Cooperation between a hierarchical set of recruitment sites targets the X chromosome for dosage compensation
- New York University, United States
Figures
Figure 1 with 2 supplements
DCC recruitment sites are defined using high resolution ChIP-seq analysis.
(A) The C. elegans dosage compensation complex (DCC) is composed of a modified condensin complex (condensin DC) which is distinguished from condensin I by the SMC-4 variant, DPY-27. The …
Figure 1—figure supplement 1
ChIP-seq data suggests that some previously defined rex-sites fail to recruit the DCC in the context of the X chromosome.
(A) SDC-2, SDC-3, DPY-30, DPY-27, and H3K4me3 ChIP-seq enrichment data is plotted for a 1 Mb window. Three previously identified rex-sites (rex-8 (rank 1), rex-28 (rank 35), and rex-29 (rank 50)) …
Figure 1—figure supplement 2
Peaks excluded by H3K4me3 overlap do not resemble strong recruitment sites.
(A) Boxplots indicating SDC-2 ChIP-seq enrichment score across each of the defined classes: strong recruitment sites (n = 17), intermediate recruitment sites (n = 16), weak recruitment sites (n = 31)…
Figure 2 with 1 supplement
Recruitment sites on the X chromosome are hierarchical
(A) Heatmaps showing ChIP-seq enrichment for the DCC recruiter proteins, SDC-2, SDC-3, and DPY-30, the condensin DC subunit DPY-27, and the histone modification H3K4me3. Heatmaps are plotted across …
Figure 2—figure supplement 1
Specific reduction in SDC-2 enrichment at weak recruitment sites in an sdc-3 null mutant.
(A) Scatterplot comparing SDC-2 ChIP-seq enrichment between wild-type (N2) and the sdc-3 null mutant. Briefly, the X chromosome was divided into 200 bp windows with a step size of 100 bp. Average …
Figure 3
SDC-2 is required for open chromatin at strong DCC recruitment sites that encode for high intrinsic nucleosome occupancy.
(A) Heatmaps showing H3 ChIP-seq enrichment across a 1 kb window centered on the 64 recruitment sites. Wild-type data (left) indicates open chromatin at all recruitment sites. Data from an sdc-2 …
Figure 4
The 12 bp recruitment motif is enriched on the X. Bound motifs are characterized by DNA-encoded nucleosome occupancy.
(A) The 12 bp recruitment motif identified using high-resolution SDC-2 ChIP-seq data. (B) Motif distribution is plotted for chromosomes I through V (shades of gray) and chromosome X (purple). Motif …
Figure 5 with 2 supplements
Clustered motifs at HOT sites distinguish the X chromosome from the autosomes.
(A) A motif cluster is defined as a strong motif (score ≥7) flanked within 200 bp by a second motif. Motif locations plotted for each of the 22 motif clusters: 17 are X- linked (11 at strong …
Figure 5—figure supplement 1
ChIP-seq data reveals that neither perfect affinity motifs nor motif clustering by itself is sufficient to explain X-specific recruitment of the DCC.
SDC-2, SDC-3, DPY-30, DPY-27, and H3K4me3 ChIP-seq enrichment is plotted across a 3 kb window centered on a representative recruitment site from each strength class, and for two autosomal loci (the …
Figure 5—figure supplement 2
Extended analysis of overlap between recruitment sites with each tested annotation.
Black box indicates presence and white box indicates absence of a given overlap. Significance of overlap is calculated using permutation test (overlapPermTest function of the regioneR package (Frucia…
Figure 6
Cooperation between recruitment sites is necessary for robust DCC recruitment.
(A) SDC-3 (left, blue) and DPY-27 (right, brown) ChIP-qPCR data is plotted for the indicated ectopic insertions. ChIP-qPCR is plotted as percent of endogenous recruitment, that is, recruitment at …
Figure 7 with 5 supplements
Multiple DCC recruitment sites establish DCC binding within defined X chromosomal domains.
(A) DPY-27 ChIP-seq data was used to calculate percent deviation from wild-type in three different recruitment site deletion strains. Briefly, the log2 ratio between wild-type and deletion strain …
Figure 7—figure supplement 1
Schematic of recruitment site deletions and ChIP-seq data demonstrating DCC recruitment in the deletion strains.
(A) Cartoon indicating location and size of the deleted recruitment sites in Figure 7. Recruitment sites are shown as light blue boxes. Strong motifs are shown in pink, weak motifs are shown in …
Figure 7—figure supplement 2
Sanger sequencing results from the wild-type and recruitment site deletion strains.
Sequence that is deleted is capitalized and in italics. Strong motifs are given in pink; weak motifs are given in blue. The rex-40 deletion bears a small insert of dpy-10 sequence, indicated in red.
Figure 7—figure supplement 3
Sliding-window analysis of DCC binding change across the X chromosome using different window sizes.
As in Figure 7, DPY-27 ChIP-seq data was used to calculate percent deviation from wild-type. Analysis was repeated for 2 Mb, 1 Mb, and 500 kb windows.
Figure 7—figure supplement 4
Control for sliding-window analysis of DCC binding change across the X chromosome.
As in Figure 7, DPY-27 ChIP-seq data was used to calculate percent deviation from wild-type. The set-4(n4600) mutation affects DCC function but not localization. TAD boundaries are shown in green. …
Figure 7—figure supplement 5
12-bp motif directionality and recruitment site interactions. .
(A) Motif directionality for all strong motifs (score ≥7) contained within annotated recruitment sites across the length of the X chromosome. The numbers in parenthesis indicate the rank of each …
Figure 8
A model for X-specific DCC targeting involves SDC-2 recognizing a small number of initial recruitment sites, followed by cooperative recruitment and DCC spreading to distinct chromosomal domains.
(A) Initial X-recognition occurs at a subset of strong recruitment sites marked by motif clustering, overlap with HOT sites, and intrinsic nucleosome occupancy encoded in DNA. We hypothesize that …
Additional files
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Source code 1
Perl and R scripts for data analysis.
Perl scripts making up the ChIP-seq analysis pipeline are provided. R script and command lines for sliding window analysis of DCC binding changes across the X chromosome are provided.
- https://doi.org/10.7554/eLife.23645.021
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Supplementary file 1
(A) Strain information.
The names and the genotypes of each strain are given in the sheet ‘strains’. Sequences for the sgRNAs and the primers used to generate the homology-mediated repair oligos are listed in the sheet ‘primers.’ Deletion and insertion coordinates as well as the inserted sequence are also indicated. (B) Data summary. The GEO accession numbers, total and mapped read counts, and antibody information are provided for the ChIP, RNA, and DNA-seq data. (C) qChIP primers. Primer sequences used for the qChIP experiments are provided. (D) Recruitment site information. The coordinates of the 64 DCC recruitment sites, the motifs contained within, and the recruitment strength category of each site are provided. (E) Motifs across the genome. Motif sequence and coordinates for all 12 bp motifs identified across the genome are provided. Supplementary file 1F: Annotation information. The annotation sources that are used in this manuscript.
- https://doi.org/10.7554/eLife.23645.022
