Different chromatin interfaces of the Drosophila dosage compensation complex revealed by high-shear ChIP-seq

Abstract

Transcriptional enhancement of X-linked genes to compensate for the sex chromosome monosomy in Drosophila males is brought about by a ribonucleoprotein assembly called Male-Specific-Lethal or Dosage Compensation Complex (MSL-DCC). This machinery is formed in male flies and specifically associates with active genes on the X chromosome. After assembly at dedicated high-affinity ''entry'' sites (HAS) on the X chromosome, the complex distributes to the nearby active chromatin. High-resolution, genome-wide mapping of the MSL-DCC subunits by chromatin immunoprecipitation (ChIP) on oligonucleotide tiling arrays suggests a rather homogenous spreading of the intact complex onto transcribed chromatin. Coupling ChIP to deep sequencing (ChIP-seq) promises to map the chromosomal interactions of the DCC with improved resolution. We present ChIP-seq binding profiles for all complex subunits, including the first description of the RNA helicase MLE binding pattern. Exploiting the preferential representation of direct chromatin contacts upon highenergy shearing, we report a surprising functional and topological separation of MSL protein contacts at three classes of chromosomal binding sites. Furthermore, precise determination of DNA fragment lengths by paired-end ChIP-seq allows decrypting of the local complex architecture. Primary contacts of MSL-2 and MLE define HAS for the DCC. In contrast, association of the DCC with actively transcribed gene bodies is mediated by MSL-3 binding to nucleosomes. We identify robust MSL-1/MOF binding at a fraction of active promoters genome-wide. Correlation analyses suggest that this association reflects a function outside dosage compensation. Our comprehensive analysis provides a new level of information on different interaction modes of a multiprotein complex at distinct regions within the genome.

Dosage Compensation inDrosophila

“…Interestingly, the MSL-dependent H4K16ac mark is more widely distributed along the X chromosome than documented MSL binding, suggesting the transient interaction of the complex with a larger fraction of the X than that observed to have stable binding (Fig. 7) Conrad et al 2012;Straub et al 2013). …”

“…Experiments to date support a model in which initial recruitment of the MSL complex to CESs generates high local concentrations that drive spreading to nearby sites of lower affinity. Recent evidence, based on high-shear ChIP-seq, suggests that MSL2 and MLE contact the HASs directly and provide a platform for the indirect association of the other subunits and roX RNA at these sites (Straub et al 2013). Movement of the complex to active genes is facilitated by the activity of MOF and MLE Morra et al 2008) and is stabilized by the binding of MSL3 to H3K36me3 ).…”

UNR facilitates the interaction of MLE with the lncRNA roX2 during Drosophila dosage compensation

“…ChIP experiments show prominent, specific interaction of MLE at HAS in S2 cells 6 . Our current data now suggest that the general RBP UNR facilitates this interaction.…”

“…Input chromatin serving as reference sample was treated accordingly. Input material (10%) and eluted DNA were subjected to RT-qPCR with SYBR green (Applied Biosystems) using HAS or promoter-specific oligonucleotides 3,6 (detailed in Supplementary Table 1). Values were normalized to internal controls and to input.…”

“…The RNA helicase MLE colocalizes with MSL2 at HAS and facilitates the incorporation of roX into the complex. The RNA is important for the distribution of the MSL-DCC along the X-chromosome, where it activates target genes 2,[5][6][7] .…”