[
International Worm Meeting,
2017]
Dosage compensation mechanisms specifically target gene regulatory complexes to the X chromosomes for transcriptional regulation. However, it remains unclear how X is specified, as the DNA sequence motifs shown to be important for binding of dosage compensation machineries are themselves not X-specific. Here, we analyzed binding of the C. elegans dosage compensation complex (DCC) through a series of experiments that include deletion and ectopic insertion of recruitment site sequences. Our data suggest that DCC recruitment is initiated by a small number of primary recruitment sites characterized by clusters of the 12-bp recruitment motif and overlap with high occupancy transcription factor target (HOT) regions, two features that fully explain X-specificity. SDC-2, the protein essential for hermaphrodite-specific recruitment of the DCC during early embryogenesis, is required to maintain open chromatin specifically at the primary recruitment sites whose DNA sequence encodes for high intrinsic nucleosome occupancy. Along the X, the primary recruitment sites are interspersed by secondary, weaker DCC recruitment sites. While insertion of a secondary recruitment element on an autosome failed to fully recruit the DCC, the same element was capable of recruitment at an ectopic locus on the X, suggesting that the function of the weaker recruitment sites are X-dependent. On the autosome, insertion of multiple recruitment elements in tandem or at a distance (>30 kb) increased DCC recruitment, demonstrating that recruitment sites cooperate over long distances. On the X, deletion of single recruitment sites resulted in reduced DCC binding across several megabases flanked by topologically associating domain (TAD) boundaries, suggesting that DCC recruitment and spreading occurs within defined X chromosomal domains. Our work illustrates a fundamental strategy for specifically targeting large chromosomal domains for co-regulation, which involves hierarchy and long-distance cooperativity between functional genomic elements.
Kramer, Maxwell, Albritton, Sarah, Kim, Jun, Street, Lena, Ragipani, Bhavana, Ercan, Sevinc, Zhang, Bo, Jimenez, David, Winterkorn, Lara
[
International Worm Meeting,
2021]
Condensins are molecular motors that compact DNA for chromosome segregation and gene regulation. In vitro experiments have begun to elucidate the mechanics of condensin function but how condensin loading and translocation along DNA controls eukaryotic chromosome structure in vivo remains poorly understood. To address this question, we took advantage of a specialized condensin, which organizes the 3D conformation of X chromosomes to mediate dosage compensation (DC) in C. elegans. Condensin DC is recruited and spreads from a small number of recruitment elements on the X chromosome (rex). We found that ectopic insertion of rex sites on an autosome leads to bidirectional spreading of the complex over hundreds of kilobases. On the X chromosome, strong rex sites contain multiple copies of a 12-bp sequence motif and act as TAD borders. Inserting a strong rex site and ectopically recruiting the complex on the X chromosome or an autosome creates a loop-anchored TAD. However, unlike the CTCF system, which controls TAD formation by cohesin, direction of the 12-bp motif does not control the specificity of loops. In an X;V fusion chromosome, condensin DC linearly spreads and increases 3D DNA contacts, but fails to form TADs in the absence of rex sites. Finally, we provide in vivo evidence for the loop extrusion hypothesis by targeting multiple dCas9-Suntag complexes to an X chromosome repeat region. Consistent with linear translocation along DNA, condensin DC accumulates at the block site. Together, our results support a model whereby strong rex sites act as insulation elements through recruitment and bidirectional spreading of condensin DC molecules and form loop-anchored TADs.