, 1360 elements; lower row, others) (15). Solid triangles and dotted triangles mark P element insertion sites that result in a solid red eye or induce a variegating phenotype, respectively. The black dot indicates the 5′ end of the P element. See Materials and Methods for detailed descriptions of the screens used to recover these lines and of the techniques used to confirm the presence of a single intact P element in each line and to map its position. In cases where a line has been recovered with a P element inserted within a gene, the gene (in green) is indicated by an asterisk. In all cases tested, the variegating phenotype is suppressed by a mutation in HP1; representative examples are shown in the pictures at the bottom, with the line crossed to the y w^67c23 line (the left-hand picture of each pair) compared to the line crossed to the y w^67c23, Su(var)2-5⁰² line (the right-hand picture of each pair).

FIG. 3.

Local deletions can induce a change in eye phenotype. (A) A high-resolution map of a ∼200-kb region from the fourth chromosome. Scale divisions are 5 kb. Genes are shown as thick horizontal arrows, with the start of transcription at the blunt end. The solid blocks below the scale bar indicate fragments of known repetitive elements, with 1360 elements in the upper row and others in the lower row. Half-arrows over the 1360 elements indicate the orientation suggested by homology (>90% over at least 50 bp) to the beginning of transcripts from the 1360 element within the Su(Ste) repeat locus (3). A PCR was used to verify the presence of the 1360 fragments shown in all lines used in this study. The copy marked with an asterisk is present in the y w^67c23 and 39C-12 lines but not in the Sb Δ2-3/TM6 or y w^67c23; net; sbd; spa^pol lines. The presence of this element was verified for individual derived lines as needed; it is present in lines 2-M626, 4-M325, and 5-M340, those lines where the P element lies close to this position. The smallest fragment of 1360 is marked with an X; see the text for discussion. In parts B to D, genomic DNA is shown as a solid black line. The number after the triangle gives the insertion site or deletion end point on the 1.2-Mb map of chromosome 4 (15). (B) Rightward deletions generated from P element mobilization in line 39C-12. (C) Rightward and leftward deletions generated from P element mobilization in line 2-M59A.R. (D) Rightward deletions generated from P element mobilization in lines derived from 39C-12, screened for a recombination event rather than a change in phenotype. Shown below are photographs of representative eyes from the starting Drosophila line 39C-12 (variegating phenotype); lines 2-M59A.R and 4-M325, derived from 39C-12 by local deletion (full red expression); and lines 5-M275 and 5-M263, derived from 2-M59A.R by local deletion (variegating phenotype). Together, the results indicate the presence of a 45- to 50-kb euchromatic domain flanked by heterochromatic domains.

FIG. 4.

Local duplications can induce a change in eye phenotype. (A) A high-resolution map of a ∼200-kb region from the fourth chromosome; symbols are the same as those described in the legend to Fig. 3. (B) Duplications generated from P element mobilization in line 39C-12. (C) A duplication generated from P element mobilization in line 2-M59A.R. (D) Duplications generated from P element mobilization in spa^pol-marked lines derived from 39C-12, screened for a recombination event rather than a change in phenotype. In each case, the extent of the sequence that is duplicated (from the proximal side of 39C-12) is shown by the overlap in the horizontal lines indicating genomic DNA. Shown below are photographs of representative eyes from the starting Drosophila lines 39C-12 (variegating phenotype) and 2-M59A.R (red-eye phenotype) and lines carrying a duplication, 6-M180 (variegating phenotype) and 4-M979 (red-eye phenotype).

FIG. 5.

Silencing is correlated with proximity to transposon 1360 fragments. The distance between the P element insertion site and the nearest 1360 element is shown for the different Drosophila lines described in the legends to Fig. 3 and 4, including those generated by independent insertion events, local deletions, and local duplications. The bar for each line starts below 0 to allow visualization of the cases where the P element is within or very close to a 1360 element. A switch from a variegating phenotype (dotted bar) to a red-eye phenotype (solid bar) is observed when the distance is greater than ca. 10 kb. See the text for a discussion of the apparent exceptions to this observation, particularly line 6-M350, marked with asterisks.

FIG. 6.

A switch in eye phenotype reflects a switch in chromatin structure. (A) Changes in chromatin structure were assessed by digestion of nuclei isolated from third-instar larvae using the restriction enzyme XbaI, as previously described (9). The DNA was then purified, digested with SalI, and size separated by gel electrophoresis, and a Southern blot was probed with the pt DNA fragment isolated from plasmid pGH19 and labeled with [³²P]dCTP. Prt, parental SalI fragment (copies not cleaved by XbaI); D XbaI and P Xba, products of cleavage at the distal and proximal sites, respectively (see map above panel A). Fly lines used as the source of nuclei are indicated above each lane. The accessibility of the proximal XbaI site was calculated as the ratio of the signal intensity from the resulting gel band relative to the sum of all three bands obtained. The percent cleavage of the proximal XbaI site, normalized to that of line 39C-X (where the P element is in euchromatin) is shown below each lane. (B) Quantitative assessment of the eye phenotype was obtained by measuring eye pigmentation. Eye pigments were extracted from adult male fly heads using acidified ethanol, and the optical density was measured at 480 nm. The mean (bar) and standard error of four or more samples (five males each) are indicated. The switch in chromatin structure is associated with restoration to 80% or more of pigment levels (normalized to 39C-X).

FIG. 7.

Silencing as a consequence of local deletions or duplications is dependent on HP1. (A) A high-resolution map of a ∼200-kb region from the fourth chromosome; symbols are those described in the legend to Fig. 3. (B) The variegating phenotype displayed by flies carrying the P element within 10 kb of a 1360 element is suppressed by mutation in HP1; representative examples are shown, with the line crossed to the y w^67c23 line (left) compared to the line crossed to the y w^67c23, Su(var)2-5⁰² line (right). Shown are the following lines: 39C-12, a transposition; 5-M340, 5-M263, 5-M244, and 5-M275, deletions; and 6-M180, a duplication. The P element in line 5-M263 does not lie close to a 1360 element but may be affected by a nearby F element (see the text).

FIG. 8.

A model for the determination of chromatin structure. A map of the 102B region is shown, with the smallest and largest rightward deletions that result in a switch from the variegating 39C-12 transgene to a red-eye phenotype indicated as demarcating a euchromatic domain. Heterochromatic domains appear to be initiated from 1360 elements (shown as bars below the map), presumably targeted by the RNAi mechanism, localizing HP1 association with concomitant H3 modification for K9 methylation. Euchromatic domains may be initiated from regions having a high concentration of histone acetyltransferase activity. Both configurations can spread until stopped by competition.