Single-cell transcriptomic signatures and gene regulatory networks modulated by Wls in mammalian midline facial formation and clefts.

Formation of highly unique and complex facial structures is controlled by genetic programs that are responsible for the precise coordination of three-dimensional tissue morphogenesis. However, the underlying mechanisms governing these processes remain poorly understood. We combined mouse genetic and genomic approaches to define the mechanisms underlying normal and defective midfacial morphogenesis. Conditional inactivation of the Wnt secretion protein Wls in Pax3-expressing lineage cells disrupted frontonasal primordial patterning, cell survival and directional outgrowth, resulting in altered facial structures, including midfacial hypoplasia and midline facial clefts. Single-cell RNA sequencing revealed unique transcriptomic atlases of mesenchymal subpopulations in the midfacial primordia, which are disrupted in the conditional Wls mutants. Differentially expressed genes and cis-regulatory sequence analyses uncovered that Wls modulates and integrates a core gene regulatory network, consisting of key midfacial regulatory transcription factors (including Msx1, Pax3 and Pax7) and their downstream targets (including Wnt, Shh, Tgfß and retinoic acid signaling components), in a mesenchymal subpopulation of the medial nasal prominences that is responsible for midline facial formation and fusion. These results reveal fundamental mechanisms underlying mammalian midfacial morphogenesis and related defects at single-cell resolution.

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion.

Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α-helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.

Non-neural surface ectodermal rosette formation and F-actin dynamics drive mammalian neural tube closure.

The mechanisms underlying mammalian neural tube closure remain poorly understood. We report a unique cellular process involving multicellular rosette formation, convergent cellular protrusions, and F-actin cable network of the non-neural surface ectodermal cells encircling the closure site of the posterior neuropore, which are demonstrated by scanning electron microscopy and genetic fate mapping analyses during mouse spinal neurulation. These unique cellular structures are severely disrupted in the surface ectodermal transcription factor Grhl3 mutants that exhibit fully penetrant spina bifida. We propose a novel model of mammalian neural tube closure driven by surface ectodermal dynamics, which is computationally visualized.

Chemically Modified Cpf1-CRISPR RNAs Mediate Efficient Genome Editing in Mammalian Cells.

CRISPR-based gene editing is a powerful technology for engineering mammalian genomes. It holds the potential as a therapeutic, although much-needed in vivo delivery systems have yet to be established. Here, using the Cpf1-crRNA (CRISPR RNA) crystal structure as a guide, we synthesized a series of systematically truncated and chemically modified crRNAs, and identify positions that are amenable to modification while retaining gene-editing activity. Modified crRNAs were designed with the same modifications that provide protection against nucleases and enable wide distribution in vivo. We show crRNAs with chemically modified terminal nucleotides are exonuclease resistant while retaining gene-editing activity. Chemically modified or DNA-substituted nucleotides at select positions and up to 70% of the crRNA DNA specificity region are also well tolerated. In addition, gene-editing activity is maintained with phosphorothioate backbone substitutions in the crRNA DNA specificity region. Finally, we demonstrate that 42-mer synthetic crRNAs from the similar CRISPR-Cas9 system are taken up by cells, an attractive property for in vivo delivery. Our study is the first to show that chemically modified crRNAs of the CRISPR-Cpf1 system can functionally replace and mediate comparable gene editing to the natural crRNA, which holds the potential for enhancing both viral- and non-viral-mediated in vivo gene editing.

Synthetic CRISPR RNA-Cas9-guided genome editing in human cells.

Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA-RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.

Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration.

HIV-1 reverse transcriptase discriminates poorly between dUTP and dTTP, and accordingly, viral DNA products become heavily uracilated when viruses infect host cells that contain high ratios of dUTP:dTTP. Uracilation of invading retroviral DNA is thought to be an innate immunity barrier to retroviral infection, but the mechanistic features of this immune pathway and the cellular fate of uracilated retroviral DNA products is not known. Here we developed a model system in which the cellular dUTP:dTTP ratio can be pharmacologically increased to favor dUTP incorporation, allowing dissection of this innate immunity pathway. When the virus-infected cells contained elevated dUTP levels, reverse transcription was found to proceed unperturbed, but integration and viral protein expression were largely blocked. Furthermore, successfully integrated proviruses lacked detectable uracil, suggesting that only nonuracilated viral DNA products were integration competent. Integration of the uracilated proviruses was restored using an isogenic cell line that had no detectable human uracil DNA glycosylase (hUNG2) activity, establishing that hUNG2 is a host restriction factor in cells that contain high dUTP. Biochemical studies in primary cells established that this immune pathway is not operative in CD4+ T cells, because these cells have high dUTPase activity (low dUTP), and only modest levels of hUNG activity. Although monocyte-derived macrophages have high dUTP levels, these cells have low hUNG activity, which may diminish the effectiveness of this restriction pathway. These findings establish the essential elements of this pathway and reconcile diverse observations in the literature.

Generation of an HIV resistant T-cell line by targeted "stacking" of restriction factors.

Restriction factors constitute a newly appreciated line of innate immune defense, blocking viral replication inside of infected cells. In contrast to these antiviral proteins, some cellular proteins, such as the CD4, CCR5, and CXCR4 cell surface receptors, facilitate HIV replication. We have used zinc finger nucleases (ZFNs) to insert a cocktail of anti-HIV restriction factors into the CCR5 locus in a T-cell reporter line, knocking out the CCR5 gene in the process. Mirroring the logic of highly active antiretroviral therapy, this strategy provides multiple parallel blocks to infection, dramatically limiting pathways for viral escape, without relying on random integration of transgenes into the genome. Because of the combination of blocks that this strategy creates, our modified T-cell lines are robustly resistant to both CCR5-tropic (R5-tropic) and CXCR4-tropic (X4-tropic) HIV-1. While zinc finger nuclease-mediated CCR5 disruption alone, which mimics the strategy being used in clinical trials, confers 16-fold protection against R5-tropic HIV, it has no effect against X4-tropic virus. Rhesus TRIM5α, chimeric human-rhesus TRIM5α, APOBEC3G D128K, or Rev M10 alone targeted to CCR5 confers significantly improved resistance to infection by both variants compared with CCR5 disruption alone. The combination of three factors targeted to CCR5 blocks infection at multiple stages, providing virtually complete protection against infection by R5-tropic and X4-tropic HIV.

Ezh2-dependent methylation in oral epithelia promotes secondary palatogenesis.

BACKGROUND: In addition to genomic risk variants and environmental influences, increasing evidence suggests epigenetic modifications are important for orofacial development and their alterations can contribute to orofacial clefts. Ezh2 encodes a core catalytic component of the Polycomb repressive complex responsible for addition of methyl marks to Histone H3 as a mechanism of repressing target genes. The role of Ezh2 in orofacial clefts remains unknown. AIMS: To investigate the epithelial role of Ezh2-dependent methylation in secondary palatogenesis. METHODS: We used conditional gene-targeting methods to ablate Ezh2 in the surface ectoderm-derived oral epithelium of mouse embryos. We then performed single-cell RNA sequencing combined with immunofluorescence and RT-qPCR to investigate gene expression in conditional mutant palate. We also employed double knockout analyses of Ezh1 and Ezh2 to address if they have synergistic roles in palatogenesis. RESULTS: We found that conditional inactivation of Ezh2 in oral epithelia results in partially penetrant cleft palate. Double knockout analyses revealed that another family member Ezh1 is dispensable in orofacial development, and it does not have synergistic roles with Ezh2 in palatogenesis. Histochemistry and single-cell RNA-seq analyses revealed dysregulation of cell cycle regulators in the palatal epithelia of Ezh2 mutant mouse embryos disrupts palatogenesis. CONCLUSION: Ezh2-dependent histone H3K27 methylation represses expression of cell cycle regulator Cdkn1a and promotes proliferation in the epithelium of the developing palatal shelves. Loss of this regulation may perturb movement of the palatal shelves, causing a delay in palate elevation which may result in failure of the secondary palate to close altogether.

Epigenetic implications in maternal diabetes and metabolic syndrome-associated risk of orofacial clefts.

Orofacial clefts (OFCs) are one of the most common types of structural birth defects. The etiologies are complicated, involving with genetic, epigenetic, and environmental factors. Studies have found that maternal diabetes and metabolic syndrome are associated with a higher risk of OFCs in offspring. Metabolic syndrome is a clustering of several disease risk factors, including hyperglycemia, dyslipidemia, obesity, and hypertension. Metabolic disease during pregnancy can increase risk of adverse outcomes and significantly influence fetal development, including orofacial formation and fusion. An altered metabolic state may contribute to developmental disorders or congenital defects including OFCs, potentially through epigenetic modulations, such as histone modification, DNA methylation, and noncoding RNA expression to alter activities of critical morphogenetic signaling or related developmental genes. This review summarizes the currently available evidence and underlying mechanisms of how the maternal metabolic syndrome is associated with OFCs in mostly human and some animal studies. It may provide a better understanding of the interactions between intrauterine metabolic status and fetal orofacial development which might be applied toward prevention and treatments of OFCs.

GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells.

Antisense oligonucleotides (ASOs) are short synthetic nucleic acids that recognize and bind to complementary RNA to modulate gene expression. It is well established that single-stranded, phosphorothioate-modified ASOs enter cells independent of carrier molecules, primarily via endocytic pathways, but that only a small portion of internalized ASO is released into the cytosol and/or nucleus, rendering the majority of ASO inaccessible to the targeted RNA. Identifying pathways that can increase the available ASO pool is valuable as a research tool and therapeutically. Here, we conducted a functional genomic screen for ASO activity by engineering GFP splice reporter cells and applying genome-wide CRISPR gene activation. The screen can identify factors that enhance ASO splice modulation activity. Characterization of hit genes uncovered GOLGA8, a largely uncharacterized protein, as a novel positive regulator enhancing ASO activity by ∼2-fold. Bulk ASO uptake is 2- to 5-fold higher in GOLGA8-overexpressing cells where GOLGA8 and ASOs are observed in the same intracellular compartments. We find GOLGA8 is highly localized to the trans-Golgi and readily detectable at the plasma membrane. Interestingly, overexpression of GOLGA8 increased activity for both splice modulation and RNase H1-dependent ASOs. Taken together, these results support a novel role for GOLGA8 in productive ASO uptake.

Consistent inhibition of HIV-1 replication in CD4+ T cells by acyclovir without detection of human herpesviruses.

Acyclovir, a nucleoside analog, is thought to be specific for the human herpesviruses because it requires a virally encoded enzyme to phosphorylate it to acyclovir monophosphate. Recently, acyclovir triphosphate was shown to be a direct inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Here, we showed that acyclovir is an inhibitor of HIV-1 replication in CD4(+) T cells from cord blood that have undetectable levels of the eight human herpesviruses. Additionally, acyclovir phosphates were detected by reverse-phase-high performance liquid chromatography (RP-HPLC) and quantified in a primer extension assay from cord blood. The data support acyclovir as an inhibitor of HIV-1 replication in herpesvirus-negative cells.

The role of Lrp6-mediated Wnt/ß-catenin signaling in the development and intervention of spinal neural tube defects in mice.

Neural tube defects (NTDs) are among the common and severe birth defects with poorly understood etiology. Mutations in the Wnt co-receptor LRP6 are associated with NTDs in humans. Either gain-of-function (GOF) or loss-of-function (LOF) mutations of Lrp6 can cause NTDs in mice. NTDs in Lrp6-GOF mutants may be attributed to altered ß-catenin-independent noncanonical Wnt signaling. However, the mechanisms underlying NTDs in Lrp6-LOF mutants and the role of Lrp6-mediated canonical Wnt/ß-catenin signaling in neural tube closure remain unresolved. We previously demonstrated that ß-catenin signaling is required for posterior neuropore (PNP) closure. In the current study, conditional ablation of Lrp6 in dorsal PNP caused spinal NTDs with diminished activities of Wnt/ß-catenin signaling and its downstream target gene Pax3, which is required for PNP closure. ß-catenin-GOF rescued NTDs in Lrp6-LOF mutants. Moreover, maternal supplementation of a Wnt/ß-catenin signaling agonist reduced the frequency and severity of spinal NTDs in Lrp6-LOF mutants by restoring Pax3 expression. Together, these results demonstrate the essential role of Lrp6-mediated Wnt/ß-catenin signaling in PNP closure, which could also provide a therapeutic target for NTD intervention through manipulation of canonical Wnt/ß-catenin signaling activities.

Local sequence targeting in the AID/APOBEC family differentially impacts retroviral restriction and antibody diversification.

Nucleic acid cytidine deaminases of the activation-induced deaminase (AID)/APOBEC family are critical players in active and innate immune responses, playing roles as target-directed, purposeful mutators. AID specifically deaminates the host immunoglobulin (Ig) locus to evolve antibody specificity, whereas its close relative, APOBEC3G (A3G), lethally mutates the genomes of retroviral pathogens such as HIV. Understanding the basis for the target-specific action of these enzymes is essential, as mistargeting poses significant risks, potentially promoting oncogenesis (AID) or fostering drug resistance (A3G). AID prefers to deaminate cytosine in WRC (W = A/T, R = A/G) motifs, whereas A3G favors deamination of CCC motifs. This specificity is largely dictated by a single, divergent protein loop in the enzyme family that recognizes the DNA sequence. Through grafting of this substrate-recognition loop, we have created enzyme variants of A3G and AID with altered local targeting to directly evaluate the role of sequence specificity on immune function. We find that grafted loops placed in the A3G scaffold all produced efficient restriction of HIV but that foreign loops in the AID scaffold compromised hypermutation and class switch recombination. Local targeting, therefore, appears alterable for innate defense against retroviruses by A3G but important for adaptive antibody maturation catalyzed by AID. Notably, AID targeting within the Ig locus is proportionally correlated to its in vitro ability to target WRC sequences rather than non-WRC sequences. Although other mechanisms may also contribute, our results suggest that local sequence targeting by AID/APOBEC3 enzymes represents an elegant example of co-evolution of enzyme specificity with its target DNA sequence.

Effect of acyclovir on HIV-1 set point among herpes simplex virus type 2-seropositive persons during early HIV-1 infection.

We evaluated whether acyclovir suppression during human immunodeficiency virus type 1 (HIV-1) acquisition reduces HIV-1 set point, increases CD4 cell counts, and selects reverse-transcriptase mutations among 76 HIV-1 seroconverters identified in a placebo-controlled trial of twice-daily acyclovir (400 mg) for the prevention of HIV acquisition in herpes simplex virus type 2 (HSV-2)-seropositive persons (HIV Prevention Trials Network study 039). We found no significant difference in plasma HIV-1 RNA levels (P =.30) or CD4 cell counts (P =.85) between the acyclovir and placebo recipients. V75I and other mutations in HIV-1 reverse transcriptase reported from in vitro acyclovir studies were not observed. In conclusion, acyclovir suppression during HIV-1 seroconversion and the subsequent 6 months does not affect HIV-1 set point.

Gene Disruption Using Chemically Modified CRISPR-Cpf1 RNA.

CRISPR-based gene editing in mammalian cells is a powerful research tool which has demonstrated efficient site-specific gene modifications and is showing promise as a therapeutic for patients with genetic diseases. Multiple different CRISPR systems have been identified, each with its own target DNA recognition sequence, expanding the editable mammalian genome. It is also now appreciated that chemically modified nucleic acids can substitute for unmodified nucleotides in guide RNAs, providing protection from exonuclease degradation and improving gene editing efficiency. CRISPR-Cpf1 unlike CRISPR-Cas9, has a substantially lower propensity for off-target genomic cleavage, making it a preferred gene editing system for many applications. Here we provide a detailed protocol for use of CRISPR-Cpf1 and chemically modified guide RNAs in cell lines, outlining the steps from designing guide RNAs to a target gene of interest, delivery and expression in cells, and analysis of gene editing events.

Expression of IL-20 Receptor Subunit ß Is Linked to EAE Neuropathology and CNS Neuroinflammation.

Considerable clinical evidence supports that increased blood-brain barrier (BBB) permeability is linked to immune extravasation of CNS parenchyma during neuroinflammation. Although BBB permeability and immune extravasation are known to be provoked by vascular endothelial growth factor-A (i.e., VEGF-A) and C-X-C motif chemokine ligand 12 (CXCL12), respectively, the mechanisms that link both processes are still elusive. The interleukin-20 (i.e., IL-20) cytokine signaling pathway was previously implicated in VEGF-mediated angiogenesis and is known to induce cellular response by way of signaling through IL-20 receptor subunit ß (i.e., IL-20RB). Dysregulated IL-20 signaling is implicated in many inflammatory pathologies, but it's contribution to neuroinflammation has yet to be reported. We hypothesize that the IL-20 cytokine, and the IL cytokine subfamily more broadly, play a key role in CNS neuroinflammation by signaling through IL-20RB, induce VEGF activity, and enhance both BBB-permeability and CXCL12-mediated immune extravasation. To address this hypothesis, we actively immunized IL-20RB-/- mice and wild-type mice to induce experimental autoimmune encephalomyelitis (EAE) and found that IL-20RB-/- mice showed amelioration of disease progression compared to wild-type mice. Similarly, we passively immunized IL-20RB-/- mice and wild-type mice with myelin-reactive Th1 cells from either IL-20RB-/- and wild-type genotype. Host IL-20RB-/- mice showed lesser disease progression than wild-type mice, regardless of the myelin-reactive Th1 cells genotype. Using multianalyte bead-based immunoassay and ELISA, we found distinctive changes in levels of pro-inflammatory cytokines between IL-20RB-/- mice and wild-type mice at peak of EAE. We also found detectable levels of all cytokines of the IL-20 subfamily within CNS tissues and specific alteration to IL-20 subfamily cytokines IL-19, IL-20, and IL-24, expression levels. Immunolabeling of CNS region-specific microvessels confirmed IL-20RB protein at the spinal cord microvasculature and upregulation during EAE. Microvessels isolated from macaques CNS tissues also expressed IL-20RB. Moreover, we identified the expression of all IL-20 receptor subunits: IL-22 receptor subunit α-1 (IL-22RA1), IL-20RB, and IL-20 receptor subunit α (IL-20RA) in human CNS microvessels. Notably, human cerebral microvasculature endothelial cells (HCMEC/D3) treated with IL-1ß showed augmented expression of the IL-20 receptor. Lastly, IL-20-treated HCMEC/D3 showed alterations on CXCL12 apicobasal polarity consistent with a neuroinflammatory status. This evidence suggests that IL-20 subfamily cytokines may signal at the BBB via IL-20RB, triggering neuroinflammation.

The HBV drug entecavir - effects on HIV-1 replication and resistance.

Entecavir, a drug approved by the Food and Drug Administration for the treatment of chronic hepatitis B virus (HBV) infection, is not believed to inhibit replication of human immunodeficiency virus type 1 (HIV-1) at clinically relevant doses. We observed that entecavir led to a consistent 1-log(10) decrease in HIV-1 RNA in three persons with HIV-1 and HBV coinfection, and we obtained supportive in vitro evidence that entecavir is a potent partial inhibitor of HIV-1 replication. Detailed analysis showed that in one of these patients, entecavir monotherapy led to an accumulation of HIV-1 variants with the lamivudine-resistant mutation, M184V. In vitro experiments showed that M184V confers resistance to entecavir. Until more is known about HIV-1-resistance patterns and their selection by entecavir, caution is needed with the use of entecavir in persons with HIV-1 and HBV coinfection who are not receiving fully suppressive antiretroviral regimens.

Cellular and developmental basis of orofacial clefts.

During craniofacial development, defective growth and fusion of the upper lip and/or palate can cause orofacial clefts (OFCs), which are among the most common structural birth defects in humans. The developmental basis of OFCs includes morphogenesis of the upper lip, primary palate, secondary palate, and other orofacial structures, each consisting of diverse cell types originating from all three germ layers: the ectoderm, mesoderm, and endoderm. Cranial neural crest cells and orofacial epithelial cells are two major cell types that interact with various cell lineages and play key roles in orofacial development. The cellular basis of OFCs involves defective execution in any one or several of the following processes: neural crest induction, epithelial-mesenchymal transition, migration, proliferation, differentiation, apoptosis, primary cilia formation and its signaling transduction, epithelial seam formation and disappearance, periderm formation and peeling, convergence and extrusion of palatal epithelial seam cells, cell adhesion, cytoskeleton dynamics, and extracellular matrix function. The latest cellular and developmental findings may provide a basis for better understanding of the underlying genetic, epigenetic, environmental, and molecular mechanisms of OFCs.

New approaches for quantitating the inhibition of HIV-1 replication by antiviral drugs in vitro and in vivo.

PURPOSE OF REVIEW: With highly active antiretroviral therapy, HIV-1 infection has become a manageable lifelong disease. Developing optimal treatment regimens requires understanding how to best measure anti-HIV activity in vitro and how drug dose-response curves generated in vitro correlate with in-vivo efficacy. RECENT FINDINGS: Several recent studies have indicated that conventional multiround infectivity assays are inferior to single cycle assays at both low and high levels of inhibition. Multiround infectivity assays can fail to detect subtle but clinically significant anti-HIV activity. The discoveries of the anti-HIV activity of the hepatitis B drug entecavir and the herpes simplex drug acyclovir were facilitated by single-round infectivity assays. Recent studies using a single-round infectivity assay have shown that a previously neglected parameter, the dose-response curve slope, is an extremely important determinant of antiviral activity. Some antiretroviral drugs have steep slopes that result in extraordinary levels of antiviral activity. The instantaneous inhibitory potential, the log reduction in infectivity in a single-round assay at clinical drug concentrations, has been proposed as a novel index for comparing antiviral activity. SUMMARY: Among in-vitro measures of antiviral activity, single-round infection assays have the advantage of measuring instantaneous inhibition by a drug. Re-evaluating the antiviral activity of approved HIV-1 drugs has shown that the slope parameter is an important factor in drug activity. Determining the instantaneous inhibitory potential by using a single-round infectivity assay may provide important insights that can predict the in-vivo efficacy of anti-HIV-1 drugs.