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.

Chromosome-wide gene-specific targeting of theDrosophiladosage compensation complex

“…Binding sites would, in this scenario, be determined by the concentration of many recognition elements in a particular DNA sequence. Our observations are compatible with the "affinities" model for targeting of the DCC (Demakova et al 2003;Fagegaltier and Baker 2004;Oh et al 2004b;Dahlsveen et al 2006). However, affinities alone fail to explain the recent observation that the MSL2 protein exhibits exceptionally stable binding to the male X chromosome, assayed by FRAP and FLIP (Straub et al 2005c), as sites of low affinity would be expected to demonstrate highly dynamic binding to the MSL complex.…”

“…Their failure suggests that DCC binding may be directed by a more complex combination of degenerate sequence motifs. A recent analysis of high-affinity sequences defined several paired motifs found enriched in MSL-binding sequences (Dahlsveen et al 2006), but these were also insufficient to predict further DCC-binding sites. To examine more complex word combinations, we used PLS regression, with which we identified several sequences that to some extent explain the observed DCC binding.…”

“…The identified motifs notably contain GA and CA dinucleotides. Sequences containing the GAGA motif appear to have an important role in attracting the DCC to the roX2 high-affinity site (Park et al 2003), and have also been found in additional high-affinity sites (Dahlsveen et al 2006). Furthermore, CA and GA dinucleotide repeats are enriched on the X chromosomes of Drosophila species exhibiting dosage compensation (Huijser et al 1987;Pardue et al 1987;Lowenhaupt et al 1989).…”

“…Accordingly, the MSL proteins were proposed to recognize the X via the ∼35 high-affinity "entry sites," from where they could spread to bind the remaining target sites by an unknown mechanism (Lyman et al 1997;Kelley et al 1999). Recent studies of flies with altered DCC concentrations, X to autosome translocations, and analysis of new "entry sites" suggest that the entry sites are simply high-affinity sites in a continuum of affinity sites dispersed along the X chromosome (Demakova et al 2003;Fagegaltier and Baker 2004;Oh et al 2004b;Dahlsveen et al 2006). The most recent model therefore proposes that X-chromosome binding is governed largely or even solely by DNA elements of a degenerate nature (for review, see Straub et al 2005a).…”

Zinc Finger Protein Zn72D Promotes Productive Splicing of the maleless Transcript

“…When MSL3, MLE, MOF, and at least one of the roX RNAs are also expressed, the complete complex forms and localizes to additional sites on the X chromosome. This was originally proposed to be the result of spreading of the complex from the ϳ35 sites to additional sites along the X chromosome (28,37,40) and was more recently proposed to be the result of the complex binding first to ϳ35 high-affinity sites and then to additional lower-affinity sites (10,12,15,20,44). MOF, a histone acetyltransferase, hyperacetylates histone H4 at lysine 16, resulting in the twofold increase in expression of X-linked genes (1,49).…”