Mouse Genome Research Articles

YY1 is a transcriptional activator of the mouse LINE-1 Tf subfamily.

Long interspersed element type 1 (LINE-1, L1) is an active autonomous transposable element in human and mouse genomes. L1 transcription is controlled by an internal RNA polymerase II promoter in the 5' untranslated region (5'UTR) of a full-length L1. It has been shown that transcription factor YY1 binds to a conserved sequence at the 5' end of the human L1 5'UTR and primarily dictates where transcription initiates. Putative YY1-binding motifs have been predicted in the 5'UTRs of two distinct mouse L1 subfamilies, Tf and Gf. Using site-directed mutagenesis, in vitro binding and gene knockdown assays, we experimentally tested the role of YY1 in mouse L1 transcription. Our results indicate that Tf, but not Gf subfamily, harbors functional YY1-binding sites in 5'UTR monomers and YY1 functions as a transcriptional activator for the mouse Tf subfamily. Activation of Tf transcription by YY1 during early embryogenesis is also supported by a reanalysis of published zygotic knockdown data. Furthermore, YY1-binding motifs are solely responsible for the synergistic interaction between Tf monomers, consistent with a model wherein distant monomers act as enhancers for mouse L1 transcription. The abundance of YY1-binding sites in Tf elements also raise important implications for gene regulation across the genome.

Molecular Interactions between Kaempferol and Apolipoprotein A-I (APOA1) of Rattus Norvegicus of Using Drug Docking

Nowadays, pharmacological research investigations are essential to the development of new medication prospects. We have created high-resolution synteny and rearrangement breakpoint maps across the human, mouse, and rat genomes using paired-end sequences from bacterial artificial chromosomes. Apolipoprotein A-I (APOA1) is often associated with a markedly elevated risk of atherosclerosis and cardiovascular disease. Several studies in the field of clinico-genetics have confirmed this fact. In this work, we employ 3D Insilico drug docking techniques to simulate kaempferol interactions with the potential mutant target protein Apolipoprotein A-I (APOA1). Kaempferol is one of the primary phytochemicals present in Hibiscus rosa-sinensis. Numerous pharmacological effects of Hibiscus rosa-sinensis have been shown to be effective in treating a wide spectrum of human ailments. We look into the connection between APOA1 and kaempferol. In post-docking tests, advanced 3D molecular visualization capabilities were utilized. Kaempferol directly reduces amino acid mutational sites, as indicated by the docking study results. The molecular 3D H-bond interaction between APOA1 and Kaempferol is illustrated using concepts of molecular dynamics techniques based on 3D views. Docking studies play a key role in pharmaceutical researches for the production of novel drug candidates. Thus, investigations on Rattus norvegicus with kaempferol plays a significant role in the pharmacological industry in designing innovative medication candidates. Lastly, we conclude that kaempferol is protective against cardiovascular diseases associated with atherosclerosis.

Golgi-localized Ring Finger Protein 121 is necessary for MYCN-driven neuroblastoma tumorigenesis

MYCN amplification predicts poor prognosis in childhood neuroblastoma. To identify MYCN oncogenic signal dependencies we performed N-ethyl-N-nitrosourea (ENU) mutagenesis on the germline of neuroblastoma-prone TH-MYCN transgenic mice to generate founders which had lost tumorigenesis. Sequencing of the mutant mouse genomes identified the Ring Finger Protein 121 (RNF121WT) gene mutated to RNFM158R associated with heritable loss of tumorigenicity. While the RNF121WT protein localised predominantly to the cis-Golgi Complex, the RNF121M158R mutation in Helix 4 of its transmembrane domain caused reduced RNF121 protein stability and absent Golgi localisation. RNF121WT expression markedly increased during TH-MYCN tumorigenesis, whereas hemizygous RNF121WT gene deletion reduced TH-MYCN tumorigenicity. The RNF121WT-enhanced growth of MYCN-amplified neuroblastoma cells depended on RNF121WT transmembrane Helix 5. RNF121WT directly bound MYCN protein and enhanced its stability. High RNF121 mRNA expression associated with poor prognosis in human neuroblastoma tissues and another MYC-driven malignancy, laryngeal cancer. RNF121 is thus an essential oncogenic cofactor for MYCN and a target for drug development.

ScNanoSeq-CUT&Tag: a single-cell long-read CUT&Tag sequencing method for efficient chromatin modification profiling within individual cells.

Chromatin modifications are fundamental epigenetic marks that determine genome functions, but it remains challenging to profile those of repetitive elements and complex genomic regions. Here, we develop scNanoSeq-CUT&Tag, a streamlined method, by adapting modified cleavage under targets and tagmentation (CUT&Tag) to the nanopore sequencing platform for genome-wide chromatin modification profiling within individual cells. We show that scNanoSeq-CUT&Tag can accurately profile histone marks and transcription factor occupancy patterns at single-cell resolution as well as distinguish different cell types. scNanoSeq-CUT&Tag efficiently maps the allele-specific chromatin modifications and allows analysis of their neighboring region co-occupancy patterns within individual cells. Moreover, scNanoSeq-CUT&Tag can accurately detect chromatin modifications for individual copies of repetitive elements in both human and mouse genomes. Overall, we prove that scNanoSeq-CUT&Tag is a valuable single-cell tool for efficiently profiling histone marks and transcription factor occupancies, especially for previously poorly studied complex genomic regions and blacklist genomic regions.

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

Exploring the genetic landscape of the brain-heart axis: A comprehensive analysis of pleiotropic effects between heart disease and psychiatric disorders

BackgroundThe genetic links between heart disease and psychiatric disorders are complex and not well understood. This study uses genome-wide association studies (GWAS) and advanced multilevel analyses to explore these connections. MethodsWe analyzed GWAS data from seven psychiatric disorders and five types of heart disease. Genetic correlations and overlaps were examined using linkage disequilibrium score regression (LDSC), high-definition likelihood (HDL), and Genetic analysis incorporating Pleiotropy and Annotation (GPA). Pleiotropic single-nucleotide variations (SNVs) were identified with pleiotropic analysis under the composite null hypothesis (PLACO) and annotated via Functional mapping and annotation of genetic associations (FUMA). Potential pleiotropic genes were identified using Multi-marker Analysis of GenoMic Annotation (MAGMA) and Summary data-based Mendelian Randomization (SMR). Mendelian randomization was employed to assess causal relationships. ResultsAmong 35 trait pairs, 32 showed significant genetic correlations or overlaps. PLACO identified 15,077 SNVs, with 287 recognized as pleiotropic loci and 20 colocalization sites. MAGMA and SMR revealed 75 potential pleiotropic genes involved in diverse pathways, including cancer, neurodevelopment, and cellular organization. Mouse Genome Informatics (MGI) queries provided evidence linking multiple genes to heart or psychiatric disorders. ConclusionsThis analysis reveals loci and genes with pleiotropic effects between heart disease and psychiatric disorders, highlighting shared biological pathways. These findings illuminate the genetic mechanisms underlying the brain-heart axis and suggest shared biological foundations for these conditions, offering potential targets for future prevention and treatment strategies.

Loss-of-function of RNA-binding protein PRRC2B causes translational defects and congenital cardiovascular malformation.

Alternative splicing generates variant forms of proteins for a given gene and accounts for functional redundancy or diversification. A novel RNA-binding protein, Pro-rich Coiled-coil Containing Protein 2B (PRRC2B), has been reported by multiple laboratories to mediate uORF-dependent and independent regulation of translation initiation required for cell cycle progression and proliferation. We identified two alternative spliced isoforms in human and mouse hearts and HEK293T cells, full-length (FL) and exon 16-excluded isoform ΔE16. A congenital heart disease-associated human mutation-mimicry knock-in of the equivalent variant in the mouse genome leads to the depletion of the full-length Prrc2b mRNA but not the alternative spliced truncated form ΔE16, does not cause any apparent structural or functional disorders. In contrast, global genetic inactivation of the PRRC2B gene in the mouse genome, nullifying both mRNA isoforms, caused patent ductus arteriosus (PDA) and neonatal lethality in mice. Bulk and single nucleus transcriptome profiling analyses of embryonic mouse hearts demonstrated a significant overall downregulation of multiple smooth muscle-specific genes in Prrc2b mutant mice resulting from reduced smooth muscle cell number. Integrated analysis of proteomic changes in Prrc2b null mouse embryonic hearts and polysome-seq and RNA-seq multi-omics analysis in human HEK293T cells uncover conserved PRRC2B-regulated target mRNAs that encode essential factors required for cardiac and vascular development. Our findings reveal the connection between alternative splicing regulation of PRRC2B, PRRC2B-mediated translational control, and congenital cardiovascular development and disorder. This study may shed light on the significance of PRRC2B in human cardiovascular disease diagnosis and treatment.

Full-length RNA transcript sequencing traces brain isoform diversity in house mouse natural populations.

The ability to generate multiple RNA transcript isoforms from the same gene is a general phenomenon in eukaryotes. However, the complexity and diversity of alternative isoforms in natural populations remain largely unexplored. Using a newly developed full-length transcript enrichment protocol with 5' CAP selection, we sequenced full-length RNA transcripts of 48 individuals from outbred populations and subspecies of Mus musculus, and from the closely related sister species Mus spretus and Mus spicilegus as outgroups. The data set represents the most extensive full-length high-quality isoform catalog at the population level to date. In total, we reliably identify 117,728 distinct isoforms, of which only 51% were previously annotated. We show that the population-specific distribution pattern of isoforms is phylogenetically informative and reflects the segregating single nucleotide polymorphism (SNP) diversity between the populations. We find that ancient housekeeping genes are a major source of the overall isoform diversity, and that the generation of alternative first exons plays a major role in generating new isoforms. Given that our data allow us to distinguish between population-specific isoforms and isoforms that are conserved across multiple populations, it is possible to refine the annotation of the reference mouse genome to a set of about 40,000 isoforms that should be most relevant for comparative functional analysis across species.

Sexually dimorphic renal expression of mouse Klotho is directed by a kidney-specific distal enhancer responsive to HNF1b

Transcription enhancers are genomic sequences regulating common and tissue-specific genes and their disruption can contribute to human disease development and progression. Klotho, a sexually dimorphic gene specifically expressed in kidney, is well-linked to kidney dysfunction and its deletion from the mouse genome leads to premature aging and death. However, the sexually dimorphic regulation of Klotho is not understood. Here, we characterize two candidate Klotho enhancers using H3K27ac epigenetic marks and transcription factor binding and investigate their functions, individually and combined, through CRISPR-Cas9 genome engineering. We discovered that only the distal (E1), but not the proximal (E2) candidate region constitutes a functional enhancer, with the double deletion not causing Klotho expression to further decrease. E1 activity is dependent on HNF1b transcription factor binding site within the enhancer. Further, E1 controls the sexual dimorphism of Klotho as evidenced by qPCR and RNA-seq. Despite the sharp reduction of Klotho mRNA, unlike germline Klotho knockouts, mutant mice present normal phenotype, including weight, lifespan, and serum biochemistry. Lastly, only males lacking E1 display more prominent acute, but not chronic kidney injury responses, indicating a remarkable range of potential adaptation to isolated Klotho loss, especially in female E1 knockouts, retaining renoprotection despite over 80% Klotho reduction.

Predicting Differentially Methylated Cytosines in TET and DNMT3 Knockout Mutants via a Large Language Model.

DNA cytosine methylation is an epigenetic marker which regulates many cellular processes. Mammalian genomes typically maintain consistent methylation patterns over time, except in specific regulatory regions like promoters and certain types of enhancers. The dynamics of DNA methylation is controlled by a complex cellular machinery, in which the enzymes DNMT3 and TET play a major role. This study explores the identification of differentially methylated cytosines (DMCs) in TET and DNMT3 knockout mutants in mice and human embryonic stem cells. We investigate (i) whether a large language model can be trained to recognize DMCs in human and mouse from the sequence surrounding the cytosine of interest, (ii) whether a classifier trained on human knockout data can predict DMCs in the mouse genome (and vice versa), (iii) whether a classifier trained on DNMT3 knockout can predict DMCs for TET knockout (and vice versa). Our study identifies statistically significant motifs associated with the prediction of DMCs each mutant, casting a new light on the understanding of DNA methylation dynamics in stem cells. Our software tool is available at https://github.com/ucrbioinfo/dmc_prediction .

Enriched G4 forming repeats in the human genome are associated with robust well-coordinated transcription and reduced cancer transcriptome variation

Non-B DNA G-quadruplex (G4) structures with guanine (G) runs of 2-4 repeats can trigger opposing experimental transcriptional impacts. Here, we employed bioinformatic algorithms to comprehensively assess correlations of steady-state RNA transcript levels with all putative G4 sequence (pG4) locations genome-wide in three mammalian genomes and in normal and tumor human tissues. The human pG4-containing gene set displays higher expression levels than the set without pG4, supporting and extending some prior observations. pG4 enrichment at transcription start sites (TSS) in human, but not chimpanzee and mouse genomes, suggests possible positive selection pressure for pG4 at human TSS, potentially driving genome rewiring and gene expression divergence between human and chimpanzee. Comprehensive bioinformatic analyses revealed lower pG4-containing gene set variability in humans and among different pG4 genes in tumors. As G4 stabilizers are under therapeutic consideration for cancer and pathogens, such distinctions between human normal and tumor G4s along with other species merit attention. Furthermore, in germline and cancer sequences, the most mutagenic pG4 mapped to regions promoting alternative DNA structures. Overall findings establish high pG4 at TSS as a human genome attribute statistically associated with robust well-coordinated transcription and reduced cancer transcriptome variation with implications for biology, model organisms, and medicine.

RNA helicase D1PAS1 resolves R-loops and forms a complex for mouse pachytene piRNA biogenesis required for male fertility.

During meiosis, RNA polymerase II transcribes pachytene piRNA precursors with unusually long and unspliced transcripts from discrete autosomal loci in the mouse genome. Despite the importance of piRNA for male fertility and a well-defined maturation process, the transcriptional machinery remains poorly understood. Here, we document that D1PAS1, an ATP-dependent RNA helicase, is critical for pachytene piRNA expression from multiple genomic loci and subsequent translocation into the cytoplasm to ensure mature piRNA biogenesis. Depletion of D1PAS1 in gene-edited mice results in the accumulation of R-loops in pachytene spermatocytes, leading to DNA-damage-induced apoptosis, disruption of piRNA biogenesis, spermatogenic arrest, and male infertility. Transcriptome, genome-wide R-loop profiling, and proteomic analyses document that D1PAS1 regulates pachytene piRNA transcript elongation and termination. D1PAS1 subsequently forms a complex with nuclear export components to ensure pachytene piRNA precursor translocation from the nucleus to the cytoplasm for processing into small non-coding RNAs. Thus, our study defines D1PAS1 as a specific transcription activator that promotes R-loop unwinding and is a critical factor in pachytene piRNA biogenesis.

GMMID: genetically modified mice information database.

Genetically engineered mouse models (GEMMs) are vital for elucidating gene function and disease mechanisms. An overwhelming number of GEMM lines have been generated, but endeavors to collect and organize the information of these GEMMs are seriously lagging behind. Only a few databases are developed for the information of current GEMMs, and these databases lack biological descriptions of allele compositions, which poses a challenge for nonexperts in mouse genetics to interpret the genetic information of these mice. Moreover, these databases usually do not provide information on human diseases related to the GEMM, which hinders the dissemination of the insights the GEMM provides as a human disease model. To address these issues, we developed an algorithm to annotate all the allele compositions that have been reported with Python programming and have developed the genetically modified mice information database (GMMID; http://www.gmmid.cn), a user-friendly database that integrates information on GEMMs and related diseases from various databases, including National Center for Biotechnology Information, Mouse Genome Informatics, Online Mendelian Inheritance in Man, International Mouse Phenotyping Consortium, and Jax lab. GMMID provides comprehensive genetic information on >70 055 alleles, 65 520 allele compositions, and ∼4000 diseases, along with biologically meaningful descriptions of alleles and allele combinations. Furthermore, it provides spatiotemporal visualization of anatomical tissues mentioned in these descriptions, shown alongside the allele compositions. Compared to existing mouse databases, GMMID considers the needs of researchers across different disciplines and presents obscure genetic information in an intuitive and easy-to-understand format. It facilitates users in obtaining complete genetic information more efficiently, making it an essential resource for cross-disciplinary researchers. Database URL: http://www.gmmid.cn.

Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development.

Chromatin domain boundaries delimited by CTCF motifs can restrict the range of enhancer action. However, disruption of domain structure often results in mild gene dysregulation and thus predicting the impact of boundary rearrangements on animal development remains challenging. Here, we tested whether structural perturbation of a chromatin domain with multiple developmental regulators can result in more acute gene dysregulation and severe developmental phenotypes. We targeted clusters of CTCF motifs in a domain of the mouse genome containing three FGF ligand genes - Fgf3, Fgf4, and Fgf15 - that regulate several developmental processes. Deletion of the 23.9kb cluster that defines the centromeric boundary of this domain resulted in ectopic interactions of the FGF genes with enhancers located across the deleted boundary that are active in the developing brain. This caused strong induction of FGF expression and perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary deletion was sufficient to cause these fully penetrant phenotypes, and strikingly, loss of a single CTCF motif within the cluster also recapitulated ectopic FGF expression and caused encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF clusters of this domain, nor to all developmental processes controlled by these three FGF genes - for example, the ability to undergo lineage specification in the blastocyst and pre-implantation development were not affected. By tracing the impact of different chromosomal rearrangements throughout mouse development, we start to uncover the determinants of phenotypic robustness and sensitivity to perturbation of chromatin boundaries. Our data show how small sequence variants at certain domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation during development and disease.

Abstract We002: A natural loss-of-function deletion of the cytohesin 1 (Cyth1) gene in BALB/cByJ mice does not impact cardiomyocyte polyploidy

The adult mammalian heart has limited regenerative capacity due in part to the low level of diploid cardiomyocytes (CMs). Multiple polymorphic genes determine the final level of diploid CMs, however, the relevant genes need to be identified. There is a large variation in CM ploidy between the highly related sister strains BALB/cJ and BALB/cByJ. We undertook an RNA-Seq comparison of neonatal ventricular heart tissue from BALB/cJ and BALB/cByJ to identify gene expression differences that might underlie this difference in CM ploidy. Cytohesin-1 (Cyth1) and Dynein heavy chain 17 (Dnah17) were particularly different in expression: in BALB/cJ (as in most other mouse strains), Cyth1 is expressed in the heart and Dnah17 isn’t expressed, whereas the opposite profile (Cyth1 off, Dnah17 on) was observed in BALB/cByJ heart. These two genes are immediately adjacent to each other in the mouse genome. We found that the basis of differential expression is a large deletion in the locus of BALB/cByJ mice spanning from the Cyth1 first intron to shortly downstream of the last Cyth1 exon. This creates a complete null Cyth1 allele and at the same time results in ectopic transcription of the downstream gene Dnah17, although this transcript is unlikely to encode a protein. Cyth1 encodes a guanine nucleotide exchange factor that was a plausible candidate for being involved in CM ploidy. Using the natural Cyth1 mutant locus plus an engineered null allele, we found no difference in CM ploidy in the absence of Cyth1 function on a cJ-cByJ F1 background or on an inbred C57BL/6J background. This study successfully identified a natural loss-of-function variant in the Cyth1 gene between BALB/cJ and BALB/cByJ but showed that this does not impact CM ploidy, at least by itself. It may underlie other phenotypic differences between these two strains.

Abstract We018: The Molecular Mechanism of Coronary Collateral Growth Induced by Repetitive Ischemia by Single Cell-RNA Sequencing

Coronary heart diseases (CHD) are the leading cause of mortality and morbidity in the United States. A well-developed coronary collateral circulation ameliorates the consequences of CHD, reducing the incidence of sudden death and infarct sizes following coronary occlusion. Stimulation of coronary collateral growth (CCG) is also an alternative therapeutic approach for patients with intractable angina pectoris. The importance of CCG is undisputable, but the process and mechanism underlying the CCG is unclear. We developed a mouse model of CCG by repetitive ischemia (RI) in adults, and the CCG was validated with contrast echo to measure the coronary blood flow 3D images of micro-CT to quantitate the CCG. In this study, we use single cell-RNA sequencing (scRNA-Seq) to analyze the molecular signaling of CCG at different time points during the RI process. We digested the hearts from RI days 5, 10, and 17 and control (naïve) mice, harvested non-cardiomyocytes, performed scRNA-Seq with 10x Genomics and sequenced with Next-Seq 500 (Illumina).The fastq files are converted to a gene count matrix using kallisto|bustools with refdata-gex-mm10-2020-A employed as the reference mouse genome. The Kallisto bustools output is then converted in R to files compatible for uploading to Cellenics, an open-source platform for single-cell RNA-Seq analysis. Dimensionality reduction is done with Principal Components Analysis (PCA) with 30 principal components. We will use UMAP to visualize cell clusters. Cellenics uses scType to annotate the cell populations automatically when building clusters of cell populations. Our preliminary data show dynamic kinetics of non-cardiomyocytes induced by RI over the timeline of CCG and different cell populations of non-cardiomyocytes and EC expression profiling varied in response to RI throughout CCG. We will validate the critical signaling in CCG and explore the potential targets to induce CCG.

Z-DNA formation in promoters conserved between human and mouse are associated with increased transcription reinitiation rates

A long-standing question concerns the role of Z-DNA in transcription. Here we use a deep learning approach DeepZ that predicts Z-flipons based on DNA sequence, structural properties of nucleotides and omics data. We examined Z-flipons that are conserved between human and mouse genomes after generating whole-genome Z-flipon maps and then validated them by orthogonal approaches based on high resolution chemical mapping of Z-DNA and the transformer algorithm Z-DNABERT. For human and mouse, we revealed similar pattern of transcription factors, chromatin remodelers, and histone marks associated with conserved Z-flipons. We found significant enrichment of Z-flipons in alternative and bidirectional promoters associated with neurogenesis genes. We show that conserved Z-flipons are associated with increased experimentally determined transcription reinitiation rates compared to promoters without Z-flipons, but without affecting elongation or pausing. Our findings support a model where Z-flipons engage Transcription Factor E and impact phenotype by enabling the reset of preinitiation complexes when active, and the suppression of gene expression when engaged by repressive chromatin complexes.

μ-Opioid receptor transcriptional variants in the murine forebrain and spinal cord

Oprm1, the gene encoding the μ-opioid receptor, has multiple reported transcripts, with a variable 3′ region and many alternative sequences encoding the C-terminus of the protein. The functional implications of this variability remain mostly unexplored, though a recurring notion is that it could be exploited by developing selective ligands with improved clinical profiles. Here, we comprehensively examined Oprm1 transcriptional variants in the murine central nervous system, using long-read RNAseq as well as spatial and single-cell transcriptomics. The results were validated with RNAscope in situ hybridization. We found a mismatch between transcripts annotated in the mouse genome (GRCm38/mm10) and the RNA-seq results. Sequencing data indicated that the primary Oprm1 transcript has a 3′ terminus located on chr10:6,860,027, which is ∼ 9.5 kilobases downstream of the longest annotated exon 4 end. Long-read sequencing confirmed that the final Oprm1 exon included a 10.2 kilobase long 3′ untranslated region, and the presence of the long variant was unambiguously confirmed using RNAscope in situ hybridization in the thalamus, striatum, cortex and spinal cord. Conversely, expression of the Oprm1 reference transcript or alternative transcripts of the Oprm1 gene was absent or close to the detection limit. Thus, the primary transcript of the Oprm1 mouse gene is a variant with a long 3′ untranslated region, which is homologous to the human OPRM1 primary transcript and encodes the same conserved C-terminal amino acid sequence.

Dose-related Mutagenic and Clastogenic Effects of Benzo[b]fluoranthene in Mouse Somatic Tissues Detected by Duplex Sequencing and the Micronucleus Assay.

We used Duplex Sequencing to study the mutagenicity of benzo[ b ]fluoranthene across the mouse genome. Dose-dependent changes in mutation frequency and spectrum quantify its role in PAH-induced carcinogenicity.