Research
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Chromosome compaction is essential for chromosome segregation during cell division and regulate 3D genome organization and gene expression during interphase. The goal of my lab is to understand the molecular mechanisms that regulate chromosome structure and function. A key feature of eukaryotic chromosomes include DNA loops, formed by the structural maintenance of chromosomes (SMC) family of proteins. SMC complexes, including condensin and cohesin are ATP dependent molecular motors that bind to and gradually enlarge chromatin loops through a process named “DNA loop extrusion”. In mammalian cells, cohesin-mediated loop extrusion forms topologically associating domains (TAD) that regulate enhancer-promoter specificity. Cohesin activity is regulated by its loaders, processivity factors, and blockers including NIPBL, WAPL, and CTCF. It is not known if condensins are similarly regulated. Recent characterization of condensin II repressor MCPH1 support the possibility. Caenorhabditis elegans, which contains both the canonical and an X-specific condensin (Figure 1), presents an ideal system to study and discover the molecular mechanisms of condensin regulation and function in vivo.
Figure 1. A) C. elegans contains both canonical condensins I and II and an X-specific condensin for dosage compensation (DC). Condensin I and DC differ by one SMC-4 variant, DPY-27. Unlike canonical condensins I and II that bind to all chromosomes, condensin DC binds specifically to the X chromosomes in hermaphrodites and reduces RNA Pol II initiation by ~2-fold across the entire chromosome. B) Our current model of condensin DC targeting is stepwise . A set of recruitment proteins (SDC &DPY-30) recognize strong recruitment elements on the X (rex) containing multiple copies of a 12-bp sequence motif. Long-distance cooperation between strong and weaker rex sites maintain specificity. Cooperative recruitment, coupled with linear spreading establishes the robust and specific binding of condensin DC across the X-chromosomes. For cooperative recruitment see Albritton et al 2017 and 2018.
Condensin DC creates loop-anchored TADs on the C. elegans X chromosomes
Condensin DC forms TADs on the X chromosomes, with eight strong recruitment sites acting as boundaries. Deleting a strong rex removes the TAD boundary and inserting a strong rex creates one. Thus, rex-condensin DC is similar to the CTCF site-cohesin in mammalian cells. In Kim, Jimenez et al 2022, we found that condensin DC differs from cohesin system in two aspects. First, unlike the CTCF motif, the orientation of the 12-bp motif is not important for loop-specificity. Second, the rex sites are also the loading sites for the complex (Figure 2). Future directions in this project include determining how rex-rex loop anchors are established and maintained. Surprisingly, maintenance of rex-rex loops do not require condensin DC. We will also address potential redundancy between SMC complexes and 3D interactions that are independent of SMCs.
Figure 2. Insertion of strong or multiple rex sites is sufficient to form a domain of condensin DC spreading and created a loop-anchored TAD on chromosome II. N2 is wild type strain without insertion. LS01 contains insertion of three recruitment elements that cooperatively recruit condensin DC ectopically to chromosome II. Top panel is the Hi-C interaction matrix, bottom panel is condensin DC binding measured by DPY-27 ChIP-seq. An ectopic loop-anchored interaction domain is created on chromosome II. For details see Kim, Jimenez et al 2022.
Topoisomerase II promotes the processivity of condensin DC loop extrusion
Having an X-specific condensin with known recruitment sites allowed us to address regulators of condensin translocation in vivo. In Morao et al 2022, we used auxin-inducible degradation of topoisomerases I and II to determine how DNA topology affects condensin translocation. TOP-2 depletion hindered condensin DC spreading and shorter Hi-C interactions (Figure 3), suggesting that DNA catenates/knots reduce processivity of condensin DC loop extrusion. TOP-1 depletion did not affect long-range spreading but resulted in accumulation of condensin DC within expressed gene bodies, suggesting a short-scale condensin translocation that is sensitive to transcription-induced supercoiling. Future directions in this project include how topoisomerases, SMCs, and Pol II interact with each other within gene bodies, addressing the effect of DNA topology on transcription.
Figure 3. Processivity of condensin DC reduces upon TOP-2 depletion. Hi-C contacts on the X chromosome are highlighted by plotting ratio of contacts on the X to autosomes. The x axis indicates the distance between contacts. Average distance of 3D contacts, ie. condensin DC mediated loops, shortens specifically upon TOP-2 depletion but not TOP-1. For details see Morao et al 2022.
An H4K20me2 demethylase regulates the dynamics of condensin DC association with the X chromosomes
Mitotic chromosomes show high levels of H4K20me1 and low levels of H4 acetylation and transcription. This is also the case for dosage-compensated X chromosomes in C. elegans. Condensin DC recruits the H4K20me2 demethylase DPY-21, increasing H4K20me1 on the X. Surprisingly, unlike the null mutant which causes full derepression of the X, a catalytically inactive DPY-21 causes a subtle increase in transcription. In collaboration with the Woehler and Preibisch labs at MDC Berlin, we performed fluorescence recovery after photobleaching (FRAP) experiments (Breimann, Moraro et al. 2022). Our results suggest that DPY-21 protein regulate the dynamics of condensin DC binding, perhaps to histone tails, which is critical for transcription repression (Figure 4).
Figure 4. The mobile fraction of condensin DC reduces in the null mutant, but not in the catalytically dead mutant of DPY-21 H4K20me2 demethylase. Mean FRAP recovery curves of DPY-27::GFP in either wild-type (green), dpy-21 null (e428, left panel) or dpy-21 catalytic (y607, right panel) mutants. Error bars denote s.e.m.. Scale bar = 5 µm. While H4K20me1 levels decrease in both mutants, only the null mutant leads to strong transcriptional repression on the X and change in dynamics of condensin DC binding. For more, see Breimann, Morao et al 2022.
Other directions / projects in the lab
Environmental control of gene regulation: Environmental control of gene expression is key for species survival. C. elegans dauer larvae is a fascinating and metabolically different state that allows worms to withstand lack of food for months. We are using genomic techniques to address if dauers compact chromatin to achieve genome-wide gene repression and how this is linked to pathways that control dauer physiology.
Evolution of dosage compensation: Most dosage compensation studies have been limited to model organisms. Yet even in the few species that were analyzed, it is clear that diverse strategies evolved by co-opting existing mechanisms of gene regulation to solve the X chromosome gene dosage problem that emerged during evolution. These strategies include silencing of one of the two X chromosomes in females (mouse), doubling the expression of the single X chromosome in males (fly), and halving the expression of both X chromosomes in hermaphrodites (worm). An intriguing question is whether species across neighboring lineages use the same strategy. We are analyzing Hi-C and chromatin features for evidence of condensin-based X chromosome repression in several sequenced nematode species representing different lineages.
Mapping chromosome segregation defects: In all eukaryotes studied to date, a common phenotype of mutants that affect chromosome compaction are DAPI-staining DNA that stretches between segregating chromosome masses in anaphase. These “anaphase bridges” are poorly characterized, in part due to the assumption that their content is invariant or random. However, this assumption may be false because previous studies have identified mutants that lead to bridges specifically enriched in centromeric, telomeric or ribosomal DNA. We are developing Bridge-seq to characterize anaphase bridges with the goal to connect them to aneuploidies and genome integrity problems mapped in genomic studies of cancers and aging.