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

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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 clusters1,2,3) and along entire chromosomes (for example X-chromosome dosage compensation4,5,6). 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 half6. 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.

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Acknowledgements

We thank A. Coulson for cosmids; S. Uzawa for technical innovation with image analysis; P. Nix for help with xol-1 XO rescue experiments; M. Ralston for statistical analysis; and R. Auty, M. Botchan, S. Brenner, T. Cline, R. Losick, E. Ralston and A. Severson for comments on the manuscript. This work was supported by an NIH grant (B.J.M.), NIH training grants (P.M. and B.K.P.), and a Netherlands Organisation for Scientific Research (NWO) Talent fellowship (J.J.). B.J.M. is an investigator and J.J. an associate of the Howard Hughes Medical Institute.

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Competing interests

Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Correspondence to Barbara J. Meyer.

Supplementary information

  1. Supplementary Notes

    This file contains Supplementary Methods, Supplementary Discussion and additional references (DOC 69 kb)

  2. Supplementary Figure 1

    This figure shows that rex arrays recapitulate DCC recruitment properties of X. DCC binding to rex-1 arrays requires the same dosage compensation proteins as binding to X chromosomes. Moreover, rex-1 sites integrated into autosomes recruit the DCC. Shown are confocal images of wild-type or dosage-compensation-defective intestinal cell nuclei carry either rex arrays or rex-1 sites integrated into autosomes. Cells were co-stained with DAPI, FISH probes, and antibodies to 3 different dosage compensation proteins. (PDF 2006 kb)

  3. Supplementary Figure 2

    This figure shows the distribution of modeled DCC recruitment sites in 30 kb segments tiled along X. The plot is juxtaposed on the recruitment map showing previously determined X regions of strong, weak, or undetectable DCC binding. (PDF 649 kb)

  4. Supplementary Figure 3

    This figure further illustrates that motifs A and B are critical for the recruitment of all DCC components to rex-1. Shown are confocal images of intestinal cell nuclei carrying either wild-type (rex-1•33) or mutant (rex-1•33mA1 or rex-1•33mB2) arrays that were co-stained with DAPI and antibodies against SDC-2 and SDC-3, the proteins critical for targeting all DCC proteins to X. (PDF 1997 kb)

  5. Supplementary Figure 4

    This figure illustrates that multiple A and B motifs collaborate to recruit the DCC. Shown are confocal images of intestinal cell nuclei carrying wild-type and mutant rex-1 – rex-4 arrays that were co-stained with DAPI, FISH probes to rex arrays, and antibodies to DPY-27. (PDF 8290 kb)

  6. Supplementary Figure 5

    This figure illustrates that rex-1 arrays compete with the X chromosome for DCC recruitment in young embryos. Shown are partial confocal projections of young embryos carrying rex-1 arrays stained with DAPI (blue), DPY-27 antibody (red), and a FISH probe (green) to identify the array. (PDF 5477 kb)

  7. Supplementary Table 1

    The table provides the DNA sequence and position (strand) of wild-type A and B motifs in rex sites and the mutant motifs used to analyze the role of motifs A and B. (DOC 60 kb)

  8. Supplementary Table 2

    Chi square analysis in this table shows significant correspondence between predictive model for DCC recruitment sites and previous DCC recruitment studies. (DOC 45 kb)

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Further reading

Figure 1: DCC recruitment elements on X ( rex sites) contain clusters of cis -acting regulatory motifs.
Figure 2: Mutational analysis of rex-1 establishes motifs A and B as cis -acting regulatory elements critical for DCC recruitment.

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