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.

Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection.

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...

Decades of investigation on genomic DNA have brought us deeper insights into its organization within the nucleus and its metabolic mechanisms. This was fueled by the parallel development of experimental techniques and has stimulated model building to simulate genome conformation in agreement with the experimental data. Here, we will discuss our recent discoveries on the chromatin units of DNA replication and DNA damage response. We will highlight their remarkable structural similarities and how both revealed themselves as clusters of nanofocal structures each on the hundred thousand base pair size range corresponding well with chromatin loop sizes. We propose that the function of these two global genomic processes is determined by the loop level organization of chromatin structure with structure dictating function.

Bacterial species able to produce proteins that are toxic against insects have been discovered at the beginning of the last century. However, up to date only two of them have been used as pesticides in mosquito control strategies targeting larval breeding sites: Bacillus thuringensis var. israelensis and Lysinibacillus sphaericus. Aiming to expand the arsenal of biopesticides, bacterial cultures from 44 soil samples were assayed for their ability to kill larvae of Aedes albopictus. A method to select, grow and test the larvicidal capability of spore-forming bacteria from each soil sample was developed. This allowed identifying 13 soil samples containing strains capable of killing Ae. albopictus larvae. Among the active isolates, one strain with high toxicity was identified as Brevibacillus laterosporus by 16S rRNA gene sequencing and by morphological characterization using transmission electron microscopy. The new isolate showed a larvicidal activity significantly higher than the B. laterosporus LMG 15441 reference strain. Its genome was phylogenomically characterized and compared to the available Brevibacillus genomes. Thus, the new isolate can be considered as a candidate adjuvant to biopesticides formulations that would help preventing the insurgence of resistance.