Mouse Genome Research Articles

FoxO-KlLF5 Pathway Switches the Flow of Macronutrients Under the Control of Insulin

KLF15 is a transcription factor that plays an important role in the activation of gluconeogenesis from amino acids as well as the suppression of lipogenesis from glucose. Although it is well known that hepatic Klf15 expression is elevated during fasting, its regulatory mechanisms have not been elucidated. Here we identified the transcription start site of liver-specific KLF15 transcript and showed that FoxO1 and FoxO3 (FoxOs) transcriptionally regulate Klf15 gene expression by directly binding to the liver-specific Klf15 promoter. To achieve this, we performed a precise in vivo promoter analysis using “in vivo Ad-luc” analytical system, combined with the genome-wide screening method “TFEL scan”, using our original cDNA library named Transcription Factor Expression Library (TFEL), which covers nearly all the transcription factors in the mouse genome. Hepatic Klf15 expression is significantly increased by the attenuation of the insulin signaling in STZ-induced insulin deficiency and insulin receptor knockdown models. In addition, we have found that FoxOs elevate the expression levels of amino acid catabolic enzymes via KLF15, resulting in accelerated amino acid metabolism during fasting. Based on these findings, we conclude that the FoxO-KLF15 pathway switches the macronutrient flow in the liver under the control of insulin.

Generation and characterization of HLA-A2 transgenic mice expressing the human TCR 1G4 specific for the HLA-A2 restricted NY-ESO-1157-165 tumor-specific peptide

BackgroundNY-ESO-1 is a tumor-specific, highly immunogenic, human germ cell antigen of the MAGE-1 family that is a promising vaccine and cell therapy candidate in clinical trial development. The mouse genome...

209-LB: RNA-Seq Analysis of Liver from Diabetic and NASH Model Mouse Treated with Streptozotocin, High-Fat Diet

Introduction: NASH is a chronic liver disease often associated with type 2 diabetes. It sometimes progresses to more serious conditions such as liver fibrosis and hepatocellular carcinoma (HCC). Therapeutic agents for NASH have not yet been developed, and an animal model with high clinical correlation is required. The STAM mouse is a NASH model mouse with a background of type 2 diabetes. This mouse model shows the same pathological progression as human NASH patients, and has been used for the evaluation of many drug candidates, as well as basic research. However the gene profile, including mutations, has not been elucidated in this model. In this study, we analyzed the RNA-seq data of STAM mice at each pathological stage and examined the clinical correlation at the gene level. Methods: NASH was induced in male mice by a single injection of streptozotocin solution 2 days after birth and feeding with high fat diet after 4 weeks of age. The mice were sacrificed and livers collected from each disease phase. RNA-Seq libraries were prepared by NEB NEXT Directional Ultra RNA Library Prep Kit (Illumina). The libraries were sequenced on Novaseq (Illumina), and the reads were aligned to mouse genome references using HISAT2. RNA-seq data analysis Sequence reads (150-bp paired read) were aligned to the mouse reference genome (GRCm38/mm10) with HISAT2 (version: 2.1.0) with default parameters. Results: First, the gene expression of the canonical pathway in NASH progression from steatosis to hepatocellular carcinoma was analyzed. Increased expression was observed in CCL2, TLR4, HSP47, αSMA, c-Myc, and cyclin D1. Since it has been reported that genetic traits are involved in the development of NASH-HCC, we next analyzed the genetic mutations in the STAM mice. The number of individuals showing mutations in Mtor, involved in Insulin signaling, increases as the disease progresses, especially in the HCC phase. Conclusion: These results indicated a clinical correlation of gene profiles in the STAM mouse. Disclosure L. P. Bui: Employee; Self; SMC Laboratories Inc. Y. Sakakibara: Employee; Self; SMC. R. Tanaka: Employee; Self; SMC Laboratories Inc. E. H. Pigney: Employee; Self; SMC Laboratories, Inc. T. Hashiguchi: Employee; Self; SMC laboratories, Inc.

A genome-wide library of MADM mice for single-cell genetic mosaic analysis.

Mosaic analysis with double markers (MADM) offers one approach to visualize and concomitantly manipulate genetically defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage, single-cell morphology and physiology, genomic imprinting phenotypes, and dissection of cell-autonomous gene functions invivo in health and disease. Yet, MADM can only be applied to <25% of all mouse genes on select chromosomes to date. To overcome this limitation, we generate transgenic mice with knocked-in MADM cassettes near the centromeres of all 19 autosomes and validate their use across organs. With this resource, >96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond a proof of principle, we apply our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We find striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division.

I4mC-EL: Identifying DNA N4-Methylcytosine Sites in the Mouse Genome Using Ensemble Learning.

As one of important epigenetic modifications, DNA N4-methylcytosine (4mC) plays a crucial role in controlling gene replication, expression, cell cycle, DNA replication, and differentiation. The accurate identification of 4mC sites is necessary to understand biological functions. In the paper, we use ensemble learning to develop a model named i4mC-EL to identify 4mC sites in the mouse genome. Firstly, a multifeature encoding scheme consisting of Kmer and EIIP was adopted to describe the DNA sequences. Secondly, on the basis of the multifeature encoding scheme, we developed a stacked ensemble model, in which four machine learning algorithms, namely, BayesNet, NaiveBayes, LibSVM, and Voted Perceptron, were utilized to implement an ensemble of base classifiers that produce intermediate results as input of the metaclassifier, Logistic. The experimental results on the independent test dataset demonstrate that the overall rate of predictive accurate of i4mC-EL is 82.19%, which is better than the existing methods. The user-friendly website implementing i4mC-EL can be accessed freely at the following.

Landscape of GPCR expression along the mouse nephron.

Kidney transport and other renal functions are regulated by multiple G protein-coupled receptors (GPCRs) expressed along the renal tubule. The rapid, recent appearance of comprehensive unbiased gene expression data in the various renal tubule segments, chiefly RNA sequencing and protein mass spectrometry data, has provided a means of identifying patterns of GPCR expression along the renal tubule. To allow for comprehensive mapping, we first curated a comprehensive list of GPCRs in the genomes of mice, rats, and humans (https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/) using multiple online data sources. We used this list to mine segment-specific and cell type-specific expression data from RNA-sequencing studies in microdissected mouse tubule segments to identify GPCRs that are selectively expressed in discrete tubule segments. Comparisons of these mapped mouse GPCRs with other omics datasets as well as functional data from isolated perfused tubule and micropuncture studies confirmed patterns of expression for well-known receptors and identified poorly studied GPCRs that are likely to play roles in the regulation of renal tubule function. Thus, we provide data resources for GPCR expression across the renal tubule, highlighting both well-known GPCRs and understudied receptors to provide guidance for future studies.

Tissue context determines the penetrance of regulatory DNA variation

Functional assessment of disease-associated sequence variation at non-coding regulatory elements is complicated by their high degree of context sensitivity to both the local chromatin and nuclear environments. Allelic profiling of DNA accessibility across individuals has shown that only a select minority of sequence variation affects transcription factor (TF) occupancy, yet low sequence diversity in human populations means that no experimental assessment is available for the majority of disease-associated variants. Here we describe high-resolution in vivo maps of allelic DNA accessibility in liver, kidney, lung and B cells from 5 increasingly diverged strains of F1 hybrid mice. The high density of heterozygous sites in these hybrids enables precise quantification of effect size and cell-type specificity for hundreds of thousands of variants throughout the mouse genome. We show that chromatin-altering variants delineate characteristic sensitivity profiles for hundreds of TF motifs. We develop a compendium of TF-specific sensitivity profiles accounting for genomic context effects. Finally, we link maps of allelic accessibility to allelic transcript levels in the same samples. This work provides a foundation for quantitative prediction of cell-type specific effects of non-coding variation on TF activity, which will facilitate both fine-mapping and systems-level analyses of common disease-associated variation in human genomes.

Single AAV-mediated CRISPR-Nme2Cas9 efficiently reduces mutant hTTR expression in a transgenic mouse model of transthyretin amyloidosis

Transthyretin (TTR) amyloidosis is a hereditary life-threatening disease characterized by deposition of amyloid fibrils. The main causes of TTR amyloidosis are mutations in the TTR gene that lead to the production of misfolded TTR protein. Reducing the production of toxic protein in the liver is a validated strategy to treat TTR amyloidosis. In this study, we established a humanized mouse model that expresses mutant human TTR (hTTR; V30M) protein in the liver to model TTR amyloidosis. Then, we compared the efficiency of reducing the expression of mutant hTTR by dual adeno-associated virus 8 (AAV8)-mediated split SpCas9 with that by single AAV8-mediated Nme2Cas9 in this model. With two gRNAs targeting different exons, dual AAV-mediated split SpCas9 system achieved efficiencies of 37% and 34% reduction of hTTR mRNA and reporter GFP expression, respectively, in the liver. Surprisingly, single AAV-mediated Nme2Cas9 treatment resulted in 65% and 71% reduction of hTTR mRNA and reporter GFP, respectively. No significant editing was identified in predicted off-target sites in the mouse and human genomes after Nme2Cas9 targeting. Thus, we provide proof of principle for using single AAV-mediated CRISPR-Nme2Cas9 to effectively reduce mutant hTTR expression invivo, which may translate into gene therapy for TTR amyloidosis.

Reduced purine biosynthesis in humans after their divergence from Neandertals.

We analyze the metabolomes of humans, chimpanzees, and macaques in muscle, kidney and three different regions of the brain. Although several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution contributes to much or all of the reduction of de novo synthesis of purines in humans.

Pervasiveness of intronless genes expressed in haploid germ cell differentiation.

BackgroundcDNA libraries derived from the brain and testis contain genes that encode almost all proteins. The brain is composed of various differentiated cells, and the testis also contains various differentiated cells, such as germ cells, and somatic cells that support germ cell differentiation, such as Sertoli and Leydig cells. Many genes appear to be expressed due to tissue complexity.MethodsThe Genome Project has sequenced the entire genomes of humans and mice. Recent research using new gene analysis technologies has found that many genes are expressed specifically in male germ cells.Main findings (Results)Functional intronless genes are significantly enriched in haploid germ cell‐specific genes.ConclusionFunctional intronless genes associated with fertility are more likely to be inherited in haploid germ cells than in somatic cells.

Learning a genome-wide score of human\u2013mouse conservation at the functional genomics level

Identifying genomic regions with functional genomic properties that are conserved between human and mouse is an important challenge in the context of mouse model studies. To address this, we develop a method to learn a score of evidence of conservation at the functional genomics level by integrating information from a compendium of epigenomic, transcription factor binding, and transcriptomic data from human and mouse. The method, Learning Evidence of Conservation from Integrated Functional genomic annotations (LECIF), trains neural networks to generate this score for the human and mouse genomes. The resulting LECIF score highlights human and mouse regions with shared functional genomic properties and captures correspondence of biologically similar human and mouse annotations. Analysis with independent datasets shows the score also highlights loci associated with similar phenotypes in both species. LECIF will be a resource for mouse model studies by identifying loci whose functional genomic properties are likely conserved.

Bayesian Identification of Transcription Factors that Regulate Aqp2 Transcription

Background Renal collecting duct and connecting tubule cells selectively express the water channel aquaporin-2 (AQP2) and Aqp2 gene transcription is strongly regulated by vasopressin. However, the transcription factors (TFs) responsible for regulation of expression of AQP2 remain largely unknown. Here, we used Bayesian data integration techniques to identify these TFs. Method The general strategy is to use Bayes’ Rule to integrate several -omic datasets to stratify a curated list of 1344 TFs present in the mouse genome with regard to probability of regulating Aqp2 gene transcription. First, existing proteomic and transcriptomic data were used to prioritize TFs according to expression level in mpkCCD and native IMCD cells. Second, we used our existing ATAC-Seq, histone H3K27-acetylation ChIP-Seq, and RNA-polymerase II ChIP-Seq data to identify enhancer regions in the CTCF loop surrounding the Aqp2 gene. The sequences within these enhancers were analyzed to identify recognized TF binding motifs within them; and these motifs were matched to TFs on the Bayesian list to identify the TFs most likely to bind Aqp2 regulatory regions. Beyond this, we use additional -omic datasets to prioritize TFs that are regulated by vasopressin. Finally, we carried out new RNA-Seq experiments mapping the time course of vasopressin induced changes in the transcriptome of mouse mpkCCD cells to further prioritize TFs that change in tandem with AQP2. Results The analysis identified 21 TFs out of 1344 in the mouse genome that are most likely to be involved in regulation of Aqp2 gene transcription. These TFs included nine that have been proposed in prior studies to play a role in Aqp2 regulation, viz. Cebpb, Elf1, Elf3, Ets1, Hes1, Jun, Junb, Nfkb1, and Sp1. The remaining 12 represent new candidates for future studies (Atf1, Cebpg, Fosl2, Irf3, Klf5, Klf6, Mef2d, Nfib, Nfyb, Nfyc, Nr2f6, Stat3). Conspicuously absent is CREB, i.e. Creb1, which has been widely proposed to mediate vasopressin-induced regulation of Aqp2 gene transcription. Instead, another CREB-like TF, Atf1, with a pKID PKA-phosphorylation domain, ranked second among all transcription factors, representing an apt target for future studies. The RNA-Seq time course experiments in mpkCCD cells showed a rapid increase in Aqp2 mRNA, within 3 hour of vasopressin exposure. This response was matched by an equally rapid increase in the abundance of the mRNA coding for Cebpb, a so-called pioneer TF, which we have shown by ChIP-seq studies to bind to an enhancer 938 bp downstream from the Aqp2 gene. Conclusions The Bayesian analysis has identified the TFs most likely to bind to these regions and likely to be regulated by vasopressin stimulation, providing a roadmap for future studies to understand regulation of Aqp2 gene expression.

C1Q‐TNF‐related peptide 8 (CTRP8) in human prostate cancer

Introduction C1Q Tumor Necrosis Factor related Peptide 8 (CTRP8) is the one of the least characterized members of the C1Q/TNF family of proteins which is engaged in diverse functions ranging from metabolism to immunity and is one of only two CTRP family members absent in the mouse genome. CTRP8 was identified in our lab as a novel ligand of the RXFP1 receptor and was found to promote invasiveness and chemoresistance to temozolomide in glioblastoma (GB)(Glogowska et al., 2013; Thanasupawat et al., 2018). We have generated high-affinity rabbit antisera to human CTRP8 to identify a selective mast cell subpopulation in human prostate cancer tissues (Hempel Sullivan et al., 2020). Mast cells are innate immune cells which are primarily known for their role in the mediation of allergic responses. They have been detected within prostate cancer tissues and serve as potential prognostic marker in prostate cancer (Johansson et al., 2010; Hempel Sullivan et al., 2020). Methods Affinity-purified rabbit polyclonal antisera directed against an epitope unique to human CTRP8 were characterized by Western blot, immunofluorescence, and immunohistochemistry in prostate cancer tissue sections and human prostate cancer tissue microarrays (TMAs). Results Of several Flag-tagged human and mouse CTRP family members transiently expressed in HEK293T cells, the CTRP8 antisera exclusively detected human CTRP8 as assessed by Western blot, demonstrating the specificity of the antisera. We studied the expression of CTRP8 in human prostate tissues. CTRP8 was expressed in a subpopulation of human mast cells distributed in prostate cancer tissues, as demonstrated by co-localization of CTRP8 immunoreactivity and toluidine blue positive mast cells in prostate cancer tissues. This was further validated by dual immunofluorescence of CTRP8 with Tryptase, a marker for mature mast cells. Initial analysis of prostate cancer tissue microarrays (TMAs) (n= 360) revealed a comparative increase in the proportion of Tryptase+/ CTRP8+ cells versus total number of mast cells (Tryptase+/ CTRP8+ and Tryptase+/ CTRP8- cells) in the extra-tumoral compartment as compared to intra-tumoral regions for Gleason grades 6 and 7 prostate cancer samples. Weak staining of CTRP8 was detected in prostate epithelial cells. The RXFP1+ human prostate cancer cell line PC3 responded to CTRP8 treatment with increased protein kinase C delta signaling and enhanced motility as determined by real-time migration assays. Conclusions Here we have identified the expression of adipokine family member CTRP8 in prostate epithelial cells and in a subpopulation of mature mast cells in patient prostate cancer tissues, thus, identifying CTRP8 as a novel player in prostate cancer and associated mast cell compartment. References Glogowska, A., et al. J. Pathol., 231, 466–479, 2013 Thanasupawat, T., et al. Mol. Oncol. 12, 1464–1479, 2018 Johansson, A., Am. J. Pathol, 177, 1031–1041, 2010 Hempel Sullivan H, et al. J. Pathol., 2020 Hempel Sullivan H, et al. Cancer Epidemiol Biomarkers Prev., 2020

An AGS associated mutation causes cellular RNA editing deficiency in mouse brain which lead to IFN pathway activation, mimicking the interferonopathy of patient brains

Abstract Objective: To determine whether and how a gene mutation found in Aicardi-Goutières syndrome (AGS), a severe autoimmune encephalopathy, is sufficient to activate the interferon signaling pathway in the brain. Background: Genetic mutations in six coding genes were found associated with AGS patients, however, none of them was demonstrated able to cause IFN pathway activation in the brain tissue, the most critical pathogenetic change of AGS. How an AGS-associated mutation activates the IFN pathway in the brain that leads to brain injury remains to be revealed. Methods: A knock-In mouse model was established through CRISPR/Cas9 gene editing approach, in which a single G&amp;gt;T nucleotide replacement was introduced into mouse genome, that encodes the change for p.K948N in mouse ADAR1 at its catalytic domain, equivalent to the p.K999N mutation found in AGS patients. We analyzed the enzyme activity of ADAR1 on the neuron specific mRNA substrates in the KI mice through RNA sequencing, the activities of IFN signaling pathway by measuring expressions of IFN stimulated inflammatory cytokines. Brain injury was observed by pathology studies. Results: The genetic mutation was confirmed. RNA transcription and protein translation were tested not affected by the mutation. The cellular RNA process in neurons was altered due to decreased enzyme activity of mutant ADAR1 on certain mRNA substrates. ISG expression were significantly increased at RNA and protein levels. Cell death in the brain was observed in the KI mice. Conclusion: A mouse model was successfully established recapitulating the genetic and innate immunologic features of AGS. An ADAR1 gene mutation is sufficient to cause IFN pathway activation in the brain through altering cellular RNA process.

GPCR‐omics of the Nephron: Mapping Receptors Along the Renal Tubule

Kidney transport and other renal functions are regulated by multiple G-protein coupled receptors (GPCRs) expressed along the renal tubule. Development of unbiased ‘-omics’ approaches and next-generation sequencing (NGS)-based techniques, including transcriptomics in microdissected renal tubules, single-cell transcriptomics, and proteomics in microdissected tubules have provided a means of identifying patterns of GPCR expression. To allow for complete GPCR mapping, we first curated a comprehensive list of GPCRs in the genomes of rats, mice and humans (https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/). From this database we mapped the expression of 758 GPCRs across fourteen nephron segments using existing transcriptomic data, providing a complete picture of GPCR expression (https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/TubuleTPM.html). We used this list to mine segment-specific and cell-type specific expression data from RNA-seq studies to identify GPCRs that are selectively expressed in discrete tubule segments. Each GPCR found to be selectively expressed was also matched to known ligands, coupled G-proteins and known physiological roles in the segment in which it is selectively expressed. Comparisons of these mapped GPCRs with other transcriptomics and proteomics datasets, functional data from isolated perfused tubules, and micro-puncture studies in the literature confirms patterns of expression for well-known receptors and identifies lesser known GPCRs that are likely to play key roles in regulation of renal tubule transport and function. This study provides data resources for GPCR expression across the renal tubule, highlighting both well-known GPCRs and understudied receptors in order to provide guidance for future experiments.

A Birst of LINE‐1 and IAP Retrotransposon Expression in the Mouse Brain Following Acute Behavioral Stress

Retrotransposons belong to mobile genetic elements that account for up to ~40% of the human genome and are propagated by being transcribed into RNA, then reverse-transcribed into DNA and inserted into a new site in the genome. Physiological regulation and possible effects of such retrotranspositions in cells of an adult organism are largely unknown. In the mouse genome LINE-1 (L1) and intracisternal A particle (IAP) are among functional and the most transpositionally active retrotransposons, capable of self-copying and insertion into DNA. Recent findings indicate that L1 is active in neuronal precursor cells during differentiation, and its de novo insertions can change expression of neuron-specific genes and cause somatic mosaicism in the adult brain. Furthermore, clinical studies have identified increased L1 expression and copy numbers in the brain of patients with psychiatric disorders. However, there has been limited understanding whether such retrotranspositions happen in the adult brain, when and how they occur, and what is their role in the CNS function. In our experiments we test the hypothesis that retrotranspositions can be activated in the brain by behavioral and particularly cognitive stimuli and that this process can influence adaptive neural plasticity. In previous experiments we showed an increase in L1 DNA copy number in hippocampus, frontal cortex and cerebellum of the mouse brain, but not in the muscular and testicular tissues after a prolonged stress. In this study we tested whether an acute stress can lead to L1 and IAP mRNA expression changes in the mouse brain. We used male C57Bl/6 mice at the age of 5 months. To assess the effect of acute stress, the animals were subjected to immobilization for 30 minutes or 2 hours. The frontal cortex and hippocampus were taken for qPCR studies. Compared to naive home cage controls, mice that underwent 30 min immobilization had significantly increased mRNA levels of L1 and IAP in frontal cortex and L1 in hippocampus. After 2 hours of immobilization IAP mRNA levels were significantly increased in both frontal cortex and hippocampus compare to control group. Therefore, L1 expression is up-regulated prior to IAP expression, perhaps, due to the differences in the transcription regulation of these two elements. Thus, we have shown that expression of both L1 and IAP is rapidly induced in several brain regions by behavioral stress. Since our previous data demonstrated a stress-induced increase of L1 and IAP DNA copy number in the same brain structures, together it points to a full retrotransposition event. We further suggest that reverse transcription involved in L1 and IAP retrotransposition by behavioral stimuli may also contribute to long-term plasticity and memory formation in the nervous system.

Transgene Mapping and Genotyping Protocol Design in a Transgenic Mouse Strain by Short‐Read Whole‐Genome Sequencing

[Aim] When creating transgenic mice, multiple lines with the same transgene are often created. For example, our laboratory has three lines of hAce2 transgenic mice (hAce2-Tg). For one of them (hAce2-Tg#16), we mapped the transgene using short-read whole-genome sequencing and designed genotyping PCR to identify the line. [Methods] We extracted genomic DNA from kidneys of hAce2-Tg#16. Using PCR-free libraries prepared with the genomic DNA, paired-end 150-bp reads were performed by DNBSEQG-400 sequencer (MGI Tech) (outsourced). In order to find the junction between the genome and the transgene, we created two search programs: one to search for matches between the transgene and each read and its complementary sequence from the front (5′ side, head-match), and the other to search for matches from the back (3′ side, tail-match). We selected reads with a match length of 5 to 145 bases. After the search, we selected reads containing the mouse genome sequence in the non-matching part by BLAST search of the Ensembl database. Based on this sequence information, we designed primers for genotyping PCR based on the flanking primer method. All animal experiments were conducted in accordance with the guidelines for animal experiments of the National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan (authorization number: DS22-55). [Results and Discussion] In a total of 882,577,032 reads (total read length: about 132 GBase), the total numbers of head-matching and tail-matching reads were 1,498 and 3,060, respectively. Among them, eight reads in the head-matching reads had the same genomic sequence. Its genomic location was in the intron of the Nrxn3 gene on chromosome 12. The transgene was truncated by 82 bases from the 3′ end. On the other hand, none of the tail-matching reads contained the neighboring genomic sequence. Genotyping PCRs with flanking primers we designed based on the sequence near the junction between transgene and genome sequences confirmed that the presence or absence of the transgene only in the line (hAce2-Tg#16). The authors have already reported examples of transgene mapping by Genomic Walking (Exp Anim 53: 103-111, 2004) and whole genome sequencing of long reads (Exp Anim 69: 279-286, 2020). This study confirmed that short-read whole-genome sequencing was also useful for mapping transgenes and designing PCR for genotyping of transgenic mice.

Variant Intronic Enhancer Controls SCN10A-short Expression and Heart Conduction.

Genetic variants in SCN10A, encoding the neuronal voltage-gated sodium channel NaV1.8, are strongly associated with atrial fibrillation, Brugada syndrome, cardiac conduction velocities, and heart rate. The cardiac function of SCN10A has not been resolved, however, and diverging mechanisms have been proposed. Here, we investigated the cardiac expression of SCN10A and the function of a variant-sensitive intronic enhancer previously linked to the regulation of SCN5A, encoding the major essential cardiac sodium channel NaV1.5. The expression of SCN10A was investigated in mouse and human hearts. With the use of CRISPR/Cas9 genome editing, the mouse intronic enhancer was disrupted, and mutant mice were characterized by transcriptomic and electrophysiological analyses. The association of genetic variants at SCN5A-SCN10A enhancer regions and gene expression were evaluated by genome-wide association studies single-nucleotide polymorphism mapping and expression quantitative trait loci analysis. We found that cardiomyocytes of the atria, sinoatrial node, and ventricular conduction system express a short transcript comprising the last 7 exons of the gene (Scn10a-short). Transcription occurs from an intronic enhancer-promoter complex, whereas full-length Scn10a transcript was undetectable in the human and mouse heart. Expression quantitative trait loci analysis revealed that the genetic variants in linkage disequilibrium with genetic variant rs6801957 in the intronic enhancer associate with SCN10A transcript levels in the heart. Genetic modification of the enhancer in the mouse genome led to reduced cardiac Scn10a-short expression in atria and ventricles, reduced cardiac sodium current in atrial cardiomyocytes, atrial conduction slowing and arrhythmia, whereas the expression of Scn5a, the presumed enhancer target gene, remained unaffected. In patch-clamp transfection experiments, expression of Scn10a-short-encoded NaV1.8-short increased NaV1.5-mediated sodium current. We propose that noncoding genetic variation modulates transcriptional regulation of Scn10a-short in cardiomyocytes that impacts NaV1.5-mediated sodium current and heart rhythm. Genetic variants in and around SCN10A modulate enhancer function and expression of a cardiac-specific SCN10A-short transcript. We propose that noncoding genetic variation modulates transcriptional regulation of a functional C-terminal portion of NaV1.8 in cardiomyocytes that impacts on NaV1.5 function, cardiac conduction velocities, and arrhythmia susceptibility.

Endonuclease enrichment TAPS for cost-effective genome-wide base-resolution DNA methylation detection.

Whole genome base-resolution methylome sequencing allows for the most comprehensive analysis of DNA methylation, however, the considerable sequencing cost often limits its applications. While reduced representation sequencing can be an affordable alternative, over 80% of CpGs in the genome are not covered. Building on our recently developed TET-assisted pyridine borane sequencing (TAPS) method, we here described endonuclease enrichment TAPS (eeTAPS), which utilizes dihydrouracil (DHU)-cleaving endonuclease digestion of TAPS-converted DNA to enrich methylated CpG sites (mCpGs). eeTAPS can accurately detect 87% of mCpGs in the mouse genome with a sequencing depth equivalent to 4× whole genome sequencing. In comparison, reduced representation TAPS (rrTAPS) detected less than 4% of mCpGs with 2.5× sequencing depth. Our results demonstrate eeTAPS to be a new strategy for cost-effective genome-wide methylation analysis at single-CpG resolution that can fill the gap between whole-genome and reduced representation sequencing.

Population and subspecies diversity at mouse centromere satellites

BackgroundMammalian centromeres are satellite-rich chromatin domains that execute conserved roles in kinetochore assembly and chromosome segregation. Centromere satellites evolve rapidly between species, but little is known about population-level diversity across these loci.ResultsWe developed a k-mer based method to quantify centromere copy number and sequence variation from whole genome sequencing data. We applied this method to diverse inbred and wild house mouse (Mus musculus) genomes to profile diversity across the core centromere (minor) satellite and the pericentromeric (major) satellite repeat. We show that minor satellite copy number varies more than 10-fold among inbred mouse strains, whereas major satellite copy numbers span a 3-fold range. In contrast to widely held assumptions about the homogeneity of mouse centromere repeats, we uncover marked satellite sequence heterogeneity within single genomes, with diversity levels across the minor satellite exceeding those at the major satellite. Analyses in wild-caught mice implicate subspecies and population origin as significant determinants of variation in satellite copy number and satellite heterogeneity. Intriguingly, we also find that wild-caught mice harbor dramatically reduced minor satellite copy number and elevated satellite sequence heterogeneity compared to inbred strains, suggesting that inbreeding may reshape centromere architecture in pronounced ways.ConclusionTaken together, our results highlight the power of k-mer based approaches for probing variation across repetitive regions, provide an initial portrait of centromere variation across Mus musculus, and lay the groundwork for future functional studies on the consequences of natural genetic variation at these essential chromatin domains.