Hua-Bing Li

Nature and function of insulator protein binding sites in the Drosophila genome

Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. Here we test this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. We find distinct combinatorial binding of insulator proteins to different classes of sites and uncover a novel type of insulator element that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. We show that insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together, these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes.

Insulators, Not Polycomb Response Elements, Are Required for Long-Range Interactions between Polycomb Targets in Drosophila melanogaster

ABSTRACT The genomic binding sites of Polycomb group (PcG) complexes have been found to cluster, forming Polycomb “bodies” or foci in mammalian or fly nuclei. These associations are thought to be driven by interactions between PcG complexes and result in enhanced repression. Here, we show that a Polycomb response element (PRE) with strong PcG binding and repressive activity cannot mediate trans interactions. In the case of the two best-studied interacting PcG targets in Drosophila, the Mcp and the Fab-7 regulatory elements, we find that these associations are not dependent on or caused by the Polycomb response elements they contain. Using functional assays and physical colocalization by in vivo fluorescence imaging or chromosome conformation capture (3C) methods, we show that the interactions between remote copies of Mcp or Fab-7 elements are dependent on the insulator activities present in these elements and not on their PREs. We conclude that insulator binding proteins rather than PcG complexes are likely to be the major determinants of the long-range higher-order organization of PcG targets in the nucleus.

A view of nuclear Polycomb bodies

Polycomb group (PcG) proteins are concentrated in nuclear foci called PcG bodies. Although some of these foci are due to the tendency of PcG binding sites in the genome to occur in linear clusters, distant PcG sites can contact one another and in some cases congregate in the same PcG body when they are repressed. Experiments using transgenes containing PcG binding sites reveal that co-localization depends on the presence of insulator elements rather than of Polycomb Response Elements (PREs) and that it can occur also when the transgenes are in the active state. A model is proposed according to which insulator proteins mediate shuttling of PcG target genes between PcG bodies when repressed to transcription factories when transcriptionally active.

Insulators target active genes to transcription factories and polycomb-repressed genes to polycomb bodies.

Polycomb bodies are foci of Polycomb proteins in which different Polycomb target genes are thought to co-localize in the nucleus, looping out from their chromosomal context. We have shown previously that insulators, not Polycomb response elements (PREs), mediate associations among Polycomb Group (PcG) targets to form Polycomb bodies. Here we use live imaging and 3C interactions to show that transgenes containing PREs and endogenous PcG-regulated genes are targeted by insulator proteins to different nuclear structures depending on their state of activity. When two genes are repressed, they co-localize in Polycomb bodies. When both are active, they are targeted to transcription factories in a fashion dependent on Trithorax and enhancer specificity as well as the insulator protein CTCF. In the absence of CTCF, assembly of Polycomb bodies is essentially reduced to those representing genomic clusters of Polycomb target genes. The critical role of Trithorax suggests that stable association with a specialized transcription factory underlies the cellular memory of the active state.

Recent advances in dynamic m6A RNA modification

The identification of m6A demethylases and high-throughput sequencing analysis of methylated transcriptome corroborated m6A RNA epigenetic modification as a dynamic regulation process, and reignited its investigation in the past few years. Many basic concepts of cytogenetics have been revolutionized by the growing understanding of the fundamental role of m6A in RNA splicing, degradation and translation. In this review, we summarize typical features of methylated transcriptome in mammals, and highlight the ‘writers’, ‘erasers’ and ‘readers’ of m6A RNA modification. Moreover, we emphasize recent advances of biological functions of m6A and conceive the possible roles of m6A in the regulation of immune response and related diseases.

The DNA-sensing AIM2 inflammasome controls radiation-induced cell death and tissue injury.

AIMing to block tissue damage Ionizing radiation kills actively dividing cells such as those in the gut and in the bone marrow. Hu et al. found a pathological role for the protein AIM2 in irradiation-induced tissue damage. AIM2 is best known for its role in sensing double-stranded DNA in the cytoplasm and alerting the body to infections. It seems that AIM2 also senses DNA damage caused by radiation and then triggers intestinal epithelial cells and bone marrow cells to die. Deficiency in AIM2 protected mice from irradiation-induced gastrointestinal syndrome and hematopoietic failure. Science, this issue p. 765 AIM2 detects radiation-induced DNA damage and triggers intestinal epithelial cell and bone marrow cell death. Acute exposure to ionizing radiation induces massive cell death and severe damage to tissues containing actively proliferating cells, including bone marrow and the gastrointestinal tract. However, the cellular and molecular mechanisms underlying this pathology remain controversial. Here, we show that mice deficient in the double-stranded DNA sensor AIM2 are protected from both subtotal body irradiation–induced gastrointestinal syndrome and total body irradiation–induced hematopoietic failure. AIM2 mediates the caspase-1–dependent death of intestinal epithelial cells and bone marrow cells in response to double-strand DNA breaks caused by ionizing radiation and chemotherapeutic agents. Mechanistically, we found that AIM2 senses radiation-induced DNA damage in the nucleus to mediate inflammasome activation and cell death. Our results suggest that AIM2 may be a new therapeutic target for ionizing radiation exposure.

Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells

Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

Inflammasome activation and metabolic disease progression

Innate pattern recognition receptors NLRs are cytosolic sensors that detect endogenous metabolic stress and form a multiprotein complex called the inflammasome, that recruits and activates caspase enzymes mediating the activation of the cytokines IL-1β and IL-18. The innate immune system and metabolic system are evolutionarily conserved, intimately integrated, and functionally dependent. In recent decades, obesity-associated metabolic diseases have been become a worldwide epidemic. Here we review recent evidence that demonstrates the important roles of NLRs and inflammasomes in response to metabolic stress in different tissues.

Chromosome Conformation Capture in Drosophila

Linear chromatin fiber is packed inside the nuclei as a complex three-dimensional structure, and the organization of the chromatin has important roles in the appropriate spatial and temporal regulation of gene expression. To understand how chromatin organizes inside nuclei, and how regulatory proteins physically interact with genes, chromosome conformation capture (3C) technique provides a powerful and sensitive tool to detect both short- and long-range DNA-DNA interaction. Here I describe the 3C technique to detect the DNA-DNA interactions mediated by insulator proteins that are closely related to PcG in Drosophila, which is also broadly applicable to other systems.

cGAS activation in phased droplets

Cyclic GMP-AMP synthase (cGAS) is activated by DNA through direct binding. Active cGAS catalyzes the production of cyclic GMP-AMP (cGAMP), and consequently activates the innate immune responses. However, the detailed biological processes are poorly understood. Du and Chen demonstrated in a recent paper published on Science that DNA binding to cGAS robustly induced the formation of liquid like droplets in which cGAS was activated. The innate immune system utilizes cGAS to detect cytosolic foreign DNA, which is essential genetic information carriers for most living organisms including viral, bacterial, and eukaryotic pathogens. Upon recognition of foreign DNA, the cytosolic sensor-cGAS triggers cGAMP production. cGAMP in turn functions as an endogenous second messenger to activate STING, leading to the production of type I interferons, as well as proinflammatory cytokines. Research in the past few years has led to significant progress toward understanding the mechanism of cytosolic DNA sensing and signaling. Although the crystal structure of cGAS alone or in complex with cytosolic foreign DNA has been resolved, the detailed in vivo biological processes remain unresolved. Recently, a physicochemical model of phase separation of fluids based on principles of polymer chemistry and soft matter physics explains a series of biological events including RNA metabolism, ribosome biogenesis, transcription, DNA damage response and signal transduction. Phase-separated biomolecular condensates produced by liquid-liquid phase separation of protein-nucleic acid-macromolecules allow rapid biological reaction of components within the dense phase. Based on this model, Du and Chen hypothesized that cGAS and cytosolic DNA interactions could lead to the formation of biomolecular condensates through liquid phase separation. To this end, they incubated fluorescently labeled cGAS protein with double-stranded DNA oligonucleotides in vitro. As expected, cGAS and DNA formed micrometer-sized liquid droplets, in which cGAS and reactants were concentrated to greatly enhance the production of cGAMP. Under physiological conditions, the formation of these droplets may have two functions: one is to concentrate reactants to amplify the signaling, and the other is to collect the invading nucleic acid molecules. cGAS also formed liquid droplets with dsRNA, but RNA did not activate cGAS to produce cGAMP, which suggests that liquid phase separation is insufficient for cGAS activation. These results confirm the validity of the previous structural studies indicating that DNA but not RNA binding induces a conformational change that activates cGAS and the following signaling. Within cells, the formation of cGAS-DNA foci was also detected. Furthermore, the cGAS-DNA foci could fuse with each other and exhibited near-complete fluorescence recovery after photobleaching, indicating that cGAS was in dynamic liquid like granules. This raises the question of whether the cGAS-DNA liquid phase separation is a dynamic and reversible process in vivo, which is important to timely terminate the activation of immune responses. Besides, the cGAS-DNA liquid like granules were distinct from cellular organelles and vesicles as evidenced by the fact that most cGAS activities were present in subcellular fractions containing the nuclear and heavy fractions. This corresponds to the physical properties of the phase separation condensates of protein-nucleic acid-macromolecules. Based on the premise that liquid phase separation is driven by multivalent interactions, valency is important for this physicochemical process. Thus, Du and Chen tested the DNA length, full-length cGAS, cytoplasmic salt concentrations and free zinc ions as important factors to regulate the formation of cGAS-DNA biomolecular condensates: (1) Long DNA has more binding sites (valency) for cGAS than short DNA, and full-length cGAS has higher valency for DNA than core-cGAS, thus long DNA and full-length cGAS phase separation are more efficient; (2) such DNA-cGAS multivalent interactions are vulnerable to cytoplasmic salt concentrations, which may be a mechanism to prevent unwanted activation of cGAS by self-DNA below a certain threshold; (3) free zinc ions facilitate cGAS activation by promoting DNA-cGAS phase transition. To a certain extent, this discovery unified our understanding of previous studies about cytosolic DNA sensing and signaling (Fig. 1). A recent study suggested that STING could function as a direct sensor for DNA. The binding affinity of STING to dsDNA was several orders of magnitude lower than that of cGAS. However, it is still necessary to explore whether STING is involved in the formation of cGAS-DNA condensates, and it will also be interesting to study details of cGAS-STING anti-infection signal pathway activation in cGAS-DNA condensates. Besides, since this study was carried out in vitro at cellular level, it is necessary to further validate these conclusions in vivo in the future. This is of particular interest given that cytosolic DNA-cGAS induces type I IFNs and other cytokines can also cause autoimmune diseases in addition to resisting infection, further investigation on the dynamics and composition of the DNA-cGAS condensates in autoimmune diseases may provide more mechanisms and therapeutic approaches. In addition, there are other immune signaling activations and transductions that may also happen in phase separated droplets, such as inflammasomes.