Cohesin depleted cells rebuild functional nuclear compartments after endomitosis

Abstract

43The human genome forms thousands of "contact domains", which are intervals of enhanced 44 contact frequency. Some, called "loop domains" are thought to form by cohesin-mediated loop 45 extrusion. Others, called "compartmental domains", form due to the segregation of active and 46 inactive chromatin into A and B compartments. Recently, Hi-C studies revealed that the 47 depletion of cohesin leads to the disappearance of all loop domains within a few hours, but 48 strengthens compartment structure. Here, we combine live cell microscopy, super-resolution 49 microscopy, Hi-C, and studies of replication timing to examine the longer-term consequences 50 of cohesin degradation in HCT-116 human colorectal carcinoma cells, tracking cells for up to 51 30 hours. Surprisingly, cohesin depleted cells proceed through an aberrant mitosis, yielding a 52 single postmitotic cell with a multilobulated nucleus. Hi-C reveals the continued disappearance 53 of loop domains, whereas A and B compartments are maintained. In line with Hi-C, microscopic 54 observations demonstrate the reconstitution of chromosome territories and chromatin 55 domains. An interchromatin channel system (IC) expands between chromatin domain clusters 56 and carries splicing speckles. The IC is lined by active chromatin enriched for RNA Pol II and 57 depleted in H3K27me3. Moreover, the cells exhibit typical early-, mid-, and late-DNA 58 replication timing patterns. Our observations indicate that the functional nuclear 59 compartmentalization can be maintained in cohesin depleted pre-and postmitotic cells.60 However, we find that replication foci -sites of active DNA synthesis -become physically 61 larger consistent with a model where cohesin dependent loop extrusion tends to compact 62 intervals of replicating chromatin, whereas their genomic boundaries are associated with 63 compartmentalization, and do not change.64 65 3 Abbreviations 66 3D FISH = 3D fluorescence in situ hybridization 67 3D SIM = 3D structured illumination microscopy 68 AID = auxin inducible degron 69 ANC / INC = active / inactive nuclear compartment 70 CT = chromosome territory 71 CD(C) = chromatin domain (cluster) 72 CTCF = CCCTC binding factor 73 DAPI = 4',6-diamidino-2-phenylindole 74 EdU = 5-Ethynyl-2'-deoxyuridine 75 Hi-C = chromosome conformation capturing combined with deep sequencing 76 IC = interchromatin compartment 77 MLN = multilobulated nucleus 78 NC = nucleosome cluster 79 PBS = phosphate buffered saline 80 PBST = phosphate buffered saline with 0.02% Tween 81 PR = perichromatin region 82 RD = replication domain 83 RL = replication labeling 84 TAD = topologically associating domain 85 86 109 strengthened, leading to the presence of compartment domains and even compartment loops110 (but no loop domains) in the treated cells [18]. Other studies, using different cell types and 111 approaches for cohesin elimination yielded similar results [19-21], (reviewed in [22]). 112Here, we study the longer-term consequences of cohesin depletion and its effects on 113 the higher order nuclear a...

Replication timing maintains the global epigenetic state in human cells

“…However, such boundaries were weak boundaries in WT and constituted a small fraction of total boundaries (Fig S8b right). Overall, these data mechanistically separate RT from TAD formation and support previous work showing essential TAD proteins Rad21 and CTCF are dispensable for global RT control ( 23 – 25 ).…”

The Interchromatin Compartment Participates in the Structural and Functional Organization of the Cell Nucleus

“…The lowest DNA density class 1 represents the IC with splicing speckles and nuclear bodies . Intensity classes 2 and 3 represent the less‐compacted chromatin of the PR, which lines the IC and shows a significant enrichment for RNAPII, and epigenetic markers indicating transcriptionally competent chromatin . In line with these assignments, RNA synthesis is most prominent within the PR .…”

“…Figure 7A illustrates the topographical context of the seven DAPI intensity classes in a single SIM section of a DAPI‐stained human fibroblast nucleus. The lowest DNA density class 1 represents the IC with splicing speckles and nuclear bodies . Intensity classes 2 and 3 represent the less‐compacted chromatin of the PR, which lines the IC and shows a significant enrichment for RNAPII, and epigenetic markers indicating transcriptionally competent chromatin .…”

“…Numerous 3D SIM studies combined with quantitative 3D image analyses revealed CDs, CDCs and CTs, the IC and the PR (compare Figure ). Readers are referred to the respective literature and recent reviews . Profound differences, however, were noted in different cell types both with regard to the width of IC channels and lacunas and the quantity and distribution of chromatin assigned to the ANC or INC.…”

Condensed chromatin behaves like a solid on the mesoscale in vitro and in living cells

“…Thus, observing relative movement between TADs does not imply that the chromatin fibers mix, which is the defining property of liquid chromatin condensates in vitro. Given that these early S-phase euchromatic replication foci associated with TAD organization persist in cells depleted of cohesion (Cremer et al, 2019), it is also possible that lower frequency interactions between chromatin fibers mediate the solid-like behavior.…”