Targeting of the dosage-compensated male X-chromosome during early Drosophila development

The essential process of dosage compensation, which corrects for the imbalance in X-linked gene expression between XX females and XY males, represents a key model for how genes are targeted for coordinated regulation. However, the mechanism by which dosage compensation complexes identify the X-chromosome during early development remained unknown because of the difficulty of sexing embryos prior to zygotic transcription. We used meiotic drive to sex Drosophila embryos prior to zygotic transcription and ChIP-seq to measure dynamics of dosage compensation factor targeting. The Drosophila Male-Specific Lethal dosage compensation complex (MSLc) requires the ubiquitous zinc-finger protein Chromatin-Linked Adaptor for MSL Proteins (CLAMP) to identify the X-chromosome. We observe a multi-stage process in which MSLc first identifies CLAMP binding sites throughout the genome followed by concentration at the strongest X-linked MSLc sites. We provide insight into the dynamic mechanism by which a large transcription complex identifies its binding sites during early development. We performed 0- to 4-h timed lays and collected and fixed embryos according to Blythe and Wieschaus (2015). We then hand-sorted embryos using a Zeiss Discovery.V8 microscope under GFP excitation using an X-CITE 120Q stereo light source. We pooled 200 (NC 11–14) to 400 (NC < 11) embryos and performed ChIP as in Blythe and Wieschaus (2015) using 3 μL of rabbit anti-CLAMP antibody per sample.