A nuclease domain fused to the Snf2 helicase confers antiphage defence in coral-associated Halomonas meridiana.

The coral reef microbiome plays a vital role in the health and resilience of reefs. Previous studies have examined phage therapy for coral pathogens and for modifying the coral reef microbiome, but defence systems against coral-associated bacteria have received limited attention. Phage defence systems play a crucial role in helping bacteria fight phage infections. In this study, we characterized a new defence system, Hma (HmaA-HmaB-HmaC), in the coral-associated Halomonas meridiana derived from the scleractinian coral Galaxea fascicularis. The Swi2/Snf2 helicase HmaA with a C-terminal nuclease domain exhibits antiviral activity against Escherichia phage T4. Mutation analysis revealed the nickase activity of the nuclease domain (belonging to PDD/EXK superfamily) of HmaA is essential in phage defence. Additionally, HmaA homologues are present in ~1000 bacterial and archaeal genomes. The high frequency of HmaA helicase in Halomonas strains indicates the widespread presence of these phage defence systems, while the insertion of defence genes in the hma region confirms the existence of a defence gene insertion hotspot. These findings offer insights into the diversity of phage defence systems in coral-associated bacteria and these diverse defence systems can be further applied into designing probiotics with high-phage resistance.

The caspase-activated DNase promotes cellular senescence.

Cellular senescence is a response to many stressful insults. DNA damage is a consistent feature of senescent cells, but in many cases its source remains unknown. Here, we identify the cellular endonuclease caspase-activated DNase (CAD) as a critical factor in the initiation of senescence. During apoptosis, CAD is activated by caspases and cleaves the genomic DNA of the dying cell. The CAD DNase is also activated by sub-lethal signals in the apoptotic pathway, causing DNA damage in the absence of cell death. We show that sub-lethal signals in the mitochondrial apoptotic pathway induce CAD-dependent senescence. Inducers of cellular senescence, such as oncogenic RAS, type-I interferon, and doxorubicin treatment, also depend on CAD presence for senescence induction. By directly activating CAD experimentally, we demonstrate that its activity is sufficient to induce senescence in human cells. We further investigate the contribution of CAD to senescence in vivo and find substantially reduced signs of senescence in organs of ageing CAD-deficient mice. Our results show that CAD-induced DNA damage in response to various stimuli is an essential contributor to cellular senescence.

The Caspase-Activated DNase drives inflammation and contributes to defense against viral infection.

Mitochondria react to infection with sub-lethal signals in the apoptosis pathway. Mitochondrial signals can be inflammatory but mechanisms are only partially understood. We show that activation of the caspase-activated DNase (CAD) mediates mitochondrial pro-inflammatory functions and substantially contributes to host defense against viral infection. In cells lacking CAD, the pro-inflammatory activity of sub-lethal signals was reduced. Experimental activation of CAD caused transient DNA-damage and a pronounced DNA damage response, involving major kinase signaling pathways, NF-κB and cGAS/STING, driving the production of interferon, cytokines/chemokines and attracting neutrophils. The transcriptional response to CAD-activation was reminiscent of the reaction to microbial infection. CAD-deficient cells had a diminished response to viral infection. Influenza virus infected CAD-deficient mice displayed reduced inflammation in lung tissue, higher viral titers and increased weight loss. Thus, CAD links the mitochondrial apoptosis system and cell death caspases to host defense. CAD-driven DNA damage is a physiological element of the inflammatory response to infection.

Corrected and republished from: "Vibrio fischeri Possesses Xds and Dns Nucleases That Differentially Influence Phosphate Scavenging, Aggregation, Competence, and Symbiotic Colonization of Squid".

Cells of Vibrio fischeri colonize the light organ of Euprymna scolopes, providing the squid bioluminescence in exchange for nutrients and protection. The bacteria encounter DNA-rich mucus throughout their transition to a symbiotic lifestyle, leading us to hypothesize a role for nuclease activity in the colonization process. In support of this, we detected abundant extracellular nuclease activity in growing cells of V. fischeri. To discover the gene(s) responsible for this activity, we screened a V. fischeri transposon mutant library for nuclease-deficient strains. Interestingly, only one strain, whose transposon insertion mapped to nuclease gene VF_1451, showed a complete loss of nuclease activity in our screens. A database search revealed that VF_1451 is homologous to the nuclease-encoding gene xds in Vibrio cholerae. However, V. fischeri strains lacking xds eventually revealed slight nuclease activity on plates upon prolonged incubation. This led us to hypothesize that a second secreted nuclease, identified through a database search as VF_0437, a homolog of V. cholerae dns, might be responsible for the residual nuclease activity. Here, we show that Xds and/or Dns are involved in essential aspects of V. fischeri biology, including natural transformation, aggregation, and phosphate scavenging. Furthermore, strains lacking either nuclease were outcompeted by the wild type for squid colonization. Understanding the specific role of nuclease activity in the squid colonization process represents an intriguing area of future research.IMPORTANCEFrom soil and water to host-associated secretions such as mucus, environments that bacteria inhabit are awash in DNA. Extracellular DNA (eDNA) is a nutritious resource that microbes dedicate significant energy to exploit. Calcium binds eDNA to promote cell-cell aggregation and horizontal gene transfer. eDNA hydrolysis impacts the construction of and dispersal from biofilms. Strategies in which pathogens use nucleases to avoid phagocytosis or disseminate by degrading host secretions are well-documented; significantly less is known about nucleases in mutualistic associations. This study describes the role of nucleases in the mutualism between Vibrio fischeri and its squid host Euprymna scolopes. We find that nuclease activity is an important determinant of colonization in V. fischeri, broadening our understanding of how microbes establish and maintain beneficial associations.

T6SS nuclease effectors in Pseudomonas syringae act as potent antimicrobials in interbacterial competition.

Type VI secretion system (T6SS) is a potent weapon employed by various Pseudomonas species to compete with neighboring microorganisms for limited nutrients and ecological niches. However, the involvement of T6SS effectors in interbacterial competition within the phytopathogen Pseudomonas syringae remains unknown. In this study, we examined two T6SS clusters in a wild-type P. syringae MB03 and verified the involvement of one cluster, namely, T6SS-1, in interbacterial competition. Additionally, our results showed that two T6SS DNase effectors, specifically Tde1 and Tde4, effectively outcompeted antagonistic bacteria, with Tde4 playing a prominent role. Furthermore, we found several cognate immunity proteins, including Tde1ia, Tde1ib, and Tde4i, which are located in the downstream loci of their corresponding effector protein genes and worked synergistically to protect MB03 cells from self-intoxication. Moreover, expression of either Tde1 or C-terminus of Tde4 in Escherichia coli cells induced DNA degradation and changes in cell morphology. Thus, our results provide new insights into the role of the T6SS effectors of P. syringae in the interbacterial competition in the natural environment. IMPORTANCE: The phytopathogen Pseudomonas syringae employs an active type VI secretion system (T6SS) to outcompete other microorganisms in the natural environment, particularly during the epiphytic growth in the phyllosphere. By examining two T6SS clusters in P. syringae MB03, T6SS-1 is found to be effective in killing Escherichia coli cells. We highlight the excellent antibacterial effect of two T6SS DNase effectors, namely, Tde1 and Tde4. Both of them function as nuclease effectors, leading to DNA degradation and cell filamentation in prey cells, ultimately resulting in cell death. Our findings deepen our understanding of the T6SS effector repertoires used in P. syringae and will facilitate the development of effective antibacterial strategies.

DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity.

Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR-Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems.

Effect of a 12-Week Walking Program Monitored by Global Physical Capacity Score (GPCS) on Circulating Cell-Free mtDNA and DNase Activity in Patients with Irritable Bowel Syndrome.

Irritable bowel syndrome (IBS) involves low-grade mucosal inflammation. Among the various approaches capable of managing the symptoms, physical activity is still under investigation. Despite its benefits, it promotes oxidative stress and inflammation. Mitochondria impacts gut disorders by releasing damage-associated molecular patterns, such as cell-free mtDNA (cf-mtDNA), which support inflammation. This study evaluated the effects of a 12-week walking program on the cf-mtDNA and DNase in 26 IBS and 17 non-IBS subjects. Pro- and anti-inflammatory cytokines were evaluated by ELISA. Digital droplet PCR was used to quantify cf-mtDNA; DNase activity was assessed using a single radial enzyme diffusion assay. PCR-RFLP was used to genotype DNASE1 rs1053874 SNP. Significantly lower IL-10 levels were found in IBS than in non-IBS individuals. Exercise reduced cf-mtDNA in non-IBS subjects but not in IBS patients. DNase activity did not correlate with the cf-mtDNA levels in IBS patients post-exercise, indicating imbalanced cf-mtDNA clearance. Different rs1053874 SNP frequencies were not found between groups. The study confirms the positive effects of regular moderate-intensity physical activity in healthy subjects and its role in cf-mtDNA release and clearance. Walking alone might not sufficiently reduce subclinical inflammation in IBS, based on imbalanced pro- and anti-inflammatory molecules. Prolonged programs are necessary to investigate their effects on inflammatory markers in IBS.

Identification and characterization of TatD DNase in planarian Dugesia japonica and its antibiofilm effect.

TatD DNase, a key enzyme in vertebrates and invertebrates, plays a pivotal role in various physiological processes. Dugesia japonica (D. japonica), a flatworm species, has remarkable regenerative capabilities and possesses a simplified immune system. However, the existence and biological functions of TatD DNase in D. japonica require further investigation. Here, we obtained the open reading frame (ORF) of DjTatD and demonstrated its conservation. The three-dimensional structure of DjTatD revealed its active site and binding mechanism. To investigate its enzymological properties, we overexpressed, purified, and characterized recombinant DjTatD (rDjTatD). We observed that DjTatD was primarily expressed in the pharynx and its expression could be significantly challenged upon stimulation with lipopolysaccharide, peptidoglycan, gram-positive and gram-negative bacteria. RNA interference results indicated that both DjTatD and DjDN2s play a role in pharyngeal regeneration and may serve as functional complements to each other. Additionally, we found that rDjTatD and recombinant T7DjTatD effectively reduce biofilm formation regardless of their bacterial origin. Together, our results demonstrated that DjTatD may be involved in the planarian immune response and pharyngeal regeneration. Furthermore, after further optimization in the future, rDjTatD and T7DjTatD can be considered highly effective antibiofilm agents.

Screening and identification of emodin as an EBV DNase inhibitor to prevent its biological functions.

BACKGROUND: The Epstein-Barr virus (EBV) is a prevalent oncovirus associated with a variety of human illnesses. BGLF5, an EBV DNase with alkaline nuclease (AN) activity, plays important roles in the viral life cycle and progression of human malignancies and has been suggested as a possible diagnostic marker and target for cancer therapy. Methods used conventionally for the detection of AN activity, radioactivity-based nuclease activity assay and DNA digestion detection by gel electrophoresis, are not suitable for screening AN inhibitors; the former approach is unsafe, and the latter is complicated. In the present study, a fluorescence-based nuclease activity assay was used to screen several natural compounds and identify an EBV DNase inhibitor. RESULTS: Fluorescence-based nuclease activity assays, in which the DNA substrate is labelled with PicoGreen dye, are cheaper, safer, and easier to perform. Herein, the results of the fluorescence-based nuclease activity assay were consistent with the results of the two conventional methods. In addition, the PicoGreen-labelling method was applied for the biochemical characterisation of viral nucleases. Using this approach, we explored EBV DNase inhibitors. After several rounds of screening, emodin, an anthraquinone derivative, was found to possess significant anti-EBV DNase activity. We verified the efficacy of emodin using the conventional DNA-cleavage assay. Furthermore, using comet assay and micronucleus formation detection, we confirmed that emodin can inhibit DNase-induced DNA damage and genomic instability. Additionally, emodin treatment inhibited EBV production. CONCLUSIONS: Using a PicoGreen-mediated nuclease activity assay, we successfully demonstrated that emodin has the potential to inhibit EBV DNase nuclease activity. Emodin also inhibits EBV DNase-related biological functions, suggesting that it is a potential inhibitor of EBV DNase.

Targeting NETs using dual-active DNase1 variants.

Background: Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Methods: Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. Results: We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Conclusion: Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states.

Elevated APE1 Dysregulates Homologous Recombination and Cell Cycle Driving Genomic Evolution, Tumorigenesis, and Chemoresistance in Esophageal Adenocarcinoma.

BACKGROUND & AIMS: The purpose of this study was to identify drivers of genomic evolution in esophageal adenocarcinoma (EAC) and other solid tumors. METHODS: An integrated genomics strategy was used to identify deoxyribonucleases correlating with genomic instability (as assessed from total copy number events in each patient) in 6 cancers. Apurinic/apyrimidinic nuclease 1 (APE1), identified as the top gene in functional screens, was either suppressed in cancer cell lines or overexpressed in normal esophageal cells and the impact on genome stability and growth was monitored in vitro and in vivo. The impact on DNA and chromosomal instability was monitored using multiple approaches, including investigation of micronuclei, acquisition of single nucleotide polymorphisms, whole genome sequencing, and/or multicolor fluorescence in situ hybridization. RESULTS: Expression of 4 deoxyribonucleases correlated with genomic instability in 6 human cancers. Functional screens of these genes identified APE1 as the top candidate for further evaluation. APE1 suppression in EAC, breast, lung, and prostate cancer cell lines caused cell cycle arrest; impaired growth and increased cytotoxicity of cisplatin in all cell lines and types and in a mouse model of EAC; and inhibition of homologous recombination and spontaneous and chemotherapy-induced genomic instability. APE1 overexpression in normal cells caused a massive chromosomal instability, leading to their oncogenic transformation. Evaluation of these cells by means of whole genome sequencing demonstrated the acquisition of changes throughout the genome and identified homologous recombination as the top mutational process. CONCLUSIONS: Elevated APE1 dysregulates homologous recombination and cell cycle, contributing to genomic instability, tumorigenesis, and chemoresistance, and its inhibitors have the potential to target these processes in EAC and possibly other cancers.

Fragmentation landscape of cell-free DNA revealed by deconvolutional analysis of end motifs.

Cell-free DNA (cfDNA) fragmentation is nonrandom, at least partially mediated by various DNA nucleases, forming characteristic cfDNA end motifs. However, there is a paucity of tools for deciphering the relative contributions of cfDNA cleavage patterns related to underlying fragmentation factors. In this study, through non-negative matrix factorization algorithm, we used 256 5' 4-mer end motifs to identify distinct types of cfDNA cleavage patterns, referred to as "founder" end-motif profiles (F-profiles). F-profiles were associated with different DNA nucleases based on whether such patterns were disrupted in nuclease-knockout mouse models. Contributions of individual F-profiles in a cfDNA sample could be determined by deconvolutional analysis. We analyzed 93 murine cfDNA samples of different nuclease-deficient mice and identified six types of F-profiles. F-profiles I, II, and III were linked to deoxyribonuclease 1 like 3 (DNASE1L3), deoxyribonuclease 1 (DNASE1), and DNA fragmentation factor subunit beta (DFFB), respectively. We revealed that 42.9% of plasma cfDNA molecules were attributed to DNASE1L3-mediated fragmentation, whereas 43.4% of urinary cfDNA molecules involved DNASE1-mediated fragmentation. We further demonstrated that the relative contributions of F-profiles were useful to inform pathological states, such as autoimmune disorders and cancer. Among the six F-profiles, the use of F-profile I could inform the human patients with systemic lupus erythematosus. F-profile VI could be used to detect individuals with hepatocellular carcinoma, with an area under the receiver operating characteristic curve of 0.97. F-profile VI was more prominent in patients with nasopharyngeal carcinoma undergoing chemoradiotherapy. We proposed that this profile might be related to oxidative stress.

Extra-viral DNA in adeno-associated viral vector preparations induces TLR9-dependent innate immune responses in human plasmacytoid dendritic cells.

Adeno-associated viral (AAV) vector suspensions produced in either human derived HEK cells or in Spodoptera frugiperda (Sf9) insect cells differ in terms of residual host cell components as well as species-specific post-translational modifications displayed on the AAV capsid proteins. Here we analysed the impact of these differences on the immunogenic properties of the vector. We stimulated human plasmacytoid dendritic cells with various lots of HEK cell-produced and Sf9 cell-produced AAV-CMV-eGFP vectors derived from different manufacturers. We found that AAV8-CMV-eGFP as well as AAV2-CMV-eGFP vectors induced lot-specific but not production platform-specific or manufacturer-specific inflammatory cytokine responses. These could be reduced or abolished by blocking toll-like receptor 9 signalling or by enzymatically reducing DNA in the vector lots using DNase. Successful HEK cell transduction by DNase-treated AAV lots and DNA analyses demonstrated that DNase did not affect the integrity of the vector but degraded extra-viral DNA. We conclude that both HEK- and Sf9-cell derived AAV preparations can contain immunogenic extra-viral DNA components which can trigger lot-specific inflammatory immune responses. This suggests that improved strategies to remove extra-viral DNA impurities may be instrumental in reducing the immunogenic properties of AAV vector preparations.

Analysis of tafazzin and deoxyribonuclease 1 like 1 transcripts and X chromosome sequencing in the evaluation of the effect of mosaicism in the TAZ gene on phenotypes in a family affected by Barth syndrome.

Barth syndrome is a rare disease affecting mitochondria structure and function in males. In our previous study, we have shown a new mutation (c.83T>A, p.Val28Glu) in the TAZ gene in two affected patients with congenital cardiomyopathy. Furthermore, women in this family had no mutations in their blood cells, whereas they only had mutations in the oral epithelial cells. The objective of the project was to evaluate the effect of intertissue mosaicisms on the Barth syndrome phenotypes, searching for another disease-related loci on chromosome X and finally to assess the consequences of the mutation. We conducted the advanced genetic study including cytogenetic research (constitutional karyotyping in blood and fibroblasts), NGS sequencing (with custom chromosome X sequencing together with the evaluation of loss of heterozygosity (LOH) and aberrations (CNV) in the whole genome) in four different tissues and sequencing of tafazzin and deoxyribonuclease 1 like 1 transcripts. The presence of deletions within the 5'untranslated region of the TAZ gene and/or the noncoding regions of the DNASE1L1 gene were detected in several tissues. Whereas, there is no intertissue mosaicism regarding point mutation in TAZ gene in all investigated tissues in female carriers. Only the male patient presented biochemical markers and neurological symptoms of Barth syndrome. All the female carriers are healthy and have normal tafazzin and deoxyribonuclease 1 like 1 transcripts in 2 analyzed tissues. The conclusion of this study is that we cannot rule out or confirm mosaicism in the noncoding regions of TAZ or DNASE1L1 genes, but this is not clinically relevant in female carriers because they are healthy. Finally, it has been proven that mutation (c.83T>A, p.Val28Glu) is responsible for disease in males in this family.

maxATAC: Genome-scale transcription-factor binding prediction from ATAC-seq with deep neural networks.

Transcription factors read the genome, fundamentally connecting DNA sequence to gene expression across diverse cell types. Determining how, where, and when TFs bind chromatin will advance our understanding of gene regulatory networks and cellular behavior. The 2017 ENCODE-DREAM in vivo Transcription-Factor Binding Site (TFBS) Prediction Challenge highlighted the value of chromatin accessibility data to TFBS prediction, establishing state-of-the-art methods for TFBS prediction from DNase-seq. However, the more recent Assay-for-Transposase-Accessible-Chromatin (ATAC)-seq has surpassed DNase-seq as the most widely-used chromatin accessibility profiling method. Furthermore, ATAC-seq is the only such technique available at single-cell resolution from standard commercial platforms. While ATAC-seq datasets grow exponentially, suboptimal motif scanning is unfortunately the most common method for TFBS prediction from ATAC-seq. To enable community access to state-of-the-art TFBS prediction from ATAC-seq, we (1) curated an extensive benchmark dataset (127 TFs) for ATAC-seq model training and (2) built "maxATAC", a suite of user-friendly, deep neural network models for genome-wide TFBS prediction from ATAC-seq in any cell type. With models available for 127 human TFs, maxATAC is the largest collection of high-performance TFBS prediction models for ATAC-seq. maxATAC performance extends to primary cells and single-cell ATAC-seq, enabling improved TFBS prediction in vivo. We demonstrate maxATAC's capabilities by identifying TFBS associated with allele-dependent chromatin accessibility at atopic dermatitis genetic risk loci.

A comprehensive Bioconductor ecosystem for the design of CRISPR guide RNAs across nucleases and technologies.

The success of CRISPR-mediated gene perturbation studies is highly dependent on the quality of gRNAs, and several tools have been developed to enable optimal gRNA design. However, these tools are not all adaptable to the latest CRISPR modalities or nucleases, nor do they offer comprehensive annotation methods for advanced CRISPR applications. Here, we present a new ecosystem of R packages, called crisprVerse, that enables efficient gRNA design and annotation for a multitude of CRISPR technologies. This includes CRISPR knockout (CRISPRko), CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), CRISPR base editing (CRISPRbe) and CRISPR knockdown (CRISPRkd). The core package, crisprDesign, offers a user-friendly and unified interface to add off-target annotations, rich gene and SNP annotations, and on- and off-target activity scores. These functionalities are enabled for any RNA- or DNA-targeting nucleases, including Cas9, Cas12, and Cas13. The crisprVerse ecosystem is open-source and deployed through the Bioconductor project ( https://github.com/crisprVerse ).

Single-Cell Chromatin Accessibility Data Combined with GWAS Improves Detection of Relevant Cell Types in 59 Complex Phenotypes.

Several disease risk variants reside on non-coding regions of DNA, particularly in open chromatin regions of specific cell types. Identifying the cell types relevant to complex traits through the integration of chromatin accessibility data and genome-wide association studies (GWAS) data can help to elucidate the mechanisms of these traits. In this study, we created a collection of associations between the combinations of chromatin accessibility data (bulk and single-cell) with an array of 201 complex phenotypes. We integrated the GWAS data of these 201 phenotypes with bulk chromatin accessibility data from 137 cell types measured by DNase-I hypersensitive sequencing and found significant results (FDR adjusted p-value ≤ 0.05) for at least one cell type in 21 complex phenotypes, such as atopic dermatitis, Graves' disease, and body mass index. With the integration of single-cell chromatin accessibility data measured by an assay for transposase-accessible chromatin with high-throughput sequencing (scATAC-seq), taken from 111 adult and 111 fetal cell types, the resolution of association was magnified, enabling the identification of further cell types. This resulted in the identification of significant correlations (FDR adjusted p-value ≤ 0.05) between 15 categories of single-cell subtypes and 59 phenotypes ranging from autoimmune diseases like Graves' disease to cardiovascular traits like diastolic/systolic blood pressure.

MilR3, a unique SARP family pleiotropic regulator in Streptomyces bingchenggensis.

Streptomyces bingchenggensis is the main industrial producer of milbemycins, which are a group of 16-membered macrocylic lactones with excellent insecticidal activities. In the past several decades, scientists have made great efforts to solve its low productivity. However, a lack of understanding of the regulatory network of milbemycin biosynthesis limited the development of high-producing strains using a regulatory rewiring strategy. SARPs (Streptomyces Antibiotic Regulatory Proteins) family regulators are widely distributed and play key roles in regulating antibiotics production in actinobacteria. In this paper, MilR3 (encoded by sbi_06842) has been screened out for significantly affecting milbemycin production from all the 19 putative SARP family regulators in S. bingchenggensis with the DNase-deactivated Cpf1-based integrative CRISPRi system. Interestingly, milR3 is about 7 Mb away from milbemycin biosynthetic gene cluster and adjacent to a putative type II PKS (the core minimal PKS encoding genes are sbi_06843, sbi_06844, sbi_06845 and sbi_06846) gene cluster, which was proved to be responsible for producing a yellow pigment. The quantitative real-time PCR analysis proved that MilR3 positively affected the transcription of specific genes within milbemycin BGC and those from the type II PKS gene cluster. Unlike previous "small" SARP family regulators that played pathway-specific roles, MilR3 was probably a unique SARP family regulator and played a pleotropic role. MilR3 was an upper level regulator in the MilR3-MilR regulatory cascade. This study first illustrated the co-regulatory role of this unique SARP regulator. This greatly enriches our understanding of SARPs and lay a solid foundation for milbemycin yield enhancement in the near future.

A DNase Type VI Secretion System Effector Requires Its MIX Domain for Secretion.

Gram-negative bacteria often employ the type VI secretion system (T6SS) to deliver diverse cocktails of antibacterial effectors into rival bacteria. In many cases, even when the identity of the delivered effectors is known, their toxic activity and mechanism of secretion are not. Here, we investigate VPA1263, a Vibrio parahaemolyticus T6SS effector that belongs to a widespread class of polymorphic effectors containing a MIX domain. We reveal a C-terminal DNase toxin domain belonging to the HNH nuclease superfamily, and we show that it mediates the antibacterial toxicity of this effector during bacterial competition. Furthermore, we demonstrate that the VPA1263 MIX domain is necessary for T6SS-mediated secretion and intoxication of recipient bacteria. These results are the first indication of a functional role for MIX domains in T6SS secretion. IMPORTANCE Specialized protein delivery systems are used during bacterial competition to deploy cocktails of toxins that target conserved cellular components. Although numerous toxins have been revealed, the activity of many remains unknown. In this study, we investigated such a toxin from the pathogen Vibrio parahaemolyticus. Our findings indicate that the toxin employs a DNase domain to intoxicate competitors. We also show that a domain used as a marker for secreted toxins is required for secretion of the toxin via a type VI secretion system.

Intrinsic bias estimation for improved analysis of bulk and single-cell chromatin accessibility profiles using SELMA.

Genome-wide profiling of chromatin accessibility by DNase-seq or ATAC-seq has been widely used to identify regulatory DNA elements and transcription factor binding sites. However, enzymatic DNA cleavage exhibits intrinsic sequence biases that confound chromatin accessibility profiling data analysis. Existing computational tools are limited in their ability to account for such intrinsic biases and not designed for analyzing single-cell data. Here, we present Simplex Encoded Linear Model for Accessible Chromatin (SELMA), a computational method for systematic estimation of intrinsic cleavage biases from genomic chromatin accessibility profiling data. We demonstrate that SELMA yields accurate and robust bias estimation from both bulk and single-cell DNase-seq and ATAC-seq data. SELMA can utilize internal mitochondrial DNA data to improve bias estimation. We show that transcription factor binding inference from DNase footprints can be improved by incorporating estimated biases using SELMA. Furthermore, we show strong effects of intrinsic biases in single-cell ATAC-seq data, and develop the first single-cell ATAC-seq intrinsic bias correction model to improve cell clustering. SELMA can enhance the performance of existing bioinformatics tools and improve the analysis of both bulk and single-cell chromatin accessibility sequencing data.