Dissecting these potential regulatory elements will help to provide a more complete picture of miR-146a transcriptional regulation. and all the other authors. Nature 489, 57–74 (2012). 9d). Yao et al. Nature 518, 317–330 (2015). La Manno, G. et al. 3e, f). 2i). n Bar graph showing the percentage of LILRB1 expression in dNK1, dNK2, and dNK3 cell subsets from healthy controls (nc = 7) and RPL patients (np = 7). After the generation of single-cell gel bead-in-emulsions (GEMs), reverse transcription was performed using a C1000 TouchTM Thermal Cycler (Bio-Rad). B cells were cultured in RPMI-1640 media with 10% (vol/vol) HI-FBS, 2 mM l-Glutamine, 55 µM β-mercaptoethanol, and 1% penicillin–streptomycin, 50 IU/mL IL-4 (200-04, Peprotech), and crosslinked CD40 ligand (130-098-775, Miltenyi Biotec). MicroRNA-146A contributes to abnormal activation of the type I interferon pathway in human lupus by targeting the key signaling proteins. PubMed Central  Methods 16, 1289–1296 (2019). Heat map displays a set of medians of normalized contact intensities calculated at different window sizes (from 2 to 50 kb), the red and blue colors in the heat map depict higher and lower interaction intensity, respectively. Prone. 177, 7303–7311 (2006). i UCSC genome browser visualization of the chromatin accessibility profiling at the IFNG locus. This result is consistent with the epigenetic modification around rs2431697 and the disparities suggest that cell lines cannot always fully mimic the context of primary cells. Primer sets and amplicons are indicated by a red arrow. Cells were routinely cultured in RPMI-1640 media with 10% FBS until ready for transfection. sgRNAs were designed using online CRISPR Design tool (crispr.mit.edu) and we selected the possible sgRNAs targeting the core DNA sequence of rs2431697 or miR-146A promoter based on high efficacy score and low potential off-target sites. We used the same gene expression matrix as used in Seurat and performed RunHarmony function in Hamony with default parameters to perform data integration. The etiology of RPL remains unknown in about 50% of cases. Periapt of Proof against Poison. Data are represented as mean ± SEM and P-values are calculated using unpaired two-tailed Student’s t-test (a–e, l–n) and paired two-tailed Student’s test (h–j). Alzheimer’s disease (AD), the most common form of dementia, is recognized as a heterogeneous disease with diverse pathophysiologic mechanisms. Langmead, B. Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Specifically, dNK1 cells were significantly decreased in RPL patients, while dNK2 cells were slightly increased and dNK3 cells were significantly increased. Med. In this study, we interrogate the molecular heterogeneity of AD by analyzing 1543 transcriptomes across five brain regions in two AD cohorts using an integrative network approach. Krivega, I. Am. We retained cells with detected gene numbers between 500 and 3000 and less than 10% mitochondrial UMIs. Finally, CRISPR activation-based modulation of this enhancer in the PBMCs of SLE patients attenuates type I interferon pathway activation by increasing miR-146a expression. ****P < 0.0001. 5, 219–219 (2014). RNA-Seq data43 were downloaded from the eQTL resources from the Gilad/Pritchard group website. To ensure lack of overlap between the discovery and replication cohorts, samples from contributing individuals who were also authors on the publications in the replication cohort were removed. PubMed  Following cleanup, the cDNA was pre-amplified and used for real-time PCR expression analysis. These authors contributed equally: Chuang Guo, Pengfei Cai, Liying Jin, Department of Oncology, The First Affiliated Hospital of USTC, Division of Molecular Medicine, Hefei National Laboratory for Physical Sciences at Microscale, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China, Chuang Guo, Pengfei Cai, Liying Jin, Qing Sha, Qiaoni Yu, Wen Zhang, Chen Jiang, Qian Liu, Dandan Zong, Kun Li, Jingwen Fang, Jun Lin, Lu Li, Zhutian Zeng, Haiming Wei & Kun Qu, HanGene Biotech, Xiaoshan Innovation Polis, Hangzhou, Zhejiang 311200, China, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, Anhui 230021, China, Fangting Lu, Yanshi Wang, Daojing Li & Xianhong Tong, CAS Center for Excellence in Molecular Cell Sciences, The CAS Key Laboratory of Innate Immunity and Chronic Disease, University of Science and Technology of China, Hefei, Anhui 230027, China, School of Data Science, University of Science and Technology of China, Hefei, Anhui 230027, China, You can also search for this author in Following 14 days of cell growth, genomic DNA of individual colonies was isolated using TransDirect Animal Tissue PCR Kit (AD201-02, Transgen Biotech), target sequence was amplified by locus-specific primer, and sequenced by Sanger sequence to screen for desired mutations. Article  5e). Maurano, M. T. et al. sgRNA lentiviral particles were produced and stably expressing KRAB-dCas9-mCherry U-937 cells were transduced as mentioned above, the cells were further selected with 1 μg/mL puromycin (ant-pr-5, Invivogene) for 72 h. CRISPRa U-937 cell line stably expressing dCas9-VP64 and MS2-P65-HSF1 fusion proteins was generated using the lenti-dCAS-VP64-Blast plasmid (Addgene 61425) and lenti-MS2-P65-HSF1-Hygro (Addgene 61426) plasmid as mentioned above. In 5E, these monsters are a force to be reckoned with. The mean gestational ages are 7.24 weeks in controls and 8.50 weeks in RPL patients. Significance was evaluated with Student’s t-test. 36, 107–117 (2012). 11, 2025 (2020). f, g Circos plot visualizing significant cis interactions. S5c, e), consistent with the aforementioned increase in cytokine-mediated signaling pathways in the dNK3 cells of RPL patients. Epidemiol. The aqueous phase was removed to extract RNA, the DNA in interphase was precipitated by 100% ethanol, and washed with 0.1 M sodium citrate and 75% ethanol to get DNA. Taken together, these data indicate that targeting this enhancer may be an effective method to modulate the IFN pathway, which is highly relevant to SLE etiopathogenesis. Results were analyzed using the comparative Ct method normalizing to a control sample and housekeeping primers. Cells were selected with 10 μg/mL Blasticidin (ant-bl-5, Invivogene) and 300 μg/mL Hygromcin (10687010, Thermo Fisher) for 1 week. Swiss Med. Quantitative reverse transcriptipn PCR (RT-PCR) reactions were performed using TB Green Premix Ex Taq reagent (RR420A, TAKARA) according to the manufacturer’s protocol and normalized by glyceraldehyde 3-phosphate dehydrogenase mRNA levels. We identified cell lineages, including NK cells, macrophages, T cells, dendritic cells, NKT cells, and immune progenitor cells based on the expression of known marker genes (Fig. Cells were transferred to 500 μL of their respective culture medium in a 48-well plate. PLoS Genet. N. Engl. PBX1 expression in uterine natural killer cells drives fetal growth. After cell lysis, 3′ and 5′ adapters are ligated to mature miRNAs. PubMed  For ncRNAs, especially for miRNAs, where the technical challenges in quantifying expression are nontrivial17, the lack of reliable readouts further impedes performing large-scale screening to define functional noncoding elements. The cells were checked regularly for mycoplasma MycoAlert Mycoplasma Detection Kit (LT07-118, Lonza). Biosafety in Microbiological and Biomedical Laboratories (BMBL) quickly became the cornerstone of biosafety practice and policy in the United States upon first publication in 1984. 4d); however, mac2 cells were highly enriched with M2 specific genes (Fig. As expected, we found a slight but not significant increase in the proportion of NK cells (P = 0.0809), a remarkable and significant decrease in macrophages (P = 0.0051), and a slight but significant increase in T cells (P = 0.0307) (Fig. Mol. Wondrous item, rare. As rs2431697 demonstrated the strongest evidence of association with SLE, we first conditioned on this marker in each cohort, under the assumption that this genetic association is due to a shared genetic effect across ancestries. 4C-seq data were analyzed using the software pipeline89,90,91, 4Cseqpipe (version 0.7), with settings: -stat_type median, -trend_resolution 2000. Trial of Anifrolumab in active systemic lupus erythematosus. GWAS and Immunochip Summary statistics from three SLE association studies40,41,42 were downloaded from immunobase.org (http://urr.cat/data/GWAS_SLE_summaryStats.zip, now also available at https://www.ebi.ac.uk/gwas/studies/GCST005831 and https://static-content.springer.com/esm/art%3A10.1038%2Fng.3496/MediaObjects/41588_2016_BFng3496_MOESM228_ESM.xlsx). If you are like me, you enjoying playing with a character build to squeeze the most utility out of them as possible. The molecular regulation of conserved gene modules during development may be distinct from that observed in disease. Saito, S., Nakashima, A., Shima, T. & Ito, M. Th1/Th2/Th17 and regulatory T-cell paradigm in pregnancy. Transl. Genet. 73, 2491–2509 (2016). STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nature 533, 95–99 (2016). Immunity 47, 1100–1113 (2017). Methods 10, 1213–1218 (2013). Revised December 2009. iii. Life Sci. S5c, d). We decipher the biological mechanism that mediates the risk for SLE conferred by rs2431697 and demonstrate that it likely alters SLE pathogenesis by regulating miR-146a expression (Fig. Zucchi, D. et al. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. e ChIP-qPCR analysis of active enhancer marks (H3K4me1 and H3K27ac) within the rs2431697-containing region in U-937, Raji, and Jurkat cells (U-937 H3K4me1: ***P < 0.0001, U-937 H3K27ac: ***P = 0.0002, Raji H3K4me1: ***P = 0.0003, Raji H3K27ac: ***P = 0.0004) (n = 3, biological replicates). 2e, g), and the dNK3 cells expressed especially high levels of the immunomodulatory IFNG gene (encoding IFN-γ) (Supplementary Fig. Front. Google Scholar. Robust 4C-seq data analysis to screen for regulatory DNA interactions. When you reach 10th level, you can’t be Charmed or Frightened by Elementals or fey, and you are immune to poison and disease. Willer, C. J., Li, Y. eQTL analysis indicates that rs2431697 is associated with miR-146a expression. Genet. After that, 300 mg Proteinase K was added for overnight incubation at 65 °C and 30 μl of RNaseA solution was added and incubated for 45 min at 37 °C. For mRNA analysis, cDNA was synthesized using PrimeScript™ RT Reagent Kit (Perfect Real Time) (RR037A, TAKARA) using 500 ng of RNA per cDNA reaction. Google Scholar. Taken together, these data indicate that the rs2431697-containing region may be a cell-type-specific enhancer regulating nearby gene expression. 6e). CAS  ADS  Biotechnol. All points are shown and bars represent means with SEM. Methods 12, 1143–1149 (2015). When abnormal fetal heart activity was observed, the patients were advised to be tested for serum β-human chorionic gonadotropin levels, with additional ultrasounds every other week. Yale. ISSN 2056-5968 (online), Single-cell profiling of the human decidual immune microenvironment in patients with recurrent pregnancy loss, https://github.com/velocyto-team/velocyto-notebooks/tree/master/python/DentateGyrus.ipynb, http://creativecommons.org/licenses/by/4.0/, https://doi.org/10.1038/s41421-020-00236-z, Single-cell RNA Sequencing Deciphers Immune Landscape of Human Recurrent Miscarriage. We summarized the RPL-associated changes in receptor/ligand interactions between immune cells and both the EVTs and stromal cells (Fig. PubMed Central  PubMed  Fresh decidual tissues were washed extensively in phosphate-buffered saline with 100 IU/mL penicillin/streptomycin and sheared into tiny pieces. 15, e1008500 (2019). Am. Briefly, dNK1, dNK2, and dNK3 subsets were sorted using the SH800S sorter (Sony). S6c). Evaluation of the TREX1 gene in a large multi-ancestral lupus cohort. Commun. Shanghai Institute of Rheumatology, Renji Hospital, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai, 200001, China, Guojun Hou, Tian Zhou, Ning Xu, Yuting Qin, Ye Ouyang, Jianyang Ma, Xinyi Zhu, Xiang Yu, Dai Dai, Huihua Ding, Jun Deng, Yuanjia Tang & Nan Shen, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai, 200032, China, Shanghai Institute of Rheumatology, China-Australia Centre for Personalized Immunology, Renji Hospital, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai, 200001, China, Shenzhen Futian Hospital for Rheumatic Diseases, Shenzhen, 518040, China, Guojun Hou, Zhihua Yin, Zhizhong Ye & Nan Shen, Division of Immunobiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, 45229, USA, Isaac T. W. Harley, Leah C. Kottyan & John B. Harley, Division of Rheumatology, School of Medicine, University of Colorado, Aurora, Colorado, 80045, USA, Department of Immunology and Microbiology, School of Medicine, University of Colorado, Aurora, Colorado, 80045, USA, Center for Autoimmune Genomics and Etiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, 45229, USA, Xiaoming Lu, Bahram Namjou, Matthew T. Weirauch, Leah C. Kottyan, John B. Harley & Nan Shen, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences(SIBS), University of Chinese Academy of Sciences, Chinese Academy of Sciences (CAS), Shanghai, 200031, China, Department of Obstetrics and Gynecology, Renji Hospital, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai, 200127, China, Shanghai Key Laboratory of Gynecologic Oncology, Renji Hospital, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai, 200127, China, Sheng Yushou Center of Cell Biology and Immunology, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University (SJTU), Shanghai, 200240, China, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45229, USA, Matthew T. Weirauch, Leah C. Kottyan, John B. Harley & Nan Shen, Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, 45229, USA, Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, 45229, USA, Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, 45229, USA, US Department of Veterans Affairs Medical Center, Cincinnati, Ohio, 45229, USA, You can also search for this author in
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