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Chromatin insulators are genetic elements implicated in the organization of chromatin and the regulation of transcription. In Drosophila, different insulator types were characterized by their locus-specific composition of insulator proteins and co-factors. Insulators mediate specific long-range DNA contacts required for the three dimensional organization of the interphase nucleus and for transcription regulation, but the mechanisms underlying the formation of these contacts is currently unknown. Here, we investigate the molecular associations between different components of insulator complexes (BEAF32, CP190 and Chromator) by biochemical and biophysical means, and develop a novel single-molecule assay to determine what factors are necessary and essential for the formation of long-range DNA interactions. We show that BEAF32 is able to bind DNA specifically and with high affinity, but not to bridge long-range interactions (LRI). In contrast, we show that CP190 and Chromator are able to mediate LRI between specifically-bound BEAF32 nucleoprotein complexes in vitro. This ability of CP190 and Chromator to establish LRI requires specific contacts between BEAF32 and their C-terminal domains, and dimerization through their N-terminal domains. In particular, the BTB/POZ domains of CP190 form a strict homodimer, and its C-terminal domain interacts with several insulator binding proteins. We propose a general model for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). This network of organized, multi-layer interactions could explain the different activities of insulators as chromatin barriers, enhancer blockers, and transcriptional regulators, and suggest a general mechanism for how insulators may shape the organization of higher-order chromatin during cell division.

At the kilo- to megabase pair scales, eukaryotic genomes are partitioned into self-interacting modules or topologically associated domains (TADs) that associate to form nuclear compartments. Here, we combine high-content super-resolution microscopies with state-of-the-art DNA-labeling methods to reveal the variability in the multiscale organization of the Drosophila genome. We find that association frequencies within TADs and between TAD borders are below ~10%, independently of TAD size, epigenetic state, or cell type. Critically, despite this large heterogeneity, we are able to visualize nanometer-sized epigenetic domains at the single-cell level. In addition, absolute contact frequencies within and between TADs are to a large extent defined by genomic distance, higher-order chromosome architecture, and epigenetic identity. We propose that TADs and compartments are organized by multiple, small-frequency, yet specific interactions that are regulated by epigenetics and transcriptional state.

Summary Although infections with virulent pathogens often induce a strong inflammatory reaction, what drives the increased immune response to pathogens compared to non-pathogenic microbes is poorly understood. One possibility is that the immune system senses the level of threat from a microorganism and augments the response accordingly. Here, focussing on cytotoxic necrotizing factor 1 (CNF1), an Escherichia coli-derived effector molecule, we showed the host indirectly sensed the pathogen by monitoring for the effector that modified RhoGTPases. CNF1 modified Rac2, which then interacted with the innate immune adaptors IMD and Rip1-Rip2 in flies and mammalian cells, respectively to drive an immune response. This response was protective and increased the ability of the host to restrict pathogen growth, thus defining a mechanism of effector-triggered immunity that contributes to how metazoans defend against microbes with pathogenic potential.

26At the kilo-to mega-base pair scales, eukaryotic genomes are partitioned into 27 self-interacting modules or topologically associated domains (TADs) that associate to 28 form nuclear compartments. Here, we combined high-content super-resolution 29 microscopies with state-of-the-art DNA labeling methods to reveal the variability in 30 the multiscale organization of the Drosophila genome. We found that association 31 frequencies within TADs and between TAD borders are below ~10%, independently 32 of TAD size, epigenetic state, or cell type. Critically, despite this large heterogeneity, 33 we were able to visualize nanometer-sized epigenetic domains at the single-cell 34 level. In addition, absolute contact frequencies within and between TADs were to a 35 large extent defined by genomic distance, higher-order chromosome architecture, 36 and epigenetic identity. We propose that TADs and compartments are organized by 37 multiple, small frequency, yet specific interactions that are regulated by epigenetics 38 and transcriptional state. 39 40 41 42 43The multi-scale organization of eukaryotic genomes defines and regulates 44 cellular identity and tissue-specific functions [1][2][3] . At the kilo-megabase scales, 45 genomes are partitioned into self-interacting modules or topologically associated 46 domains (TADs) 4-6 . TAD formation seems to require specific looping interactions 47 between TAD borders 7,8 , while the association of TADs can lead to the formation of 48 active/repressed compartments 9 . These structural levels were often seen as highly 49 stable over time, however, recent single-cell Hi-C studies have reported different 50 degrees of heterogeneity 10,11 . Other studies have reported that genomes also display 51 stochasticity in their association with the nuclear lamina 12 , in the formation of 52 chromosome territory neighborhoods 13 , and in gene kissing 14 . However, access to 53 single-cell absolute probability contact measurements between loci and efficient 54 detection of low-frequency, long-range interactions are essential to quantify the 55 stochastic behaviour of chromatin at different scales. 56Here, we combined high-content super-resolution microscopy with state-of-57 the-art DNA labeling methods to reveal the variability in the multiscale organization of 58 chromosomes in different cell-types and developmental stages in Drosophila. 59 Remarkably, we found that stochasticity is present at all levels of chromosome 60 architecture, but is locally modulated by sequence and epigenetic state. Contacts 61 between consecutive TAD borders were infrequent, independently of TAD size, 62 epigenetic state, or cell type. Moreover, long-range contact probabilities between 63 non-consecutive borders, the overall folding of chromosomes, and the clustering of 64 epigenetic domains into active/repressed compartments displayed different degrees 65 of stochasticity that globally depended on cell-type. Overall, our results show that 66 contacts between and within TADs are rare, but can be epigenetical...

Fanconi Anemia (FA) is a genetic disorder characterized by elevated cancer susceptibility and pro-inflammatory cytokine production. Using SLX4FANCP deficiency as a working model, we questioned the trigger for chronic inflammation in FA. We found that absence of SLX4 caused cytoplasmic DNA accumulation, including sequences deriving from active Long INterspersed Element-1 (LINE-1), triggering the cGAS-STING pathway to elicit interferon (IFN) expression. In agreement, absence of SLX4 leads to upregulated LINE-1 retrotransposition. Importantly, similar results were obtained with the FANCD2 upstream activator of SLX4. Furthermore, treatment of FA cells with the Tenofovir reverse transcriptase inhibitor (RTi), that prevents endogenous retrotransposition, decreased both accumulation of cytoplasmic DNA and pro-inflammatory signaling. Collectively, our data suggest a contribution of endogenous RT activities to the generation of immunogenic cytoplasmic nucleic acids responsible for inflammation in FA. The additional observation that RTi decreased pro-inflammatory cytokine production induced by DNA replication stress-inducing drugs further demonstrates the contribution of endogenous RTs to sustaining chronic inflammation. Altogether, our data open perspectives in the prevention of adverse effects of chronic inflammation in tumorigenesis.

Inflammation is an essential part of immunity against pathogens and tumors but can promote disease if not tightly regulated. Self and non-self-nucleic acids can trigger inflammation, through recognition by the cyclic GMP-AMP (cGAMP) synthetase (cGAS) and subsequent activation of the stimulator of interferon genes (STING) protein. Here, we show that RNA:DNA hybrids can be detected by cGAS and that the Lysyl-tRNA synthetase (LysRS) inhibits STING activation through two complementary mechanisms. First, LysRS interacts with RNA:DNA hybrids, delaying recognition by cGAS and impeding cGAMP production. Second, RNA:DNA hybrids stimulate LysRS-dependent production of diadenosine tetraphosphate (Ap4A) that in turn attenuates STING-dependent signaling. We propose a model whereby these mechanisms cooperate to buffer STING activation. Consequently, modulation of the LysRS-Ap4A axis in vitro or in vivo interferes with inflammatory responses. Thus, altogether, we establish LysRS and Ap4A as pharmacological targets to control STING signaling and treat inflammatory diseases.

NOD2 (nucleotide-binding oligomerization domain containing 2)is an important cytosolic pattern recognition receptor that activates NF-B and other immune effector pathways such as autophagy and antigen presentation. Despite its intracellular localization, NOD2 participates in sensing of extracellular microbes such as Staphylococcus aureus. NOD2 ligands similar to the minimal synthetic ligand muramyl dipeptide (MDP) are generated by internalization and processing of bacteria in hydrolytic phagolysosomes. However, how these derived ligands exit this organelle and access the cytosol to activate NOD2 is poorly understood. Here, we address how phagosome-derived NOD2 ligands access the cytosol in human phagocytes. Drawing on data from Drosophila phagosomes, we identify an evolutionarily conserved role of SLC15A transporters, Drosophila Yin and PEPT2, as MDP transporters in fly and human phagocytes, respectively. We show that PEPT2 is highly expressed by human myeloid cells. Ectopic expression of both Yin and PEPT2 increases the sensitivity of NOD2-dependent NF-B activation. Additionally, we show that PEPT2 associates with phagosome membranes. Together, these data identify Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated transporters that are likely to be of particular importance in delivery of bacteria-derived ligands generated in phagosomes to cytosolic sensors recruited to the vicinity of these organelles.