Spatial and temporal organization of the genome: Current state and future aims of the 4D nucleome project.

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.

Two contrasting classes of nucleolus-associated domains in mouse fibroblast heterochromatin.

In interphase eukaryotic cells, almost all heterochromatin is located adjacent to the nucleolus or to the nuclear lamina, thus defining nucleolus-associated domains (NADs) and lamina-associated domains (LADs), respectively. Here, we determined the first genome-scale map of murine NADs in mouse embryonic fibroblasts (MEFs) via deep sequencing of chromatin associated with purified nucleoli. We developed a Bioconductor package called NADfinder and demonstrated that it identifies NADs more accurately than other peak-calling tools, owing to its critical feature of chromosome-level local baseline correction. We detected two distinct classes of NADs. Type I NADs associate frequently with both the nucleolar periphery and the nuclear lamina, and generally display characteristics of constitutive heterochromatin, including late DNA replication, enrichment of H3K9me3, and little gene expression. In contrast, Type II NADs associate with nucleoli but do not overlap with LADs. Type II NADs tend to replicate earlier, display greater gene expression, and are more often enriched in H3K27me3 than Type I NADs. The nucleolar associations of both classes of NADs were confirmed via DNA-FISH, which also detected Type I but not Type II probes enriched at the nuclear lamina. Type II NADs are enriched in distinct gene classes, including factors important for differentiation and development. In keeping with this, we observed that a Type II NAD is developmentally regulated, and present in MEFs but not in undifferentiated embryonic stem (ES) cells.

Mitogen Kinase Kinase (MKK7) Controls Cytokine Production In Vitro and In Vivo in Mice.

Mitogen kinase kinase 4 (MKK4) and mitogen kinase kinase 7 (MKK7) are members of the MAP2K family that can activate downstream mitogen-activated protein kinases (MAPKs). MKK4 has been implicated in the activation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, while MKK7 has been reported to activate only JNK in response to different stimuli. The stimuli, as well as the cell type determine which MAP2K member will mediate a given response. In various cell types, MKK7 contributes to the activation of downstream MAPKs, JNK, which is known to regulate essential cellular processes, such as cell death, differentiation, stress response, and cytokine secretion. Previous studies have also implicated the role of MKK7 in stress signaling pathways and cytokine production. However, little is known about the degree to which MKK4 and MKK7 contribute to innate immune responses in macrophages or during inflammation in vivo. To address this question and to elucidate the role of MKK4 and MKK7 in macrophage and in vivo, we developed MKK4- and MKK7-deficient mouse models with tamoxifen-inducible Rosa26 CreERT. This study reports that MKK7 is required for JNK activation both in vitro and in vivo. Additionally, we demonstrated that MKK7 in macrophages is necessary for lipopolysaccharide (LPS)-induced cytokine production, M1 polarization, and migration, which appear to be a major contributor to the inflammatory response in vivo. Conversely, MKK4 plays a significant, but minor role in cytokine production in vivo.

New frontiers: discovering cilia-independent functions of cilia proteins.

In most vertebrates, mitotic spindles and primary cilia arise from a common origin, the centrosome. In non-cycling cells, the centrosome is the template for primary cilia assembly and, thus, is crucial for their associated sensory and signaling functions. During mitosis, the duplicated centrosomes mature into spindle poles, which orchestrate mitotic spindle assembly, chromosome segregation, and orientation of the cell division axis. Intriguingly, both cilia and spindle poles are centrosome-based, functionally distinct structures that require the action of microtubule-mediated, motor-driven transport for their assembly. Cilia proteins have been found at non-cilia sites, where they have distinct functions, illustrating a diverse and growing list of cellular processes and structures that utilize cilia proteins for crucial functions. In this review, we discuss cilia-independent functions of cilia proteins and re-evaluate their potential contributions to "cilia" disorders.

Perspective: "Current understanding of NADs dynamics and mechanisms of Disease".

Chromatin architecture is essential for gene regulation, and multiple levels of the 3D chromatin organization exhibit dynamic changes during organismal development and cell differentiation. Heterochromatin, termed compartment B in Hi-C datasets, is a phase-separating gene-silencing form of chromatin, preferentially located at the two nuclear sites, nuclear (lamina-associate chromatin domains, LADs) and nucleoli (nucleoli-associated chromatin domains, NADs) peripheries. LADs and NADs contain both interchangeable and location-specific chromatin domains. Recent studies suggest striking dynamics in LADs and NADs during the differentiation of embryonic stem cells into neural progenitors and neurons. Here we discuss recent advances in understanding NADs changes during neuronal differentiation and future questions on how NADs integrity can contribute to healthy neurodevelopment and neurodevelopment diseases.

TNF-alpha promotes cilia elongation via mixed lineage kinases signaling in mouse fibroblasts and human RPE-1 cells.

The primary cilium is a characteristic feature of most non-immune cells and functions as an environmental signal transduction sensor. The defects in primary cilium have profound effects on the developmental program, including the maturation of retinal epithelium. The ciliary length is tightly regulated during ciliogenesis, but the impact of inflammation on ciliary length remains elusive. The current study investigates the outcome of inflammatory stimuli for the primary cilium length in retinal epithelium cells and mouse embryonic fibroblasts. Here, we report that exposure to the pro-inflammatory cytokine TNF-alpha elongates cilia in a mixed-lineage kinase (MLK)-dependent manner. Pro-inflammatory stimuli such as bacterial LPS and interferon-gamma have similar effects on ciliary length. In contrast, febrile condition-mimicking heat stress dramatically reduced the number of ciliated cells regardless of TNF-alpha exposure but did not shorten TNF-induced elongation, suggesting distinct but rapid effects of inflammatory stresses on ciliogenesis.

Inflammatory stress-mediated chromatin changes underlie dysfunction in endothelial cells.

Inflammatory stresses underlie endothelial dysfunction and contribute to the development of chronic cardiovascular disorders such as atherosclerosis and vascular fibrosis. The initial transcriptional response of endothelial cells to pro-inflammatory cytokines such as TNF-alpha is well established. However, very few studies uncover the effects of inflammatory stresses on chromatin architecture. We used integrative analysis of ATAC-seq and RNA-seq data to investigate chromatin alterations in human endothelial cells in response to TNF-alpha and febrile-range heat stress exposure. Multi-omics data analysis suggests a correlation between the transcription of stress-related genes and endothelial dysfunction drivers with chromatin regions exhibiting differential accessibility. Moreover, microscopy identified the dynamics in the nuclear organization, specifically, the changes in a subset of heterochromatic nucleoli-associated chromatin domains, the centromeres. Upon inflammatory stress exposure, the centromeres decreased association with nucleoli in a p38-dependent manner and increased the number of transcripts from pericentromeric regions. Overall, we provide two lines of evidence that suggest chromatin alterations in vascular endothelial cells during inflammatory stresses.

Nucleolus and centromere TSA-Seq reveals variable localization of heterochromatin in different cell types.

Genome differential positioning within interphase nuclei remains poorly explored. We extended and validated TSA-seq to map genomic regions near nucleoli and pericentric heterochromatin in four human cell lines. Our study confirmed that smaller chromosomes localize closer to nucleoli but further deconvolved this by revealing a preference for chromosome arms below 36-46 Mbp in length. We identified two lamina associated domain subsets through their differential nuclear lamina versus nucleolar positioning in different cell lines which showed distinctive patterns of DNA replication timing and gene expression across all cell lines. Unexpectedly, active, nuclear speckle-associated genomic regions were found near typically repressive nuclear compartments, which is attributable to the close proximity of nuclear speckles and nucleoli in some cell types, and association of centromeres with nuclear speckles in hESCs. Our study points to a more complex and variable nuclear genome organization than suggested by current models, as revealed by our TSA-seq methodology.

Mitogen-activated protein kinase kinase 7 in inflammatory, cancer, and neurological diseases.

Stress-activated mitogen-activated protein kinase kinase 7 (MKK7) is a member of the dual-specificity mitogen-activated protein kinase family. In the human body, MKK7 controls essential physiological processes, including but not limited to proliferation and differentiation in multiple tissues and organs. MKK7, along with the MKK4 pathway, has been implicated in stress-activated activities and biological events that are mediated by c-Jun N-terminal kinase (JNK) signaling. Although numerous studies have been performed to identify the role of JNK in multiple biological processes, there are limited publications that focus on dissecting the independent role of MKK7. Recent research findings have spurred testing via in vivo genetically deficient models, uncovering previously undocumented JNK-independent functions of MKK7. Here we discuss both JNK-dependent and-independent functions of MKK7 in vivo. This review summarizes the role of MKK7 in inflammation, cytokine production, cancer, and neurological diseases.

Stress as a Chromatin Landscape Architect.

The exponential development of methods investigating different levels of spatial genome organization leads to the appreciation of the chromatin landscape's contribution to gene regulation and cell fate. Multiple levels of 3D chromatin organization include chromatin loops and topologically associated domains, followed by euchromatin and heterochromatin compartments, chromatin domains associated with nuclear bodies, and culminate with the chromosome territories. 3D chromatin architecture is exposed to multiple factors such as cell division and stress, including but not limited to mechanical, inflammatory, and environmental challenges. How exactly the stress exposure shapes the chromatin landscape is a new and intriguing area of research. In this mini-review, the developments that motivate the exploration of this field are discussed.

Viral Infection or IFN-α Alters Mitotic Spindle Orientation by Modulating Pericentrin Levels.

Congenital microcephaly occurs in utero during Zika virus (ZIKV) infection. The single-gene disorder, Majewski osteodysplastic primordial dwarfism type II (MOPDII), also leads to microcephaly and is concomitant with a decrease in the centrosomal protein, pericentrin (PCNT). This protein is a known contributor of mitotic spindle misorientation and ultimately, microcephaly. Similar to MOPDII, either viral infection or interferon (IFN)-α exposure reduced PCNT levels at the mitotic spindle poles. We unexpectedly found that infection of cells with any one of a diverse set of viruses, such as ZIKV, dengue virus, cytomegalovirus, influenza A virus, or hepatitis B virus, or treatment of cells with the anti-viral cytokine, IFN-α, produced mitotic spindle misorientation. These findings demonstrate a related mechanism for the development of microcephaly in viral infection, the host's antiviral IFN response, and primordial dwarfism.

New dimensions of asymmetric division in vertebrates.

Traditionally, we imagine that cell division gives rise to two identical daughter cells. Nevertheless, all cell divisions, to some degree, display asymmetry. Asymmetric cell division is defined as the generation of two daughter cells with different physical content and/or developmental potential. Several organelles and cellular components including the centrosome, non-coding RNA, chromatin, and recycling endosomes are involved in the process of asymmetric cell division. Disruption of this important process is known to induce profound defects in development, the immune response, regeneration of tissues, aging, and cancer. Here, we discuss recent advances that expand our understanding of the mechanisms and consequences of asymmetric cell division in vertebrate organisms.

The Centrosome, a Multitalented Renaissance Organelle.

The centrosome acts as a microtubule-organizing center (MTOC) from the G1 to G2 phases of the cell cycle; it can mature into a spindle pole during mitosis and/or transition into a cilium by elongating microtubules (MTs) from the basal body on cell differentiation or cell cycle arrest. New studies hint that the centrosome functions in more than MT organization. For instance, it has recently been shown that a specific substructure of the centrosome-the mother centriole appendages-are required for the recycling of endosomes back to the plasma membrane. This alone could have important implications for a renaissance in our understanding of the development of primary cilia, endosome recycling, and the immune response. Here, we review newly identified roles for the centrosome in directing membrane traffic, the immunological synapse, and the stress response.

Human basal body basics.

In human cells, the basal body (BB) core comprises a ninefold microtubule-triplet cylindrical structure. Distal and subdistal appendages are located at the distal end of BB, where they play indispensable roles in cilium formation and function. Most cells that arrest in the G0 stage of the cell cycle initiate BB docking at the plasma membrane followed by BB-mediated growth of a solitary primary cilium, a structure required for sensing the extracellular environment and cell signaling. In addition to the primary cilium, motile cilia are present in specialized cells, such as sperm and airway epithelium. Mutations that affect BB function result in cilia dysfunction. This can generate syndromic disorders, collectively called ciliopathies, for which there are no effective treatments. In this review, we focus on the features and functions of BBs and centrosomes in Homo sapiens.

The Centrosome Undergoes Plk1-Independent Interphase Maturation during Inflammation and Mediates Cytokine Release.

Cytokine production is a necessary event in the immune response during inflammation and is associated with mortality during sepsis, autoimmune disorders, cancer, and diabetes. Stress-activated MAP kinase signaling cascades that mediate cytokine synthesis are well established. However, the downstream fate of cytokines before they are secreted remains elusive. We report that pro-inflammatory stimuli lead to recruitment of pericentriolar material, specifically pericentrin and γ-tubulin, to the centrosome. This is accompanied by enhanced microtubule nucleation and enrichment of the recycling endosome component FIP3, all of which are hallmarks of centrosome maturation during mitosis. Intriguingly, centrosome maturation occurs during interphase in an MLK-dependent manner, independent of the classic mitotic kinase, Plk1. Centrosome disruption by chemical prevention of centriole assembly or genetic ablation of pericentrin attenuated interleukin-6, interleukin-10, and MCP1 secretion, suggesting that the centrosome is critical for cytokine production. Our results reveal a function of the centrosome in innate immunity.

Centrosome-intrinsic mechanisms modulate centrosome integrity during fever.

The centrosome is critical for cell division, ciliogenesis, membrane trafficking, and immunological synapse function. The immunological synapse is part of the immune response, which is often accompanied by fever/heat stress (HS). Here we provide evidence that HS causes deconstruction of all centrosome substructures primarily through degradation by centrosome-associated proteasomes. This renders the centrosome nonfunctional. Heat-activated degradation is centrosome selective, as other nonmembranous organelles (midbody, kinetochore) and membrane-bounded organelles (mitochondria) remain largely intact. Heat-induced centrosome inactivation was rescued by targeting Hsp70 to the centrosome. In contrast, Hsp70 excluded from the centrosome via targeting to membranes failed to rescue, as did chaperone inactivation. This indicates that there is a balance between degradation and chaperone rescue at the centrosome after HS. This novel mechanism of centrosome regulation during fever contributes to immunological synapse formation. Heat-induced centrosome inactivation is a physiologically relevant event, as centrosomes in leukocytes of febrile patients are disrupted.

CPEB regulation of TAK1 synthesis mediates cytokine production and the inflammatory immune response.

The cytoplasmic-element-binding (CPEB) protein is a sequence-specific RNA-binding protein that regulates cytoplasmic polyadenylation-induced translation. In mouse embryo fibroblasts (MEFs) lacking CPEB, many mRNAs encoding proteins involved in inflammation are misregulated. Correlated with this aberrant translation in MEFs, a macrophage cell line depleted of CPEB and treated with lipopolysaccharide (LPS) to stimulate the inflammatory immune response expresses high levels of interleukin-6 (IL-6), which is due to prolonged nuclear retention of NF-κB. Two proteins involved in NF-κB nuclear localization and IL-6 expression, IκBα and transforming growth factor beta-activated kinase 1 (TAK1), are present at excessively low and high steady-state levels, respectively, in LPS-treated CPEB-depleted macrophages. However, only TAK1 has an altered synthesis rate that is CPEB dependent and CPEB/TAK1 double depletion alleviates high IL-6 production. Peritoneal macrophages isolated from CPEB knockout (KO) mice treated with LPS in vitro also have prolonged NF-κB nuclear retention and produce high IL-6 levels. LPS-injected CPEB KO mice secrete prodigious amounts of IL-6 and other proinflammatory cytokines and exhibit hypersensitivity to endotoxic shock; these effects are mitigated when the animals are also injected with (5Z)-7-oxozeaenol, a potent and specific inhibitor of TAK1. These data show that CPEB control of TAK1 mRNA translation mediates the inflammatory immune response.

Role of the mixed-lineage protein kinase pathway in the metabolic stress response to obesity.

Saturated free fatty acid (FFA) is implicated in the metabolic response to obesity. In vitro studies indicate that FFA signaling may be mediated by the mixed-lineage protein kinase (MLK) pathway that activates cJun NH2-terminal kinase (JNK). Here, we examined the role of the MLK pathway in vivo using a mouse model of diet-induced obesity. The ubiquitously expressed MLK2 and MLK3 protein kinases have partially redundant functions. We therefore compared wild-type and compound mutant mice that lack expression of MLK2 and MLK3. MLK deficiency protected mice against high-fat-diet-induced insulin resistance and obesity. Reduced JNK activation and increased energy expenditure contribute to the metabolic effects of MLK deficiency. These data confirm that the MLK pathway plays a critical role in the metabolic response to obesity.

Analysis of properties of small heat shock protein Hsp25 in MAPK-activated protein kinase 2 (MK2)-deficient cells: MK2-dependent insolubilization of Hsp25 oligomers correlates with susceptibility to stress.

Small heat shock proteins (sHsps) exist in dynamic oligomeric complexes and display diverse biological functions ranging from chaperone properties to modulator of apoptosis. So far, the role of stress-dependent phosphorylation of mammalian sHsps for its structure and function has been analyzed by using various phosphorylation site mutants overexpressed in different cell types as well as by non-exclusive inhibitors of the p38 MAPK cascade. Here we investigate the role of phosphorylation of endogenous sHsp in a genetic model lacking the major Hsp25 kinase, the MAP kinase-activated protein kinase MK2. We demonstrate that in MK2-deficient fibroblasts, where no stress-dependent phosphorylation of Hsp25 at Ser86 and no in vitro binding to 14-3-3 was detectable, stress-dependent disaggregation of endogenous Hsp25 complexes is impared and kinetics of arsenite-dependent, H2O2-dependent, and sublethal heat shock-induced insolubilization of Hsp25 is delayed. Similarly, green fluorescent protein-tagged Hsp25 shows retarded subcellular accumulation into stress granules in MK2-deficient cells after arsenite treatment. Decreased insolubilization of Hsp25 in MK2-deficient cells correlates with increased resistance against arsenite, H2O2, and sublethal heat shock treatment and with decreased apoptosis. In contrast, after severe, lethal heat shock MK2-deficient embryonic fibroblasts cells show fast and complete insolubilization of Hsp25 independent of MK2 and no increased stress resistance. Hence, MK2-dependent formation of insoluble stress granules and irreversible cell damage by oxidative stresses and sublethal heat shock correlate and only upon severe, lethal heat shock MK2-independent processes could determine insolubilization of Hsp25 and are more relevant for cellular stress damage.