SAFB restricts contact domain boundaries associated with L1 chimeric transcription.

Long interspersed element-1 (LINE-1 or L1) comprises 17% of the human genome, continuously generates genetic variations, and causes disease in certain cases. However, the regulation and function of L1 remain poorly understood. Here, we uncover that L1 can enrich RNA polymerase IIs (RNA Pol IIs), express L1 chimeric transcripts, and create contact domain boundaries in human cells. This impact of L1 is restricted by a nuclear matrix protein scaffold attachment factor B (SAFB) that recognizes transcriptionally active L1s by binding L1 transcripts to inhibit RNA Pol II enrichment. Acute inhibition of RNA Pol II transcription abolishes the domain boundaries associated with L1 chimeric transcripts, indicating a transcription-dependent mechanism. Deleting L1 impairs domain boundary formation, and L1 insertions during evolution have introduced species-specific domain boundaries. Our data show that L1 can create RNA Pol II-enriched regions that alter genome organization and that SAFB regulates L1 and RNA Pol II activity to preserve gene regulation.

Neutrophil Targeting Platform Reduces Neutrophil Extracellular Traps for Improved Traumatic Brain Injury and Stroke Theranostics.

Traumatic brain injuries (TBI) and stroke are major causes of morbidity and mortality in both developing and developed countries. The complex and heterogeneous pathophysiology of TBI and cerebral ischemia-reperfusion injury (CIRI), in addition to the blood-brain barrier (BBB) resistance, is a major barrier to the advancement of diagnostics and therapeutics. Clinical data showed that the severity of TBI and stroke is positively correlated with the number of neutrophils in peripheral blood and brain injury sites. Furthermore, neutrophil extracellular traps (NETs) released by neutrophils correlate with worse TBI and stroke outcomes by impairing revascularization and vascular remodeling. Therefore, targeting neutrophils to deliver NETs inhibitors to brain injury sites and reduce the formation of NETs can be an optimal strategy for TBI and stroke therapy. Herein, the study designs and synthesizes a reactive oxygen species (ROS)-responsive neutrophil-targeting delivery system loaded with peptidyl arginine deiminase 4 (PAD4) inhibitor, GSK484, to prevent the formation of NETs in brain injury sites, which significantly inhibited neuroinflammation and improved neurological deficits, and improved the survival rate of TBI and CIRI. This strategy may provide a groundwork for the development of targeted theranostics of TBI and stroke.

CRISPR Activation Reverses Haploinsufficiency and Functional Deficits Caused by TTN Truncation Variants.

BACKGROUND: TTN truncation variants (TTNtvs) are the most common genetic lesion identified in individuals with dilated cardiomyopathy, a disease with high morbidity and mortality rates. TTNtvs reduce normal TTN (titin) protein levels, produce truncated proteins, and impair sarcomere content and function. Therapeutics targeting TTNtvs have been elusive because of the immense size of TTN, the rarity of specific TTNtvs, and incomplete knowledge of TTNtv pathogenicity. METHODS: We adapted CRISPR activation using dCas9-VPR to functionally interrogate TTNtv pathogenicity and develop a therapeutic in human cardiomyocytes and 3-dimensional cardiac microtissues engineered from induced pluripotent stem cell models harboring a dilated cardiomyopathy-associated TTNtv. We performed guide RNA screening with custom TTN reporter assays, agarose gel electrophoresis to quantify TTN protein levels and isoforms, and RNA sequencing to identify molecular consequences of TTN activation. Cardiomyocyte epigenetic assays were also used to nominate DNA regulatory elements to enable cardiomyocyte-specific TTN activation. RESULTS: CRISPR activation of TTN using single guide RNAs targeting either the TTN promoter or regulatory elements in spatial proximity to the TTN promoter through 3-dimensional chromatin interactions rescued TTN protein deficits disturbed by TTNtvs. Increasing TTN protein levels normalized sarcomere content and contractile function despite increasing truncated TTN protein. In addition to TTN transcripts, CRISPR activation also increased levels of myofibril assembly-related and sarcomere-related transcripts. CONCLUSIONS: TTN CRISPR activation rescued TTNtv-related functional deficits despite increasing truncated TTN levels, which provides evidence to support haploinsufficiency as a relevant genetic mechanism underlying heterozygous TTNtvs. CRISPR activation could be developed as a therapeutic to treat a large proportion of TTNtvs.

Guilt by association: EcDNA as a mobile transactivator in cancer.

Extrachromosomal DNA (ecDNA), first described in the 1960s, is emerging as a prevalent but poorly characterized oncogenic alteration in cancer. ecDNA is a reservoir for oncogene amplification and is associated with an aggressive tumor phenotype and poor patient outcome. Despite the long-held knowledge of its existence, little is known about how ecDNA affects tumor cell behavior. Recent data reveal that ecDNA hubs are mobile transcriptional enhancers which can transactivate gene expression through chromatin interactions. Given its prevalence, structural complexity, and unequal segregation into daughter cells, ecDNA can offer selective growth advantages, contribute to intratumor heterogeneity (ITH), and accelerate tumor evolution. Future technology development is expected to transform the current paradigm for studying ecDNA and lead to therapeutic strategies targeting ecDNA vulnerabilities.

Oncogenic extrachromosomal DNA functions as mobile enhancers to globally amplify chromosomal transcription.

Extrachromosomal, circular DNA (ecDNA) is emerging as a prevalent yet less characterized oncogenic alteration in cancer genomes. We leverage ChIA-PET and ChIA-Drop chromatin interaction assays to characterize genome-wide ecDNA-mediated chromatin contacts that impact transcriptional programs in cancers. ecDNAs in glioblastoma patient-derived neurosphere and prostate cancer cell cultures are marked by widespread intra-ecDNA and genome-wide chromosomal interactions. ecDNA-chromatin contact foci are characterized by broad and high-level H3K27ac signals converging predominantly on chromosomal genes of increased expression levels. Prostate cancer cells harboring synthetic ecDNA circles composed of characterized enhancers result in the genome-wide activation of chromosomal gene transcription. Deciphering the chromosomal targets of ecDNAs at single-molecule resolution reveals an association with actively expressed oncogenes spatially clustered within ecDNA-directed interaction networks. Our results suggest that ecDNA can function as mobile transcriptional enhancers to promote tumor progression and manifest a potential synthetic aneuploidy mechanism of transcription control in cancer.

Sox2 controls neural stem cell self-renewal through a Fos-centered gene regulatory network.

The Sox2 transcription factor is necessary for the long-term self-renewal of neural stem cells (NSCs). Its mechanism of action is still poorly defined. To identify molecules regulated by Sox2, and acting in mouse NSC maintenance, we transduced, into Sox2-deleted NSC, genes whose expression is strongly downregulated following Sox2 loss (Fos, Jun, Egr2), individually or in combination. Fos alone rescued long-term proliferation, as shown by in vitro cell growth and clonal analysis. Furthermore, pharmacological inhibition by T-5224 of FOS/JUN AP1 complex binding to its targets decreased cell proliferation and expression of the putative target Suppressor of cytokine signaling 3 (Socs3). Additionally, Fos requirement for efficient long-term proliferation was demonstrated by the reduction of NSC clones capable of long-term expansion following CRISPR/Cas9-mediated Fos inactivation. Previous work showed that the Socs3 gene is strongly downregulated following Sox2 deletion, and its re-expression by lentiviral transduction rescues long-term NSC proliferation. Fos appears to be an upstream regulator of Socs3, possibly together with Jun and Egr2; indeed, Sox2 re-expression in Sox2-deleted NSC progressively activates both Fos and Socs3 expression; in turn, Fos transduction activates Socs3 expression. Based on available SOX2 ChIPseq and ChIA-PET data, we propose a model whereby Sox2 is a direct activator of both Socs3 and Fos, as well as possibly Jun and Egr2; furthermore, we provide direct evidence for FOS and JUN binding on Socs3 promoter, suggesting direct transcriptional regulation. These results provide the basis for developing a model of a network of interactions, regulating critical effectors of NSC proliferation and long-term maintenance.

Sox2-Dependent 3D Chromatin Interactomes in Transcription, Neural Stem Cell Proliferation and Neurodevelopmental Diseases.

In our article, we asked whether Sox2, a transcription factor important in brain development and disease, is involved in gene regulation through its action on long-range interactions between promoters and distant enhancers. Our findings highlight that Sox2 shapes a genome-wide network of promoter-enhancer interactions, acting by direct binding to these elements. Sox2 loss affects the three-dimensional (3D) genome and decreases the activity of a subset of genes involved in Sox2-bound interactions. At least one of such downregulated genes, Socs3, is critical for long-term neural stem cell maintenance. These results point to the possibility of identifying a transcriptional network downstream to Sox2, and involved in neural stem cell maintenance. In addition, interacting Sox2-bound enhancers are often connected to genes which are relevant, in man, to neurodevelopmental disease; this may facilitate the detection of functionally relevant mutations in regulatory elements in man, contributing to neural disease.

Mapping the Global Chromatin Connectivity Network for Sox2 Function in Neural Stem Cell Maintenance.

The SOX2 transcription factor is critical for neural stem cell (NSC) maintenance and brain development. Through chromatin immunoprecipitation (ChIP) and chromatin interaction analysis (ChIA-PET), we determined genome-wide SOX2-bound regions and Pol II-mediated long-range chromatin interactions in brain-derived NSCs. SOX2-bound DNA was highly enriched in distal chromatin regions interacting with promoters and carrying epigenetic enhancer marks. Sox2 deletion caused widespread reduction of Pol II-mediated long-range interactions and decreased gene expression. Genes showing reduced expression in Sox2-deleted cells were significantly enriched in interactions between promoters and SOX2-bound distal enhancers. Expression of one such gene, Suppressor of Cytokine Signaling 3 (Socs3), rescued the self-renewal defect of Sox2-ablated NSCs. Our work identifies SOX2 as a major regulator of gene expression through connections to the enhancer network in NSCs. Through the definition of such a connectivity network, our study shows the way to the identification of genes and enhancers involved in NSC maintenance and neurodevelopmental disorders.

The calcium-dependent kinase OsCPK24 functions in cold stress responses in rice.

Calcium-dependent protein kinases (CPKs) are serine/threonine protein kinases that function in plant stress responses. Although CPKs are recognized as key messengers in signal transduction, the specific roles of CPKs and the molecular mechanisms underlying their activity remain largely unknown. Here, we characterized the function of OsCPK24, a cytosol-localized calcium-dependent protein kinase in rice. OsCPK24 was universally and highly expressed in rice plants and was induced by cold treatment. Whereas OsCPK24 knockdown plants exhibited increased sensitivity to cold compared to wild type (WT), OsCPK24-overexpressing plants exhibited increased cold tolerance. Plants overexpressing OsCPK24 exhibited increased accumulation of proline (an osmoprotectant) and glutathione (an antioxidant) and maintained a higher GSH/GSSG (reduced glutathione to oxidized glutathione) ratio during cold stress compared to WT. In addition to these effects in response to cold stress, we observed the kinase activity of OsCPK24 varied under different calcium concentrations. Further, OsCPK24 phosphorylated OsGrx10, a glutathione-dependent thioltransferase, at rates modulated by changes in calcium concentration. Together, our results support the hypothesis that OsCPK24 functions as a positive regulator of cold stress tolerance in rice, a process mediated by calcium signaling and involving phosphorylation and the inhibition of OsGrx10 to sustain higher glutathione levels.

Processes Underlying a Reproductive Barrier in indica-japonica Rice Hybrids Revealed by Transcriptome Analysis.

In rice (Oryza sativa), hybrids between indica and japonica subspecies are usually highly sterile, which provides a model system for studying postzygotic reproductive isolation. A killer-protector system, S5, composed of three adjacent genes (ORF3, ORF4, and ORF5), regulates female gamete fertility of indica-japonica hybrids. To characterize the processes underlying this system, we performed transcriptomic analyses of pistils from rice variety Balilla (BL), Balilla with transformed ORF5+ (BL5+) producing sterile female gametes, and Balilla with transformed ORF3+ and ORF5+ (BL3+5+) producing fertile gametes. RNA sequencing of tissues collected before (MMC), during (MEI), and after (AME) meiosis of the megaspore mother cell detected 19,269 to 20,928 genes as expressed. Comparison between BL5+ and BL showed that ORF5+ induced differential expression of 8,339, 6,278, and 530 genes at MMC, MEI, and AME, respectively. At MMC, large-scale differential expression of cell wall-modifying genes and biotic and abiotic response genes indicated that cell wall integrity damage induced severe biotic and abiotic stresses. The processes continued to MEI and induced endoplasmic reticulum (ER) stress as indicated by differential expression of ER stress-responsive genes, leading to programmed cell death at MEI and AME, resulting in abortive female gametes. In the BL3+5+/BL comparison, 3,986, 749, and 370 genes were differentially expressed at MMC, MEI, and AME, respectively. Large numbers of cell wall modification and biotic and abiotic response genes were also induced at MMC but largely suppressed at MEI without inducing ER stress and programed cell death , producing fertile gametes. These results have general implications for the understanding of biological processes underlying reproductive barriers.

Pathogenic Events in a Nonhuman Primate Model of Oral Poliovirus Infection Leading to Paralytic Poliomyelitis.

Despite a great deal of prior research, the early pathogenic events in natural oral poliovirus infection remain poorly defined. To establish a model for study, we infected 39 macaques by feeding them single high doses of the virulent Mahoney strain of wild type 1 poliovirus. Doses ranging from 107 to 109 50% tissue culture infective doses (TCID50) consistently infected all the animals, and many monkeys receiving 108 or 109 TCID50 developed paralysis. There was no apparent difference in the susceptibilities of the three macaque species (rhesus, cynomolgus, and bonnet) used. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia, and virus was isolated from tonsils, gut mucosa, and draining lymph nodes. Viral replication proteins were detected in both epithelial and lymphoid cell populations expressing CD155 in the tonsil and intestine, as well as in spinal cord neurons. Necrosis was observed in these three cell types, and viral replication in the tonsil/gut was associated with histopathologic destruction and inflammation. The sustained response of neutralizing antibody correlated temporally with resolution of viremia and termination of virus shedding in oropharynges and feces. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), extending previous studies of poliovirus pathogenesis in humans. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis and to assess the efficacy of candidate antiviral drugs and new vaccines.IMPORTANCE Early pathogenic events of poliovirus infection remain largely undefined, and there is a lack of animal models mimicking natural oral human infection leading to paralytic poliomyelitis. All 39 macaques fed with single high doses ranging from 107 to 109 TCID50 Mahoney type 1 virus were infected, and many of the monkeys developed paralysis. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia; tonsil, mesentery lymph nodes, and intestinal mucosa served as major target sites of viral replication. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), thereby supplementing historical reconstructions of poliovirus pathogenesis. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis, candidate antiviral drugs, and the efficacy of new vaccines.

Tuberculous pleurisy drives marked effector responses of γδ, CD4+, and CD8+ T cell subpopulations in humans.

Although tuberculous pleurisy (TP) presumably involves a hypersensitivity reaction, there is limited evidence indicating overreactive effector responses of γδ T cells and αß T cells and their interrelation with Foxp3(+) Tregs in pleural and other compartments. We found that TP induced reciprocal representations of Foxp3(+) Tregs and Mtb phosphoantigen-specific Vγ2Vδ2 T cells in different anatomic compartments. Patients with TP exhibited appreciable numbers of "proliferating" Ki-67(+) Vγ2Vδ2 T cells in the airway where Foxp3(+) Tregs were not dominant, whereas striking increases in Foxp3(+) Tregs in the blood and pleural compartments coincided with low frequencies of Vγ2Vδ2 T cells. Interestingly, anti-tuberculosis chemotherapy control of Mtb infection in patients with TP reversed reciprocal representations of Foxp3(+) Tregs and proliferating Vγ2Vδ2 T cells. Surprisingly, despite high-level Foxp3(+) Tregs, TP appeared to drive overreactive responses of IFN-γ-producing Vγ2Vδ2, CD4(+)CD25(+), and CD8(+)CD25(+) T effector subpopulations, whereas IL-22-producing Vγ2Vδ2 T cells increased subtly. Th1 effector responses were sustained despite remarkable declines in Foxp3(+) Tregs at 1 mo after the treatment. Overreactive T effector responses of Mtb-reactive γδ T cells, αß CD25(+)CD4(+), and CD25(+)CD8(+) T cell subpopulations appear to be immune features for TP. Increased Foxp3(+) Tregs might be responsive to overreactive TP but unable to influence T effector responses despite having an inverse relation with proliferating Vγ2Vδ2 T cells.