Clustered DNA motifs mark X chromosomes for repression by a dosage compensation complex

Abstract

Gene expression in metazoans is regulated not only at the level of individual genes but also in a coordinated manner across large chromosomal domains (for example centromeres, telomeres and imprinted gene clusters) and along entire chromosomes (for example X-chromosome dosage compensation). The primary DNA sequence usually specifies the regulation of individual genes, but the nature of cis-acting information that controls genes over large regions has been elusive: higher-order DNA structure, specific histone modifications, subnuclear compartmentalization and primary DNA sequence are possibilities. One paradigm of chromosome-wide gene regulation is Caenorhabditis elegans dosage compensation in which a large dosage compensation complex (DCC) is targeted to both X chromosomes of hermaphrodites to repress transcript levels by half. This essential process equalizes X-linked gene expression between the sexes (XO males and XX hermaphrodites). Here we report the discovery and dissection of cis-acting sites that mark nematode X chromosomes as targets for gene repression by the DCC. These rex (recruitment element on X) sites are widely dispersed along X and reside in promoters, exons and intergenic regions. rex sites share at least two distinct motifs that act in combination to recruit the DCC. Mutating these motifs severely reduces or abolishes DCC binding in vivo, demonstrating the importance of primary DNA sequence in chromosome-wide regulation. Unexpectedly, the motifs are not enriched on X, but altering motif numbers within rex sites demonstrates that motif co-occurrence in unusually high densities is essential for optimal DCC recruitment. Thus, X-specific repression is established through sequences not specific to X. The distribution of common motifs provides the foundation for repression along an entire chromosome.

Figure 1. DCC recruitment elements on X (rex sites) contain clusters of cis-acting regulatory motifs

a, DCC recruitment map of the C. elegans X chromosome. The positions of dissected rex sites (yellow) are indicated relative to previously determined X regions that strongly recruit (dark green), weakly recruit (light green) or fail to recruit (red) the DCC when detached from X. b, Positions of cis-regulatory motifs A (red circles) and B (blue squares) and their corresponding Z-scores in each full-strength rex site (241 bp for rex-1, 147 bp for rex-2, 115 bp for rex-3, and 411 bp for rex-4). Z-scores for every nucleotide position were calculated as (raw score minus mean score)/(standard deviation of all scores). Raw scores were generated by comparing every window of seven or eight nucleotides to position weight matrices (PWMs) for motif A and motif B. Within a rex site, individual A and B motifs were named according to their relative scores against the PWMs, with A₁ and B₁ being the best scoring instances per site (see Supplementary Table 1). c, Models describing cis-acting regulatory motifs A and B based on PWMs. d, Plot showing correspondence between a predictive model for DCC recruitment (see the text) and previous X-chromosome-wide recruitment data. The percentage of predicted positive 30-kb segments is significantly greater in strongly (A, D and G; dark green) and weakly (B and F; light green) recruiting X regions than in non-recruiting regions (C and E; red) (P<0.004; see Supplementary Table 2). The percentage of positive windows predicted for the entire X is 30% (grey line).

Figure 2. Mutational analysis of rex-1 establishes motifs A and B as cis-acting regulatory elements critical for DCC recruitment

Confocal images of intestinal cell nuclei (4,6-diamidino-2-phenylindole (DAPI) stain, blue) carrying wild-type (WT; a, d, f, g) or mutant (b, c, e, h) rex arrays (fluorescence in situ hybridization, green) co-stained with DPY-27 antibodies (red). DPY-27 binding to X requires all other known DCC components. The recruitment strength category (see the text for description) of each array is indicated by a number (0-3) at the right. a, Arrays containing a cloned 33-bp rex-1 fragment (1A,1B motifs) recruit the DCC robustly. b, c, Mutating either motif A (b) or motif B (c)in rex-1·33 abolishes DCC recruitment. (See also Supplementary Fig. 1.) d, Extending rex-1 to 60 bp adds a second motif A and markedly increases recruitment strength to the degree that arrays begin to outcompete the X for DCC binding, as demonstrated by the weaker X chromosome staining relative to array staining. e, When both A motifs in rex-1·60 are mutated, DCC recruitment to the arrays is severely reduced, and X chromosome staining is restored. f, g, rex-1·241 (f) and rex-1·148 (g) each contain four A motifs as well as two or one B motifs, respectively; both exhibit maximum recruitment strength, completely outcompeting the X for DCC staining. h, Loss of the only B motif in rex-1·148 (rex-1·148 mB₂) reduces recruitment but does not eliminate it. i, Map of rex-1 fragments used in this paper. Red circles represent A motifs; blue squares represent B motifs. Stronger recruitment is indicated by darker green. Scale bar, 2 μm.