Predicting environmentally responsive transgenerational differential DNA methylated regions (epimutations) in the genome using a hybrid deep-machine learning approach

Background Deep learning is an active bioinformatics artificial intelligence field that is useful in solving many biological problems, including predicting altered epigenetics such as DNA methylation regions. Deep learning (DL) can learn an informative representation that addresses the need for defining relevant features. However, deep learning models are computationally expensive, and they require large training datasets to achieve good classification performance. Results One approach to addressing these challenges is to use a less complex deep learning network for feature selection and Machine Learning (ML) for classification. In the current study, we introduce a hybrid DL-ML approach that uses a deep neural network for extracting molecular features and a non-DL classifier to predict environmentally responsive transgenerational differential DNA methylated regions (DMRs), termed epimutations, based on the extracted DL-based features. Various environmental toxicant induced epigenetic transgenerational inheritance sperm epimutations were used to train the model on the rat genome DNA sequence and use the model to predict transgenerational DMRs (epimutations) across the entire genome. Conclusion The approach was also used to predict potential DMRs in the human genome. Experimental results show that the hybrid DL-ML approach outperforms deep learning and traditional machine learning methods.

The rat genome database (RGD) facilitates genomic and phenotypic data integration across multiple species for biomedical research

Model organism research is essential for discovering the mechanisms of human diseases by defining biologically meaningful gene to disease relationships. The Rat Genome Database (RGD, (https://rgd.mcw.edu)) is a cross-species knowledgebase and the premier online resource for rat genetic and physiologic data. This rich resource is enhanced by the inclusion and integration of comparative data for human and mouse, as well as other human disease models including chinchilla, dog, bonobo, pig, 13-lined ground squirrel, green monkey, and naked mole-rat. Functional information has been added to records via the assignment of annotations based on sequence similarity to human, rat, and mouse genes. RGD has also imported well-supported cross-species data from external resources. To enable use of these data, RGD has developed a robust infrastructure of standardized ontologies, data formats, and disease- and species-centric portals, complemented with a suite of innovative tools for discovery and analysis. Using examples of single-gene and polygenic human diseases, we illustrate how data from multiple species can help to identify or confirm a gene as involved in a disease and to identify model organisms that can be studied to understand the pathophysiology of a gene or pathway. The ultimate aim of this report is to demonstrate the utility of RGD not only as the core resource for the rat research community but also as a source of bioinformatic tools to support a wider audience, empowering the search for appropriate models for human afflictions.

Characterization of an active LINE-1 in the naked mole-rat genome

Naked mole-rats (NMRs, Heterocephalus glaber) are the longest-living rodent species. A reason for their long lifespan is pronounced cancer resistance. Therefore, researchers believe that NMRs have unknown secrets of cancer resistance and seek to find them. Here, to reveal the secrets, we noticed a retrotransposon, long interspersed nuclear element 1 (L1). L1s can amplify themselves and are considered endogenous oncogenic mutagens. Since the NMR genome contains fewer L1-derived sequences than other mammalian genomes, we reasoned that the retrotransposition activity of L1s in the NMR genome is lower than those in other mammalian genomes. In this study, we successfully cloned an intact L1 from the NMR genome and named it NMR-L1. An L1 retrotransposition assay using the NMR-L1 reporter revealed that NMR-L1 was active retrotransposon, but its activity was lower than that of human and mouse L1s. Despite lower retrotrasposition activity, NMR-L1 was still capable of inducing cell senescence, a tumor-protective system. NMR-L1 required the 3′ untranslated region (UTR) for retrotransposition, suggesting that NMR-L1 is a stringent-type of L1. We also confirmed the 5′ UTR promoter activity of NMR-L1. Finally, we identified the G-quadruplex structure of the 3′ UTR, which modulated the retrotransposition activity of NMR-L1. Taken together, the data indicate that NMR-L1 retrotranspose less efficiently, which may contribute to the cancer resistance of NMRs.

Novel roles of an intragenic G-quadruplex in controlling microRNA expression and cardiac function

Simultaneous dysregulation of multiple microRNAs (miRs) affects various pathological pathways related to cardiac failure. In addition to being potential cardiac disease-specific markers, miR-23b/27b/24-1 were reported to be responsible for conferring cardiac pathophysiological processes. In this study, we identified a conserved guanine-rich RNA motif within the miR-23b/27b/24-1 cluster that can form an RNA G-quadruplex (rG4) in vitro and in cells. Disruption of this intragenic rG4 significantly increased the production of all three miRs. Conversely, a G4-binding ligand tetrandrine (TET) stabilized the rG4 and suppressed miRs production in human and rodent cardiomyocytes. Our further study showed that the rG4 prevented Drosha-DGCR8 binding and processing of the pri-miR, suppressing the biogenesis of all three miRs. Moreover, CRISPR/Cas9-mediated G4 deletion in the rat genome aberrantly elevated all three miRs in the heart in vivo, leading to cardiac contractile dysfunction. Importantly, loss of the G4 resulted in reduced targets for the aforementioned miRs critical for normal heart function and defects in the L-type Ca2+ channel-ryanodine receptor (LCC-RyR) coupling in cardiomyocytes. Our results reveal a novel mechanism for G4-dependent regulation of miR biogenesis, which is essential for maintaining normal heart function.

Establishment of a Cre-rat resource for creating conditional and physiological relevant models of human diseases

The goal of this study is to establish a Cre/loxP rat resource for conditional and physiologically predictive rat models of human diseases. The laboratory rat (R. norvegicus) is a central experimental animal in several fields of biomedical research, such as cardiovascular diseases, aging, infectious diseases, autoimmunity, cancer models, transplantation biology, inflammation, cancer risk assessment, industrial toxicology, pharmacology, behavioral and addiction studies, and neurobiology. Up till recently, the ability of creating genetically modified rats has been limited compared to that in the mouse mainly due to lack of genetic manipulation tools and technologies in the rat. Recent advances in nucleases, such as CRISPR/Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR associated protein 9), as well as TARGATT™ integrase system enables fast, efficient and site-specific introduction of exogenous genetic elements into the rat genome. Here, we report the generation of a collection of tissue-specific, inducible transgenic Cre rats as tool models using TARGATT™, CRISPR/Cas9 and random transgenic approach. More specifically, we generated Cre driver rat models that allow controlled gene expression or knockout (conditional models) both temporally and spatially through the Cre-ERT2/loxP system. A total of 10 Cre rat lines and one Cre reporter/test line were generated, including eight (8) Cre lines for neural specific and two (2) lines for cardiovascular specific Cre expression. All of these lines have been deposited with the Rat Resource and Research Center and provide a much-needed resource for the bio-medical community who employ rat models for their studies of human diseases.

Abstract 12677: Impaired Cardiomyocyte Contractility to Angiotensin-(1-12) in a Humanized Angiotensinogen Model of Hypertension

Background: Angiotensin-(1-12) [Ang-(1-12)] is a chymase-dependent source for Angiotensin II (Ang II) inotropic activity that may be impaired in a model of sustained hypertension with high cardiac Ang II content due to insertion of the human angiotensinogen (AGT) gene in the rat genome. Accordingly, we evaluated the effects of Ang-(1-12) and Ang II on myocyte contractility and [Ca 2+ ] i regulation in 9 adult male transgenic rats expressing the human AGT gene [TGR(hAGT)L1623)] and 9 SD controls. Methods: We compared LV myocyte contraction, relaxation and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to Ang-(1-12) (4x10 -6 M) and Ang II (10 -6 M) in freshly isolated LV myocytes. Results: Myocyte contraction (dL/dt max , 109.6 vs 127.9 μm/s), relaxation (dR/dt max , 95.3 vs 107.5 μm/s) and [Ca 2+ ] iT (0.15 vs 0.24) were depressed in TGR(hAGT)L1623 rats compared to SD controls. Moreover, cell contractile and [Ca 2+ ] iT responses following exposure to Ang-(1-12) or Ang II were markedly blunted. In SD myocytes, versus baseline, Ang II or Ang-(1-12) superfusion produced significant increases in dL/dt max [Ang II: 44% vs Ang-(1-12): 34%], dR/dt max (33% vs 26%) and [Ca 2+ ] iT (31% vs 25%). Importantly, the magnitude of the responses to the two agents in TGR(hAGT)L1623 myocytes was significantly reduced. Versus the changes in SD myocytes, Ang-(1-12) caused significantly less increases in dL/dt max (22%), dR/dt max (16%) and [Ca 2+ ] iT (15%) in TGR(hAGT)L1623 myocytes . Ang II also caused similar significantly attenuated increases in dL/dt max (27%), dR/dt max (25%) and [Ca 2+ ] iT (23%). The Ang-(1-12)-induced inotropic effects were completely prevented in the presence of the inhibitory cAMP analog, Rp-cAMPS (10 –4 M, 2 hours) in both SD and TGR(hAGT)L1623 myocytes, but were further augmented only intransgenic rats after incubation of myocytes with the G i inhibitor, pertussis toxin (PTX, 2 μg/ml, 36°C, 5 hours). Conclusions: Ang-(1-12) stimulates LV myocyte contractile function and [Ca 2+ ] iT in both SD and TGR(hAGT)L1623 rats. Furthermore, we now show that the blunted inotropic responses to Ang-(1-12) and Ang II in rats expressing the human AGT gene is mediated through a cAMP-dependent mechanism that is coupled to both stimulatory G and inhibitory PTX-sensitive G proteins.

Rat models of human diseases and related phenotypes: a systematic inventory of the causative genes

The rat has been used for a long time as the model of choice in several biomedical disciplines. Numerous inbred strains have been isolated, displaying a wide range of phenotypes and providing many models of human traits and diseases. Rat genome mapping and genomics was considerably developed in the last decades. The availability of these resources has stimulated numerous studies aimed at discovering disease genes by positional identification. Numerous rat genes have now been identified that underlie monogenic or complex diseases and remarkably, these results have been translated to the human in a significant proportion of cases, leading to the identification of novel human disease susceptibility genes, helping in studying the mechanisms underlying the pathological abnormalities and also suggesting new therapeutic approaches. In addition, reverse genetic tools have been developed. Several genome-editing methods were introduced to generate targeted mutations in genes the function of which could be clarified in this manner [generally these are knockout (KO) mutations]. Furthermore, even when the human gene causing a disease is identified, mutated rat strains (in particular KO strains) were created to analyze the gene function and the disease pathogenesis. Today, about 300 rat genes have been identified as underlying diseases or playing a key role in critical biological processes that are altered in diseases. This article provides the reader with an inventory of these genes.

Rat models of human diseases and related phenotypes: a systematic inventory of the causative genes

The rat has been used for a long time as the model of choice in several biomedical disciplines. Numerous inbred strains have been isolated, displaying a wide range of phenotypes and providing many models of human traits and diseases. Rat genome mapping and genomics was considerably developed in the last decades. The availability of these resources has stimulated numerous studies aimed at discovering disease genes by positional identification. Numerous rat genes have now been identified that underlie monogenic or complex diseases and remarkably, these results have been translated to the human in a significant proportion of cases, leading to the identification of novel human disease susceptibility genes, helping in studying the mechanisms underlying the pathological abnormalities and also suggesting new therapeutic approaches. In addition, reverse genetic tools have been developed. Several genome-editing methods were introduced to generate targeted mutations in genes the function of which could be clarified in this manner [generally these are knockout (KO) mutations]. Furthermore, even when the human gene causing a disease is identified, mutated rat strains (in particular KO strains) were created to analyze the gene function and the disease pathogenesis. Today, about 300 rat genes have been identified as underlying diseases or playing a key role in critical biological processes that are altered in diseases. This article provides the reader with an inventory of these genes.

The Year of the Rat: The Rat Genome Database at 20: a multi-species knowledgebase and analysis platform

Formed in late 1999, the Rat Genome Database (RGD, https://rgd.mcw.edu) will be 20 in 2020, the Year of the Rat. Because the laboratory rat, Rattus norvegicus, has been used as a model for complex human diseases such as cardiovascular disease, diabetes, cancer, neurological disorders and arthritis, among others, for >150 years, RGD has always been disease-focused and committed to providing data and tools for researchers doing comparative genomics and translational studies. At its inception, before the sequencing of the rat genome, RGD started with only a few data types localized on genetic and radiation hybrid (RH) maps and offered only a few tools for querying and consolidating that data. Since that time, RGD has expanded to include a wealth of structured and standardized genetic, genomic, phenotypic, and disease-related data for eight species, and a suite of innovative tools for querying, analyzing and visualizing this data. This article provides an overview of recent substantial additions and improvements to RGD’s data and tools that can assist researchers in finding and utilizing the data they need, whether their goal is to develop new precision models of disease or to more fully explore emerging details within a system or across multiple systems.