Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically defined, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity.

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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...

The cellular mechanisms required to ensure homeostasis of the hematopoietic niche and the ability of this niche to support hematopoiesis upon stress remain elusive. We here identify Wnt5a in Osterix+ mesenchymal progenitor and stem cells (MSPCs) as a critical factor for niche-dependent hematopoiesis. Mice lacking Wnt5a in MSPCs suffer from stress-related bone marrow (BM) failure and increased mortality. Niche cells devoid of Wnt5a show defective actin stress fiber orientation due to an elevated activity of the small GTPase CDC42. This results in incorrect positioning of autophagosomes and lysosomes, thus reducing autophagy and increasing oxidative stress. In MSPCs from patients from BM failure states which share features of peripheral cytopenia and hypocellular BM, we find similar defects in actin stress fiber orientation, reduced and incorrect colocalization of autophagosomes and lysosomes, and CDC42 activation. Strikingly, a short pharmacological intervention to attenuate elevated CDC42 activation in vivo in mice prevents defective actin-anchored autophagy in MSPCs, salvages hematopoiesis and protects against lethal cytopenia upon stress. In summary, our study identifies Wnt5a as a restriction factor for niche homeostasis by affecting CDC42-regulated actin stress-fiber orientation and autophagy upon stress. Our data further imply a critical role for autophagy in MSPCs for adequate support of hematopoiesis by the niche upon stress and in human diseases characterized by peripheral cytopenias and hypocellular BM.

Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences that was enabled by the development of chemoselective reactions for the site‐specific modification of proteins. Antibody‐oligonucleotide conjugates combine the antibody's target specificity with the reversible, sequence‐encoded binding properties of oligonucleotides like DNAs or peptide nucleic acids (PNAs), allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub‐tag® technology in combination with Cu‐catalyzed azide‐alkyne cycloaddition for the site‐specific conjugation of single DNA and PNA strands to an eGFP‐binding nanobody. We show binding of the conjugate to recombinant eGFP and subsequent sequence‐specific annealing of fluorescently labelled imager strands. Furthermore, we reversibly stain eGFP‐tagged proteins in human cells, thus demonstrating the suitability of our conjugation strategy to generate antibody‐oligonucleotides for reversible immunofluorescence imaging.