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.
Highlights
A mechanism for selecting and marking an entire chromosome for coordinate regulation is central to the process of dosage compensation
We found that fluorescence in situ hybridization (FISH) signals for DBF1 and DBF7 overlapped with MSL1 staining in the mof1 background and in females carrying one copy of the NOPU insert (Figure 3)
Recruitment of Partial Complexes To investigate the ability of the dosage compensation complex (DCC) binding fragment’’ (DBF) to recruit partial and non-functional DCC complexes, we looked for anti-MSL1 staining in the msl mutant backgrounds
Summary
A mechanism for selecting and marking an entire chromosome for coordinate regulation is central to the process of dosage compensation. In the worm Caenorhabditis elegans, gene expression from both X chromosomes in hermaphrodites is reduced to about half when compared to expression of the single X chromosomes in males (reviewed in [4]) This is achieved by the binding of a multi-subunit dosage compensation complex (DCC) along the entire length of both X chromosomes. In this case, the selection of the chromosomes is not due to a single locus, but is thought to involve multiple recognition sites for the DCC [5,6]. The selection of the X in flies uses a system with similarities to both mechanisms mentioned above, relying in part on the expression and accumulation of two non-coding RNAs from the X and in part on the presence of specific recruitment sites distinguishing the X from other chromosomes [12,13]
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