Systematic Chromatin Architecture Analysis inXenopus tropicalisReveals Conserved Three-Dimensional Folding Principles of Vertebrate Genomes

Abstract

23Metazoan genomes are folded into 3D structures in interphase nuclei. 24 However, the molecular mechanism remains unknown. Here, we show that 25 topologically associating domains (TADs) form in two waves during Xenopus 26 tropicalis embryogenesis, first at zygotic genome activation and then as the 27 expression of CTCF and Rad21 is elevated. We also found TAD structures 28 continually change for at least three times during development. Surprisingly, 29 the directionality index is preferentially stronger on one side of TADs where 30 orientation-biased CTCF and Rad21 binding are observed, a conserved 31 pattern that is found in human cells as well. Depletion analysis revealed CTCF, 32 Rad21, and RPB1, a component of RNAPII, are required for the establishment 33 of TADs. Overall, our work shows that Xenopus is a powerful model for 34 chromosome architecture analysis. Furthermore, our findings indicate that 35 cohesin-mediated extrusion may anchor at orientation-biased CTCF binding 36 sites, supporting a CTCF-anchored extrusion model as the mechanism for 37 TAD establishment. 38 39 40 41 42 3 KEYWORDS 43 Hi-C, Topologically associating domain (TAD), CTCF, Orientation-biased 44 binding, Cohesin-mediated extrusion, Directionality, Zygotic genome activation 45 (ZGA), RNAPII, Xenopus tropicalis, Genome assembly 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 Interphase chromosomes are partitioned into topologically associating 65 domains (TADs) 1-4 which segregate into compartments of active or repressive 66 chromatins 5-7 . TAD structures are relatively stable and resilient to 67 environmental perturbations 8,9 . Chromosome architecture at the TAD level is 68 also evolutionarily conserved in eukaryotic species 4,10,11 . Disruption of TAD 69 borders leads to developmental disorders and even tumorigenesis, thus 70 underlining the importance of 3D genome organization in gene regulation 12-15 . 71 The establishment of chromatin architecture during embryogenesis provides 72 an initial spatial frame which may guide proper genome organization, 73 chromatin interaction and gene regulation in following development and 74 differentiation processes 16 . The timing of de novo TADs formation during 75 development has been examined in Drosophila, mouse, zebrafish, and 76 human 17-21 . TAD structures form at zygotic genome activation (ZGA) and 77 continually consolidate during early embryo development in fruit fly, mouse and 78 human 17,19-21 . However, in zebrafish, TADs already exist before ZGA and are 79 lost after ZGA before being reestablished in later developmental stages 18 . This 80 difference raises the question if the process of TAD formation is evolutionarily 81 conserved. 82 Cohesin complex-mediated DNA loop extrusion was recently reported in 83 several in vitro studies 22,23 and proposed as a functional mechanism 84 underlying TAD establishment 24-26 . In cultured cells, the deletion of cohesin 85 5 Rad21 alone is enough to abolish the establishment of TADs 27 . In addition, 86 CTCF, WAPL, and PDS5 proteins p...

CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression

“…Accordingly, we show here that CTCF is also required for chromatin structure in zebrafish embryos (Fig. 1), extending these conclusions to vertebrates and in agreement with a recent report showing that CTCF knockdown in Xenopus embryos altered chromatin structure 41 .…”

“…1a-d). These data confirmed that CTCF is essential for 3D chromosome organization in zebrafish embryos, as described for other vertebrates including mammals and frogs 5,15,41 . Next, we analyzed A and B compartments in wild-type and ctcf −/− embryos and, although we found a similar distribution of AB compartments, we detected increased AB interactions and decreased compartmentalization strength in the mutants (Extended Data Fig.…”

“…1h), suggesting that they may be formed by CTCF-independent mechanisms. Therefore, we conclude that CTCF is essential for the establishment of most chromatin loops in zebrafish embryos, similarly to other vertebrates 5,41 .…”

CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression

“…1a-d). These data confirmed that CTCF is essential for 3D chromosome organization in zebrafish embryos, as described for other vertebrates including mammals and frogs 5,15,43 . Next, we analyzed A and B compartments in wild-type and ctcf -/embryos and, although we found a similar distribution of AB compartments, we detected increased AB interactions and decreased compartmentalization strength in the mutants ( Supplementary Fig.…”

CTCF as a boundary factor for cohesin-mediated loop extrusion: evidence for a multi-step mechanism

“…Fourth, although TADs and loops were initially reported to be cell-type invariant [ 11 , 132 ], it has now become clear that cell differentiation is associated with widespread changes to TAD and loop organization [ 133 – 140 ]. Consistently, mutating RBRs in CTCF only disrupts a subset of loops [ 36 , 37 ] and CTCF binding can be regulated [ 141 , 142 ].…”