The pausing zone and control of RNA polymerase II elongation by Spt5: Implications for the pause-release model.

The pause-release model of transcription proposes that 40-100 bases from the start site RNA Pol II pauses, followed by release into productive elongation. Pause release is facilitated by the PTEFb phosphorylation of the RNA Pol II elongation factor, Spt5. We mapped paused polymerases by eNET-seq and found frequent pausing in zones that extend ∼0.3-3 kb into genes even when PTEFb is inhibited. The fraction of paused polymerases or pausing propensity declines gradually over several kb and not abruptly as predicted for a discrete pause-release event. Spt5 depletion extends pausing zones, suggesting that it promotes the maturation of elongation complexes to a low-pausing state. The expression of mutants after Spt5 depletion showed that phosphomimetic substitutions in the CTR1 domain diminished pausing throughout genes. By contrast, mutants that prevent the phosphorylation of the Spt5 RNA-binding domain strengthened pausing. Thus, distinct Spt5 phospho-isoforms set the balance between pausing and elongation.

Widespread Backtracking by RNA Pol II Is a Major Effector of Gene Activation, 5' Pause Release, Termination, and Transcription Elongation Rate.

In addition to phosphodiester bond formation, RNA polymerase II has an RNA endonuclease activity, stimulated by TFIIS, which rescues complexes that have arrested and backtracked. How TFIIS affects transcription under normal conditions is poorly understood. We identified backtracking sites in human cells using a dominant-negative TFIIS (TFIISDN) that inhibits RNA cleavage and stabilizes backtracked complexes. Backtracking is most frequent within 2 kb of start sites, consistent with slow elongation early in transcription, and in 3' flanking regions where termination is enhanced by TFIISDN, suggesting that backtracked pol II is a favorable substrate for termination. Rescue from backtracking by RNA cleavage also promotes escape from 5' pause sites, prevents premature termination of long transcripts, and enhances activation of stress-inducible genes. TFIISDN slowed elongation rates genome-wide by half, suggesting that rescue of backtracked pol II by TFIIS is a major stimulus of elongation under normal conditions.

Control of RNA Pol II Speed by PNUTS-PP1 and Spt5 Dephosphorylation Facilitates Termination by a "Sitting Duck Torpedo" Mechanism.

Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA polymerase II (Pol II) elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, Pol II decelerates from >2 kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a model for termination, the "sitting duck torpedo" mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert Pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homolog NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.

Dynamic turnover of paused Pol II complexes at human promoters.

Paused RNA polymerase II (Pol II) that piles up near most human promoters is the target of mechanisms that control entry into productive elongation. Whether paused Pol II is a stable or dynamic target remains unresolved. We report that most 5' paused Pol II throughout the genome is turned over within 2 min. This process is revealed under hypertonic conditions that prevent Pol II recruitment to promoters. This turnover requires cell viability but is not prevented by inhibiting transcription elongation, suggesting that it is mediated at the level of termination. When initiation was prevented by triptolide during recovery from high salt, a novel preinitiated state of Pol II lacking the pausing factor Spt5 accumulated at transcription start sites. We propose that Pol II occupancy near 5' ends is governed by a cycle of ongoing assembly of preinitiated complexes that transition to pause sites followed by eviction from the DNA template. This model suggests that mechanisms regulating the transition to productive elongation at pause sites operate on a dynamic population of Pol II that is turning over at rates far higher than previously suspected. We suggest that a plausible alternative to elongation control via escape from a stable pause is by escape from premature termination.

MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination.

Viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity. However, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, arthritogenic alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the draining lymph node and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease is more severe. In contrast to prior studies implicating a key role for lymph node macrophages in limiting viral dissemination, these findings exemplify a previously unrecognized arbovirus-scavenging role for lymphatic endothelial cells and improve our mechanistic understanding of viremia control during arthritogenic alphavirus infection.

RNA Pol II Dynamics Modulate Co-transcriptional Chromatin Modification, CTD Phosphorylation, and Transcriptional Direction.

Eukaryotic genes are marked by conserved post-translational modifications on the RNA pol II C-terminal domain (CTD) and the chromatin template. How the 5'-3' profiles of these marks are established is poorly understood. Using pol II mutants in human cells, we found that slow transcription repositioned specific co-transcriptionally deposited chromatin modifications; histone H3 lysine 36 trimethyl (H3K36me3) shifted within genes toward 5' ends, and histone H3 lysine 4 dimethyl (H3K4me2) extended farther upstream of start sites. Slow transcription also evoked a hyperphosphorylation of CTD Ser2 residues at 5' ends of genes that is conserved in yeast. We propose a "dwell time in the target zone" model to explain the effects of transcriptional dynamics on the establishment of co-transcriptionally deposited protein modifications. Promoter-proximal Ser2 phosphorylation is associated with a longer pol II dwell time at start sites and reduced transcriptional polarity because of strongly enhanced divergent antisense transcription at promoters. These results demonstrate that pol II dynamics help govern the decision between sense and divergent antisense transcription.

Precise gene models using long-read sequencing reveal a unique poly(A) signal in Giardia lamblia.

During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3' UTR of an mRNA, thus determining much of its post-transcriptional fate. Using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding Giardia lamblia cleavage sites for over 2600 genes. We find that G. lamblia uses an AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how G. lamblia lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes with evidence of alternative polyadenylation. These results suggest that despite pared-down cleavage and polyadenylation machinery, 3' end formation still appears to be an important regulatory step for gene expression in G. lamblia.

Accuracy and resolution of conventional and additively manufactured silicone elastomers as applied in maxillofacial therapies.

STATEMENT OF PROBLEM: Silicone elastomers are becoming more readily available for additive manufacturing, which may be advantageous for fabricating maxillofacial prostheses. However, the properties of three-dimensionally (3D) printed silicone as compared with conventionally processed silicone have not been well studied. PURPOSE: The purpose of this in vitro study was to compare the dimensional accuracy and surface resolution of additively manufactured with conventional room-temperature vulcanized (RTV) silicones. MATERIAL AND METHODS: A custom aluminum mold was used to generate hand-spatulated specimens (A103 and VerSilTal-50F, n=20). A computer-aided design and computer-aided manufacturing workflow was used to generate additively manufactured specimens (Sil30 and TrueSil, n=20). Digital surface scans of each specimen were recorded; a scan of the mold served as the control. Surface dimensions were measured with a digital metrology software program, while digital overlays were made using a 3D processing software program. The surface resolution of the specimens was assessed by analyzing 4 topographical landmarks (flat surfaces, raised lines, domes, and scribed lines) with a visual qualitative grading scale. The data were analyzed with 1-way analysis of variance, followed by a Student-Newman-Keuls post hoc test (α=.05). RESULTS: The specimens demonstrated statistical differences in trueness and precision (P<.001). The TrueSil specimens showed the largest deviation in measurements of trueness and precision (up to -1.374%). The other specimens yielded percentage mean differences that were more consistently within the range of the American Dental Association International Organization for Standardization standard for elastomers. The manually fabricated specimens yielded more consistently ideal ratings for resolution than did the additively manufactured ones, with the Sil30 specimens receiving the most Charlie (not clinically acceptable) ratings. CONCLUSIONS: Numerical differences between each specimen and the control were considered negligible for maxillofacial applications. Notable discrepancies related to the quality of resolution, wherein the benchtop-manufactured specimens consistently generated better results compared with additively manufactured ones. Other factors, such as resiliency, odor, and cost, posed limitations in justifying the use of silicones in a direct-to-print workflow.

Effects of Transcription Elongation Rate and Xrn2 Exonuclease Activity on RNA Polymerase II Termination Suggest Widespread Kinetic Competition.

The torpedo model of transcription termination asserts that the exonuclease Xrn2 attacks the 5'PO4-end exposed by nascent RNA cleavage and chases down the RNA polymerase. We tested this mechanism using a dominant-negative human Xrn2 mutant and found that it delayed termination genome-wide. Xrn2 nuclease inactivation caused strong termination defects downstream of most poly(A) sites and modest delays at some histone and U snRNA genes, suggesting that the torpedo mechanism is not limited to poly(A) site-dependent termination. A central untested feature of the torpedo model is that there is kinetic competition between the exonuclease and the pol II elongation complex. Using pol II rate mutants, we found that slow transcription robustly shifts termination upstream, and fast elongation extends the zone of termination further downstream. These results suggest that kinetic competition between elongating pol II and the Xrn2 exonuclease is integral to termination of transcription on most human genes.

Resident mesenchymal vascular progenitors modulate adaptive angiogenesis and pulmonary remodeling via regulation of canonical Wnt signaling.

Adaptive angiogenesis is necessary for tissue repair, however, it may also be associated with the exacerbation of injury and development of chronic disease. In these studies, we demonstrate that lung mesenchymal vascular progenitor cells (MVPC) modulate adaptive angiogenesis via lineage trace, depletion of MVPC, and modulation of ß-catenin expression. Single cell sequencing confirmed MVPC as multipotential vascular progenitors, thus, genetic depletion resulted in alveolar simplification with reduced adaptive angiogenesis. Following vascular endothelial injury, Wnt activation in MVPC was sufficient to elicit an emphysema-like phenotype characterized by increased MLI, fibrosis, and MVPC driven adaptive angiogenesis. Lastly, activation of Wnt/ß-catenin signaling skewed the profile of human and murine MVPC toward an adaptive phenotype. These data suggest that lung MVPC drive angiogenesis in response to injury and regulate the microvascular niche as well as subsequent distal lung tissue architecture via Wnt signaling.

Technique to Fabricate Custom Nasal Pillows and Monoblock Mandibular Advancement Device Using A Digital Workflow for Combination Therapy of Obstructive Sleep Apnea.

Combination therapy using continuous positive airway pressure and oral appliance therapy is an effective non-surgical treatment for obstructive sleep apnea. However, the laboratory expense and additional chairside time prevent it from being a preferred option. This article describes a technique for fabricating custom nasal pillows and monoblock mandibular advancement device with potential lower cost and accelerated timeline using a digital workflow.

Effect of Toothbrushing on Surface Color of Ceramic-polymer Materials: An In Vitro Study.

AIM AND OBJECTIVE: The purpose of this study was to examine the effects of toothbrushing on the change in color of extrinsic characterization of ceramic-polymer materials. MATERIALS AND METHODS: Two ceramic-polymer materials (CeraSmart, GC; Enamic, VITA) and one lithium-disilicate material (IPS e.max CAD; Ivoclar Vivadent) were tested. Specimens of each material were prepared, characterized, and glazed per manufacturer's instructions. The treated surface of the blocks were then brushed in a toothpaste slurry with artificial saliva using a toothbrush machine with a soft toothbrush. Commission Internationale de L'Eclairage (CIE) L*a*b* values were recorded with a spectrophotometer at baseline and at 3, 6, 9, and 12 simulated years of brushing (7,300 strokes/year). A mean change in color (ΔE*) and standard deviation was determined for each group and brushing interval. Data were analyzed with a two-way repeated measures ANOVA examining the effects of toothbrushing the ceramic materials on ΔE* over time (α = 0.05). RESULTS: The difference in the ΔE* between CeraSmart and Enamic was significant at 3 years, while the differences between them were not significant at 6, 9, and 12 years of simulated brushing. The ΔE* of IPS e.max CAD was significantly lower than CeraSmart and Enamic at all time points (all p < 0.0001) except for the comparison with Enamic at 3 years. CONCLUSION: The extrinsic stains on the ceramic-polymer materials may be more susceptible to change from simulated toothbrushing compared to the lithium-disilicate material. CLINICAL SIGNIFICANCE: Toothbrushing may change the color of extrinsic characterization of ceramic-polymer materials. However, the change may remain clinically imperceptible to the naked eye (ΔE* > 1.0) for nearly 6 equivalent years of brushing.

An Investigation of the Effect of Modifying and Reusing Impression Copings on Transfer of Implant Analog Position and Angulation.

AIM: The aim of this research was to determine whether sterilization and reutilization of impression copings had an impact on the accuracy of casts made for multiimplant restorations. MATERIALS AND METHODS: Four master casts embedded with five implant analogs were fabricated. Polyvinyl siloxane (PVS) impressions of the master cast with copings attached to the analogs were made and poured in dental stone. The impression copings were subjected to cleaning and sterilization. These processes were repeated 30 cycles for each of the two groups of five impression copings: one without modification and one with modification that included air abrasion and PVS adhesive. A coordinate measuring machine (CMM) was used to measure relative angles and distances between the reference analog and analogs. The relative angles and distances measured on the stone casts were compared to the master resin cast to obtain positional and angular displacements. RESULTS: For impression copings that were not modified, a significant difference was detected for both positional and angular displacements. For impression copings that were modified, a significant change was observed only for positional displacement. The maximum discrepancies measured for positional and angular displacements after 30 cycles of reuse were only 81 µm and 0.46°, respectively, regardless of the modification. CONCLUSION: Within the limitations of this study, unmodified impression copings that have undergone 30 cycles of cleaning and sterilization appeared to incur more impression inaccuracy than those impression copings that were modified by airborne-particle abrasion and PVS adhesive. CLINICAL SIGNIFICANCE: Impression copings used in this study can likely be recycled up to 30 times without reducing the accuracy of the impression to a level that may be considered clinically significant.

Assessing the Accuracy of Casting and Additive Manufacturing Techniques for Fabrication of a Complete Palatal Coverage Metal Framework.

PURPOSE: To provide information regarding the accuracy of additive manufacturing in comparison to conventional casting, specifically for fabrication of complete palatal coverage metal frameworks. MATERIALS AND METHODS: Three additive manufacturing techniques were tested: selective laser melting (SLM), electron beam melting (EBM), and computer-aided design/cast (CADcast), with conventional casting as the control. Both the SLM and EBM groups were tested pre- and post-finishing, for a total of six test groups (n = 10/group). A digital master design was used as the standard to which all frameworks were digitally compared by best-fit analysis, which generated root mean square values using proprietary software. A one-way ANOVA was conducted to test for statistical differences among materials, followed by a post-hoc multiple comparison test (Tukey's test HSD). Surface roughness for one framework arbitrarily selected from each group was analyzed using a profilometer. RESULTS: There was a significant difference in accuracy among the materials (F = 99.79, p < 0.0001). A post-hoc Tukey test indicated that CADcast differed significantly from the other five materials (i.e., most accurate, p < 0.01). EBM prefinished and EBM finished were both significantly different from the other materials (i.e., least accurate). Color mapping images help visualize the differences between each framework compared to the master design. The surface roughness values ranged from 22 to 63.5 µm, with CADcast being the smoothest, and EBM prefinished the roughest. CONCLUSIONS: CADcast and SLM techniques were as or more accurate than the conventional technique for producing an uncomplicated framework design. Further investigation is recommended regarding the surface roughness of additive manufacturing products and potential biological complications.

Effect of Split-File Digital Workflow on Crown Margin Adaptation.

PURPOSE: Computer-aided design/computer-aided manufacturing (CAD/CAM) is becoming increasingly integrated into dental practice workflow at a pace that exceeds scientific validation. The aim of this study is to evaluate a complete digital split-file protocol relative to segmental digital and analog techniques for restoring a single maxillary anterior edentulous space with custom abutment and crown. MATERIALS AND METHODS: Four treatment workflows were assessed: complete digital (CD), segmental digital (SD), milled wax (AM), and heat pressed and hand waxed (AH) and heat pressed. The CD workflow "split" an abutment and crown into separate files to fabricate a zirconia abutment and both zirconia/lithium disilicate crown restorations. The SD workflow scanned the existing abutment for design of segmental restorations in zirconia, lithium disilicate, and milled wax (AM). The AH specimens were conventionally hand waxed. Both the AM and AH specimens were heat pressed with lithium disilicate. All restorations were evaluated with standardized measurements using scanning electron microscopy (SEM) as manufactured without internal adjustments and after manual adjustment. The number of adjustments, adjustment time, and location of adjustments were recorded. One-way ANOVA with repeated measures was used to report geometric means with 95% confidence intervals. RESULTS: The mean marginal gap after adjustment of the CD group was 69 µm, with an upper bound (UB) of 79 µm and a lower bound (LB) of 60 µm. SD group mean was 26 µm with an UB of 31 µm and LB of 22 µm. The AM group mean was 32 µm, with an UB of 49 µm and a LB of 20 µm; AH group mean of 26 µm with an UB of 34 µm and a LB of 20 µm. The SD, AM, and AH workflows were statistically similar (p = 1.000), and the CD workflow was statistically greater than the other three (p < 0.001). CONCLUSIONS: The split-file (CD) protocol results in marginal gap size within clinical standards after adjustment; however, 52 of the 60 digitally produced restorations showed a horizontal marginal offset that required adjustment for proper contours.

Analysis of TFAP2A mutations in Branchio-Oculo-Facial Syndrome indicates functional complexity within the AP-2α DNA-binding domain.

Multiple lines of evidence indicate that the AP-2 transcription factor family has an important regulatory function in human craniofacial development. Notably, mutations in TFAP2A, the gene encoding AP-2α, have been identified in patients with Branchio-Oculo-Facial Syndrome (BOFS). BOFS is an autosomal-dominant trait that commonly presents with facial clefting, eye defects and branchial skin anomalies. Examination of multiple cases has suggested either simple haploinsufficiency or more complex genetic causes for BOFS, especially as the clinical manifestations are variable, with no clear genotype-phenotype correlation. Mutations occur throughout TFAP2A, but mostly within conserved sequences within the DNA contact domain of AP-2α. However, the consequences of the various mutations for AP-2α protein function have not been evaluated. Therefore, it remains unclear if all BOFS mutations result in similar changes to the AP-2α protein or if they each produce specific alterations that underlie the spectrum of phenotypes. Here, we have investigated the molecular consequences of the mutations that localize to the DNA-binding region. We show that although individual mutations have different effects on DNA binding, they all demonstrate significantly reduced transcriptional activities. Moreover, all mutant derivatives have an altered nuclear:cytoplasmic distribution compared with the predominantly nuclear localization of wild-type AP-2α and several can exert a dominant-negative activity on the wild-type AP-2α protein. Overall, our data suggest that the individual TFAP2A BOFS mutations can generate null, hypomorphic or antimorphic alleles and that these differences in activity, combined with a role for AP-2α in epigenetic events, may influence the resultant pathology and the phenotypic variability.

Regulation of V-nitrogenase genes in Anabaena variabilis by RNA processing and by dual repressors.

Anabaena variabilis ATCC 29413 fixes nitrogen in specialized cells called heterocysts using either a Mo-nitrogenase or a V-nitrogenase. V-nitrogenase structural genes, vnfDGK, as well as vnfEN form an operon with ava4025, located upstream of vnfDG that is repressed by fixed nitrogen and by Mo. The ava4025-vnfDGKEN operon is under the control of a Mo-repressible promoter located nearly 600 bp upstream of ava4025. Levels of vnfDG transcript were about 500-fold higher than ava4025, the first gene of the operon. This may be the result of RNA processing at a site 87 bp upstream of vnfDG that was initially identified as the transcription start site. A strain with a deletion in the coding region of ava4025 grew diazotrophically with Mo or with V. Two similar proteins, VnfR1 and VnfR2, whose genes are located some distance from the ava4025-vnfDGKEN operon, each repressed transcription from the ava4025-vnfDGKEN promoter and a mutant lacking both VnfR1 and VnfR2 made the V-nitrogenase in the presence of Mo. Overexpression of the V-nitrogenase in the double vnfR1 vnfR2 mutant resulted in decreased activity of the Mo-nitrogenase. VnfR1 bound specifically, in vitro, to a region upstream of the ava4025 promoter.

A specific and portable gene expression program underlies antigen archiving by lymphatic endothelial cells.

Antigens from protein subunit vaccination traffic from the tissue to the draining lymph node, either passively via the lymph or carried by dendritic cells at the local injection site. Lymph node (LN) lymphatic endothelial cells (LEC) actively acquire and archive foreign antigens, and archived antigen can be released during subsequent inflammatory stimulus to improve immune responses. Here, we answer questions about how LECs achieve durable antigen archiving and whether there are transcriptional signatures associated with LECs containing high levels of antigen. We used single cell sequencing in dissociated LN tissue to quantify antigen levels in LEC and dendritic cell populations at multiple timepoints after immunization, and used machine learning to define a unique transcriptional program within archiving LECs that can predict LEC archiving capacity in independent data sets. Finally, we validated this modeling, showing we could predict antigen archiving from a transcriptional dataset of CHIKV infected mice and demonstrated in vivo the accuracy of our prediction. Collectively, our findings establish a unique transcriptional program in LECs that promotes antigen archiving that can be translated to other systems.

Chikungunya virus infection disrupts lymph node lymphatic endothelial cell composition and function via MARCO.

Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we found that, during the first 24 hours of infection, CHIKV RNA accumulated in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response - including recruitment of myeloid cells to the LN - was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.

Chikungunya virus infection disrupts lymph node lymphatic endothelial cell composition and function via MARCO.

Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we find that during the first 24 h of infection, CHIKV RNA accumulates in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response, including recruitment of myeloid cells to the LN, was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, we find that antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.