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[
International Worm Meeting,
2015]
Multi-subunit condensin complexes are essential for chromosome condensation during both mitosis and meiosis and have also been implicated as having important roles in transcriptional regulation during interphase. Metazoans contain two condensin complexes, I and II, which specifically localize to different chromosome regions where they perform different functions. Caenorhabditis elegans contains a third condensin complex, Condensin DC, whose localization is uniquely restricted to the hermaphrodite X chromosome where it acts as part of the Dosage Compensation Complex (DCC) to repress X-transcription. The regulatory mechanisms by which Condensin DC is targeted specifically to the X chromosome are not yet fully understood.As part of the DCC, Condensin DC interacts with at least four other non-condensin proteins, including two zinc-finger-containing proteins, SDC-2 and SDC-3, which act to recruit Condensin DC to approximately 100 recruitment sites across the X-chromosome. Evidence suggests that this initial targeting is sequence-dependent. Sites of initial recruitment, termed recruitment elements on X (rex), are enriched for a 10bp recruitment motif. Our analyses indicate that this motif is four times enriched on the X-chromosome as compared to autosomes and is often clustered at rex-sites. However, the motif is not unique to the X-chromosome; both the X chromosome and the autosomes contain many perfect matches that are not bound by the DCC. Further, we show that insertion of a rex-site in single-copy onto an autosome fails to detectably recruit DCC. Increasing the number of inserted rex-site copies overcomes the inability recruit on autosomes. We conclude that motif sequence, while important for DCC recruitment, is not sufficient to recruit the complex on its own. We hypothesize that chromosomal context of the X chromosome facilitates the specificity of DCC recruitment. .
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[
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.
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[
International Worm Meeting,
2013]
In C. elegans, the dosage compensation complex is targeted specifically to both of the hermaphroditic X chromosomes to decrease X-linked transcription by half. This complex is homologous to subunits of the evolutionarily conserved condensins that play key roles in chromosome condensation. The dosage compensation complex condensin IDC has been shown to initially bind to specific recruitment sites on the X and then spread along the chromosome. These recruitment sites are enriched for a previously reported DNA sequence motif. Performing ChIP-seq analyses, we identified a core part of the condensin IDC motif enriched at binding sites of the canonical condensin II. Both the condensin IDC and condensin II motif are not sufficient to explain all condensin binding specificity. Approximately 12% and 25% of the condensin IDC and condensin II binding sites contain the motif, respectively. In addition, at a moderate motif match cutoff, 8% of the motifs across the genome are actually bound by each condensin. To identify additional factors that specify condensin IDC binding to the X specific recruitment sites, we analyzed the chromatin context of bound and unbound motif sites by comparing different histone modifications from the modENCODE project. Employing a machine learning approach, we found that condensins favor an active chromatin environment and that specific chromatin marks are associated with each condensin type. Our results provide a first step into the identification of distinct factors that facilitate specific binding of condensins to the DNA, supporting a model in which active chromatin marks have a high predictive value for condensin binding.
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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.
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[
International Worm Meeting,
2021]
The WD-40 repeat protein CDC-20 is the co-activator of the Anaphase Promoting Complex/Cyclosome (APC/C), the E3 ubiquitin ligase responsible for mitotic exit, as well as a core subunit of the mitotic checkpoint complex that restrains APC/C activity when chromosomes are not yet attached to the mitotic spindle. CDC-20 rapidly fluxes through the kinetochore region of chromosomes via association with a conserved binding motif, known as the ABBA motif, in BUB-1. CDC-20 flux through kinetochores is essential for mitotic checkpoint activation, which delays anaphase onset until all chromosomes attach to microtubules, and for promoting anaphase onset following kinetochore-microtubule attachment (Kim and Lara-Gonzalez et al, 2017, Genes and Development 31:1089-1094). Here, we investigate the regulation of BUB-1-dependent recruitment of CDC-20 to kinetochores. We found that mutating a conserved Polo-like Kinase 1 (PLK-1) docking site in BUB-1 eliminated CDC-20 kinetochore recruitment to the same extent as mutating the ABBA motif; in addition, the peak kinetochore recruitment of the mutant BUB-1 was reduced to ~50% of wildtype levels, likely due to a role for BUB-1-bound PLK-1 in promoting kinetochore recruitment of BUB-1. To address if the defect in CDC-20 kinetochore recruitment was a consequence of reduced BUB-1 kinetochore localization or was due to BUB-1-docked PLK-1 regulating the ABBA motif-CDC-20 interaction, we selectively mutated the BUB-1-associated protein BUB-3 to impair its recognition of the phosphorylated kinetochore scaffold KNL-1. The BUB-3 phospho-recognition mutant reduced peak BUB-1 kinetochore recruitment to ~30% of wildtype levels; however the consequences on anaphase onset and CDC-20 kinetochore localization were significantly less severe than observed for PLK-1-docking mutant BUB-1. These observations support a model in which BUB-1-associated PLK-1 is essential for CDC-20 kinetochore recruitment, either by direct phospho-regulation of the ABBA motif or by controlling access of the ABBA motif to CDC-20. Our current experiments are focused on distinguishing between these two potential mechanisms.