Targeting determinants of dosage compensation in Drosophila

Abstract

The dosage compensation complex (DCC) in Drosophila melanogaster is responsible for up-regulating transcription from the single male X chromosome to equal the transcription from the two X chromosomes in females. Visualization of the DCC, a large ribonucleoprotein complex, on male larval polytene chromosomes reveals that the complex binds selectively to many interbands on the X chromosome. The targeting of the DCC is thought to be in part determined by DNA sequences that are enriched on the X. So far, lack of knowledge about DCC binding sites has prevented the identification of sequence determinants. Only three binding sites have been identified to date, but analysis of their DNA sequence did not allow the prediction of further binding sites. We have used chromatin immunoprecipitation to identify a number of new DCC binding fragments and characterized them in vivo by visualizing DCC binding to autosomal insertions of these fragments, and we have demonstrated that they possess a wide range of potential to recruit the DCC. By varying the in vivo concentration of the DCC, we provide evidence that this range of recruitment potential is due to differences in affinity of the complex to these sites. We were also able to establish that DCC binding to ectopic high-affinity sites can allow nearby low-affinity sites to recruit the complex. Using the sequences of the newly identified and previously characterized binding fragments, we have uncovered a number of short sequence motifs, which in combination may contribute to DCC recruitment. Our findings suggest that the DCC is recruited to the X via a number of binding sites of decreasing affinities, and that the presence of high- and moderate-affinity sites on the X may ensure that lower-affinity sites are occupied in a context-dependent manner. Our bioinformatics analysis suggests that DCC binding sites may be composed of variable combinations of degenerate motifs.

Figure 1. The Drosophila X Chromosome Contains a Hierarchy of DCC Binding Sites

Polytene chromosomes from Drosophila larvae showing the DNA stained in blue and anti-MSL1 staining in red. (A) The DCC binds in a defined pattern to the X chromosome in wild-type males (top panel). Mutations in mof, msl-3, and mle lead to partial or non-functioning complexes (cartoons) binding to a subset of sites. Anti-MSL-1 staining of X chromosomes from females expressing MSL2 and homozygous for the mof¹, msl-3¹, and mle¹ mutations are shown. (B) The same hierarchy of sites can be seen in females expressing different concentrations of MSL2. The NOPU and SXB1–2 constructs give rise to decreasing levels of MSL2 expression [27], and this leads to the recruitment of the DCC to decreasing numbers of sites on both X chromosomes. These sites are the same as those found in the mutant backgrounds described in (A) [28]. Females homozygous for the NOPU construct (top panel) have a DCC binding pattern quite similar to wild-type males, lacking only a few sites.

Figure 2. Identification of New DCC Binding Fragments Using Chromatin IP

(A) Subsection of genomic P1 phage clone filter, showing 120 clones out of a total of 9,216, hybridized with MSL1 ChIP probe (upper panel), then stripped and re-hybridized to the mock IP control (lower panel). Two examples of clones showing MSL1 enrichment chosen for further analysis are indicated by red circles. Note that clones are always spotted in duplicate on the membrane. (B) Southern blots of digested P1 clone DNA hybridized to MSL1 ChIP (left panel) or mock ChIP (right panel). Red boxes highlight the bands enriched in the MSL1 ChIP chosen for cloning, known as DBF1–DBF14. Size in kilobases is indicated to the left of the figure. Lane order and restriction digests are as follows: Lane 1, DBF1 (BglII); 2, DBF3 (XhoI); 3, DBF5 (XhoI); 4, DBF6 (ApaLI); 5, DBF7 (BamHI); 6, DBF9 (XhoI); 7, DBF10 (EcoRI); 8, DBF11 (ApaLI); 9, DBF12 (EcoRI); 10, DBF13 (EcoRI); 11, DBF14 (EcoRI); and 12, autosomal DNA control P1 clone (EcoRI). Note that clones DBF2, DBF4, and DBF8 (false positives) have been omitted from this figure. P1 phage clone identifiers are listed in the Materials and Methods section.

Figure 3. The Endogenous Loci of the DCC Binding Fragments Overlap with MSL1 In Vivo

Immuno-FISH experiments using the identified DBFs as probes (FISH signals in green) and staining with anti-MSL1 antibodies to visualize DCC binding (in red). Each panel consists of images of the FISH signal (top), the anti-MSL1 signal (middle), and the merge (bottom). The FISH signals from DBF9, DBF6, DBF5, and DBF12 overlap with the anti-MSL1 signals in the msl-3¹ mutant background (w/w; msl-3¹, [w+ Hsp83 MSL2]). DBF11 overlaps partially with MSL1 staining in this background. DBF1 and DBF7 overlap with weak anti-MSL signals in the mof¹ mutant background (mof¹/mof¹; +; [w+ Hsp83 MSL2]/+). FISH signals from the endogenous loci of DBF3, DBF10, and DBF13 do not overlap with MSL1 staining in either the msl-3¹or mof¹ background. However, DBF10 does overlap with MSL1 staining in females carrying two copies of the NOPU insert (2 × NOPU, Figure1), whereas DBF3 and DBF13 overlap with MSL staining in wild-type males (WT).

Figure 4. Recruitment of the DCC to DBF Insertions in Wild-Type Males

Polytene chromosomes with FISH signals in green and anti-MSL1 signals in red showing examples of DBF insertions recruiting the DCC in wild-type males. The cytological position of DBF inserts was estimated from FISH experiments (upper image in each panel). Recruitment of the DCC was demonstrated with immunoflourescence using anti-MSL1 antibodies (lower image in each panel). Examples from all DBF inserts that showed recruitment in wild-type males are shown. A summary of the complete analysis can be found in Table 2.

Figure 5. Several DCC Binding Fragments Can Recruit the Complex When MSL2 Levels Are Limiting

Examples of DCC recruitment to DBF inserts in females carrying one copy of the SXB1–2 or NOPU MSL2 expression constructs (see Figure 1). Anti-MSL1 staining is shown in red and DNA staining in blue. Arrows indicate the position of the inserts and X indicates the X chromosome. Inserts DBF12-99EF (A), DBF12-85A (B), DBF9-96C (C), DBF6-100A (E), and DBF5-95C (G) recruit the DCC at lower levels of MSL2 in the SXB1–2 background, whereas inserts DBF9B-98F (D), DBF6-63C (F), DBF5-91F (H), DBF7-88E (I), DBF7-93B (J), DBF1-26A (K), and DBF9A-96D (L) recruit the complex in the NOPU background. For a summary of the complete results, see Table 2.

Figure 6. Increasing the Levels of the DCC Increases Recruitment to Low-Affinity Binding Fragments

Polytene chromosomes showing FISH signals (top panels, green) and anti-MSL1 staining (red) in the genetic backgrounds indicated. Arrows indicate the position of the inserts and arrowheads indicate autosomal sites of DCC binding in the MSL1 and MSL2 over-expression background. Recruitment of the DCC to the DBF11-85D insert is enhanced when the levels of DCC are increased by over-expression of MSL1 and MSL2 compared to wild-type males. The DBF3-96B insert does not recruit the DCC in wild-type males, but recruitment can be seen when MSL1 and MSL2 are over-expressed.

Figure 7. High-Affinity DCC Binding Fragments Recruit Partial and Inactive Complexes in msl-3¹ and mof¹ Mutants

Examples of DCC recruitment to DBF inserts in females expressing MSL2 and carrying mutations in msl-3¹ (w/w; msl-3¹, [w+ Hsp83 MSL2]/+) or mof¹ (mof¹/mof¹; +; [w+ Hsp83 MSL2]/+). Anti-MSL1 staining is shown in red and DNA staining in blue. Arrows indicate the position of the inserts and X indicates the X chromosome. Inserts DBF12-B-99EF (A), DBF12-B-85A (B), DBF9-B-96C (C), DBF9B-98F (D), DBF6-100A (E), and DBF5-95C (G) recruit partial complexes in the msl-3¹ mutant. Inserts DBF6-63C (F), DBF5-91F (H), and DBF7-88E (I) recruit inactive complexes in the mof¹ mutant background.

Figure 8. Minimal Spreading from High-Affinity Sites to Low-Affinity Autosomal Sites

Examples of two inserts, DBF9-96C (A–C) and DBF5-91F (D–F), that cause occasional spreading of the DCC to one additional band next to the insertion site in wild-type males (A and D). When complex levels are increased by over-expression of MSL1 and MSL2, the secondary DCC binding sites appear as part of the set of autosomal binding sites for the complex (B and E) and are present even in the absence of the DBF insert (C and F). Anti-MSL1 staining is shown in red and DNA staining in blue. Small inserts in (A) and (E) show the FISH signals (green) corresponding to the P element insertion. Arrows indicate the position of the inserts and arrowheads indicate autosomal sites of DCC binding.

Figure 9. DNA Sequence Logos of Representative Motifs Putatively Involved in DCC Binding

Logos (generated using the WebLogo software) represent degenerate motifs based on related pairs of elements from Table S1. The logos were generated by aligning all the words of related elements from specific pairs: (A) pairs 5, 6, 7, 8, 9, and 10; (B) pairs 11, 12, 13, and 14; (C) pairs 17, 18, 19, and 20; (D) 21, 22, and 23; (E) pairs 15 and 16; and (F) pairs 1, 2, and 3. Pairs 4 and 24 were not included. Motifs Ia–Ie all contain GAGA-related sequences. Motifs IIa and IIb as well as IIIa–IIIc are also related to each other.