(A) ChIP-seq profiles of Cbr SDC-2 and Cbr DPY-27 binding to X chromosomes. ChIP-seq experiments were performed using an anti-FLAG antibody to immunoprecipitate SDC-2 from a strain encoding FLAG-tagged SDC-2, and the same anti-FLAG antibody was used in ChIP-seq experiments to immunoprecipitate DPY-27 from a strain encoding FLAG-tagged DPY-27. The control IgG ChIP-seq profile on X is also shown. Peaks that correspond to recruitment elements on X (rex sites), as determined by the assay in (B), are indicated in orange above the ChIP-seq profiles. RPKM is the abbreviation for reads per kilobase per million reads mapped. (B) Assay performed in vivo to determine whether DNAs from ChIP-seq peaks recruit the DCC when detached from X. XX embryos carrying extrachromosomal arrays with multiple copies of DNA from a ChIP-seq peak in (A) were stained with a DNA FISH probe to the array (red) and DPY-27 antibody (green). If the DNA from a peak failed to recruit the DCC, DPY-27 staining would identify X chromosomes but not the array. If DNA from a peak encoded a recruitment site (rex site), DPY-27 staining would co-localize with the array and the X chromosome. In the merged image, the array would appear yellow and the X chromosome would appear green. Often, an array carries enough copies of a rex site that it titrates most of the DCC from X, and only the array itself shows evidence of DCC binding, appearing yellow in the merged image. In that case, the X chromosome is not detectable by DPY-27 antibody staining. XX strains carrying rex arrays that titrate the DCC from X cannot be propagated due to the defect in dosage compensation caused by DCC titration. (C) C. briggsae rex sites recruit the C. briggsae DCC but not the C. elegans DCC. Shown is a C. briggsae or C. elegans XX gut nucleus carrying an extrachromosomal array containing multiple copies of the C. briggsae DCC recruitment site rex-8. Nuclei were stained with appropriate species-specific C. briggsae or C. elegans antibodies to the DCC subunit DPY-27 (green), DAPI (gray), and an array FISH probe (red). In C. briggsae, DPY-27 bound to arrays in about 40% of the 52 scored nuclei carrying a Cbr rex-8 array, and the DCC was titrated from X. In C. elegans, DPY-27 bound to arrays in 0% of the 27 scored nuclei carrying a Cbr rex-8 array, and DPY-27 binding to the C. elegans X was evident. Scale bar, 5 μm. (D) C. elegans rex sites do not recruit the C. briggsae DCC. Shown is a C. elegans or C. briggsae XX gut nucleus carrying an extrachromosomal array containing multiple copies of the C. elegans recruitment site rex-33 with three MEX motifs (ln[P] scores of −13.13,–15.33, –15.35). Nuclei were stained with C. elegans or C. briggsae antibodies to DCC subunit DPY-27 (green), DAPI (gray), and an array FISH probe (red). In C. elegans, DPY-27 bound to arrays in 100% of the 63 scored nuclei carrying a Cel rex-33 array, and the DCC was titrated from X. In C. briggsae, DPY-27 bound to arrays in 0% of the 53 scored nuclei carrying a Cel rex-33 array, but did bind to Cbr X chromosomes in the same nuclei (Table 2). Scale bar, 5 μm. (E) Quantification of exemplary Cbr recruitment assays in vivo using extrachromosomal arrays containing multiple copies of DNA from Cbr DCC ChIP-seq peaks that define rex sites. Data are shown for DPY-27 recruitment to DNA from four strong Cbr ChIP-seq peaks and a control region of DNA lacking a DCC peak (flat one containing the gene mom-1). Shown are the locations of the sites on X, the total number of embryonic nuclei scored for DPY-27 recruitment to the array, and the percent of nuclei recruiting the DCC. Arrays carrying rex sites recruit the DCC but arrays carrying the control flat region fail to recruit the DCC. Results of DCC recruitment assays in vivo for all rex sites are presented in Table 2.