Characterization of a strain-specific CD-1 reference genome reveals potential inter- and intra-strain functional variability.

BACKGROUND: CD-1 is an outbred mouse stock that is frequently used in toxicology, pharmacology, and fundamental biomedical research. Although inbred strains are typically better suited for such studies due to minimal genetic variability, outbred stocks confer practical advantages over inbred strains, such as improved breeding performance and low cost. Knowledge of the full genetic variability of CD-1 would make it more useful in toxicology, pharmacology, and fundamental biomedical research. RESULTS: We performed deep genomic DNA sequencing of CD-1 mice and used the data to identify genome-wide SNPs, indels, and germline transposable elements relative to the mm10 reference genome. We used multiple genome-wide sequencing data types and previously published CD-1 SNPs to validate our called variants. We used the called variants to construct a strain-specific CD-1 reference genome, which we show can improve mappability and reduce experimental biases from genome-wide sequencing data derived from CD-1 mice. Based on previously published ChIP-seq and ATAC-seq data, we find evidence that genetic variation between CD-1 mice can lead to alterations in transcription factor binding. We also identified a number of variants in the coding region of genes which could have effects on translation of genes. CONCLUSIONS: We have identified millions of previously unidentified CD-1 variants with the potential to confound studies involving CD-1. We used the identified variants to construct a CD-1-specific reference genome, which can improve accuracy and reduce bias when aligning genomics data derived from CD-1 mice.

Single nucleus multiome analysis of the prefrontal cortex from C9orf72 ALS/FTD patients illuminates pathways affected during disease progression.

Repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and familial frontotemporal dementia (ALS/FTD). To identify molecular defects that take place in the dorsolateral frontal cortex of patients with C9orf72 ALS/FTD, we compared healthy controls with C9orf72 ALS/FTD donor samples staged based on the levels of cortical phosphorylated TAR DNA binding protein (pTDP-43), a neuropathological hallmark of disease progression. We identified distinct molecular changes in different cell types that take place during disease progression. These alterations include downregulation of nuclear and mitochondrial ribosomal protein genes in early disease stages that become upregulated as the disease progresses. High ratios of premature oligodendrocytes expressing low levels of genes encoding major myelin protein components are characteristic of late disease stages and may represent a unique signature of C9orf72 ALS/FTD. Microglia with increased reactivity and astrocyte specific transcriptome changes in genes involved in glucose/glycogen metabolism are also associated with disease progression. Late stages of C9orf72 ALS/FTD correlate with sequential changes in the regulatory landscape of several genes in glial cells, namely MBP/MAG/MOG in oligodendrocytes, CD83/IRF8 in microglia, and GLUT1/GYS2/AGL in astrocytes. Only layer 2-3 cortical projection neurons with high expression of CUX2/LAMP5 are significantly reduced in C9orf72 ALS/FTD patients with respect to controls. Our findings reveal previously unknown progressive functional changes in cortical cells of C9orf72 ALS/FTD patients that shed light on the mechanisms underlying the pathology of this disease.

Expansion of the genotypic and phenotypic spectrum of CTCF-related disorder guides clinical management: 43 new subjects and a comprehensive literature review.

Monoallelic variants of CTCF cause an autosomal dominant neurodevelopmental disorder with a wide range of features, including impacts on the brain, growth, and craniofacial development. A growing number of subjects with CTCF-related disorder (CRD) have been identified due to the increased application of exome sequencing, and further delineation of the clinical spectrum of CRD is needed. Here, we examined the clinical features, including facial profiles, and genotypic spectrum of 107 subjects with identified CTCF variants, including 43 new and 64 previously described subjects. Among the 43 new subjects, 23 novel variants were reported. The cardinal clinical features in subjects with CRD included intellectual disability/developmental delay (91%) with speech delay (65%), motor delay (53%), feeding difficulties/failure to thrive (66%), ocular abnormalities (56%), musculoskeletal anomalies (53%), and behavioral problems (52%). Other congenital anomalies were also reported, but none of them were common. Our findings expanded the genotypic and phenotypic spectrum of CRD that will guide genetic counseling, management, and surveillance care for patients with CRD. Additionally, a newly built facial gestalt on the Face2Gene tool will facilitate prompt recognition of CRD by physicians and shorten a patient's diagnostic odyssey.

Recruitment of CTCF to an Fto enhancer is responsible for transgenerational inheritance of BPA-induced obesity.

The mechanisms by which environmentally-induced epiphenotypes are transmitted transgenerationally in mammals are poorly understood. Here we show that exposure of pregnant mouse females to bisphenol A (BPA) results in obesity in the F2 progeny due to increased food intake. This epiphenotype can be transmitted up to the F6 generation. Analysis of chromatin accessibility in sperm of the F1-F6 generations reveals alterations at sites containing binding motifs for CCCTC-binding factor (CTCF) at two cis-regulatory elements (CREs) of the Fto gene that correlate with transmission of obesity. These CREs show increased interactions in sperm of obese mice with the Irx3 and Irx5 genes, which are involved in the differentiation of appetite-controlling neurons. Deletion of the CTCF site in Fto results in mice that have normal food intake and fail to become obese when ancestrally exposed to BPA. The results suggest that epigenetic alterations of Fto can lead to the same phenotypes as genetic variants.

The Cupid shuffle: Do enhancers prefer specific promoters?

Two recent reports (Martinez-Ara et al., 2022; Bergman et al., 2022) explore the compatibility between enhancers and promoters and find that enhancers preferentially activate promoters with low intrinsic activity rather than favoring housekeeping or cell-type-specific promoters.

Beyond Genes: Germline Disruption in the Etiology of Autism Spectrum Disorders.

Investigations into the etiology of autism spectrum disorders have been largely confined to two realms: variations in DNA sequence and somatic developmental exposures. Here we suggest a third route-disruption of the germline epigenome induced by exogenous toxicants during a parent's gamete development. Similar to cases of germline mutation, these molecular perturbations may produce dysregulated transcription of brain-related genes during fetal and early development, resulting in abnormal neurobehavioral phenotypes in offspring. Many types of exposures may have these impacts, and here we discuss examples of anesthetic gases, tobacco components, synthetic steroids, and valproic acid. Alterations in parental germline could help explain some unsolved phenomena of autism, including increased prevalence, missing heritability, skewed sex ratio, and heterogeneity of neurobiology and behavior.

Is developmental synchrony enabled by CTCF residence time?

Depletion of CTCF in cultured cells has minor effects on transcription whereas its mutation leads to embryonic lethality and developmental defects. In a recent issue of Nature Cell Biology, Soochit et al. (2021) show that the residence time of CTCF on DNA may explain its critical role in cell differentiation.

Exposure to sevoflurane results in changes of transcription factor occupancy in sperm and inheritance of autism†.

One in 54 children in the United States is diagnosed with autism spectrum disorder. De novo germline and somatic mutations cannot account for all cases of autism spectrum disorder, suggesting that epigenetic alterations triggered by environmental exposures may be responsible for a subset of autism spectrum disorder cases. Human and animal studies have shown that exposure of the developing brain to general anesthetic agents can trigger neurodegeneration and neurobehavioral abnormalities, but the effects of general anesthetics on the germline have not been explored in detail. We exposed pregnant mice to sevoflurane during the time of embryonic development when the germ cells undergo epigenetic reprogramming and found that more than 38% of the directly exposed F1 animals exhibit impairments in anxiety and social interactions. Strikingly, 44-47% of the F2 and F3 animals, which were not directly exposed to sevoflurane, show the same behavioral problems. We performed ATAC-seq and identified more than 1200 differentially accessible sites in the sperm of F1 animals, 69 of which are also present in the sperm of F2 animals. These sites are located in regulatory regions of genes strongly associated with autism spectrum disorder, including Arid1b, Ntrk2, and Stmn2. These findings suggest that epimutations caused by exposing germ cells to sevoflurane can lead to autism spectrum disorder in the offspring, and this effect can be transmitted through the male germline inter- and transgenerationally.

Novel mRNAs 3' end-associated cis-regulatory elements with epigenomic signatures of mammalian enhancers in the Arabidopsis genome.

The precise spatial and temporal control of gene expression requires the coordinated action of genomic cis-regulatory elements (CREs), including transcriptional enhancers. However, our knowledge of enhancers in plants remains rudimentary and only a few plant enhancers have been experimentally defined. Here, we screened the Arabidopsis thaliana genome and identified >1900 unique candidate CREs that carry the genomic signatures of mammalian enhancers. These were termed putative enhancer-like elements (PEs). Nearly all PEs are intragenic and, unexpectedly, most associate with the 3' ends of protein-coding genes. PEs are hotspots for transcription factor binding and harbor motifs resembling cleavage/polyadenylation signals, potentially coupling 3' end processing to the transcriptional regulation of other genes. Hi-C data showed that 24% of PEs are located at regions that can interact intrachromosomally with other protein-coding genes and, surprisingly, many of these target genes interact with PEs through their 3' UTRs. Examination of the genomes of 1135 sequenced Arabidopsis accessions showed that PEs are conserved. Our findings suggest that the identified PEs may serve as transcriptional enhancers and sites for mRNA 3' end processing, and constitute a novel group of CREs in Arabidopsis.

An Overview of Methodologies in Studying lncRNAs in the High-Throughput Era: When Acronyms ATTACK!

The discovery of pervasive transcription in eukaryotic genomes provided one of many surprising (and perhaps most surprising) findings of the genomic era and led to the uncovering of a large number of previously unstudied transcriptional events. This pervasive transcription leads to the production of large numbers of noncoding RNAs (ncRNAs) and thus opened the window to study these diverse, abundant transcripts of unclear relevance and unknown function. Since that discovery, recent advances in high-throughput sequencing technologies have identified a large collection of ncRNAs, from microRNAs to long noncoding RNAs (lncRNAs). Subsequent discoveries have shown that many lncRNAs play important roles in various eukaryotic processes; these discoveries have profoundly altered our understanding of the regulation of eukaryotic gene expression. Although the identification of ncRNAs has become a standard experimental approach, the functional characterization of these diverse ncRNAs remains a major challenge. In this chapter, we highlight recent progress in the methods to identify lncRNAs and the techniques to study the molecular function of these lncRNAs and the application of these techniques to the study of plant lncRNAs.

Long Noncoding RNAs in Plants.

The eukaryotic genomes are pervasively transcribed. In addition to protein-coding RNAs, thousands of long noncoding RNAs (lncRNAs) modulate key molecular and biological processes. Most lncRNAs are found in the nucleus and associate with chromatin, but lncRNAs can function in both nuclear and cytoplasmic compartments. Emerging work has found that many lncRNAs regulate gene expression and can affect genome stability and nuclear domain organization both in plant and in the animal kingdom. Here, we describe the major plant lncRNAs and how they act, with a focus on research in Arabidopsis thaliana and our emerging understanding of lncRNA functions in serving as molecular sponges and decoys, functioning in regulation of transcription and silencing, particularly in RNA-directed DNA methylation, and in epigenetic regulation of flowering time.

Small RNAs: essential regulators of gene expression and defenses against environmental stresses in plants.

Eukaryotic genomes produce thousands of diverse small RNAs (smRNAs), which play vital roles in regulating gene expression in all conditions, including in survival of biotic and abiotic environmental stresses. SmRNA pathways intersect with most of the pathways regulating different steps in the life of a messenger RNA (mRNA), starting from transcription and ending at mRNA decay. SmRNAs function in both nuclear and cytoplasmic compartments; the regulation of mRNA stability and translation in the cytoplasm and the epigenetic regulation of gene expression in the nucleus are the main and best-known modes of smRNA action. However, recent evidence from animal systems indicates that smRNAs and RNA interference (RNAi) also participate in the regulation of alternative pre-mRNA splicing, one of the most crucial steps in the fast, efficient global reprogramming of gene expression required for survival under stress. Emerging evidence from bioinformatics studies indicates that a specific class of plant smRNAs, induced by various abiotic stresses, the sutr-siRNAs, has the potential to target regulatory regions within introns and thus may act in the regulation of splicing in response to stresses. This review summarizes the major types of plant smRNAs in the context of their mechanisms of action and also provides examples of their involvement in regulation of gene expression in response to environmental cues and developmental stresses. In addition, we describe current advances in our understanding of how smRNAs function in the regulation of pre-mRNA splicing. WIREs RNA 2016, 7:356-381. doi: 10.1002/wrna.1340 For further resources related to this article, please visit the WIREs website.

Stress-induced endogenous siRNAs targeting regulatory intron sequences in Brachypodium.

Exposure to abiotic stresses triggers global changes in the expression of thousands of eukaryotic genes at the transcriptional and post-transcriptional levels. Small RNA (smRNA) pathways and splicing both function as crucial mechanisms regulating stress-responsive gene expression. However, examples of smRNAs regulating gene expression remain largely limited to effects on mRNA stability, translation, and epigenetic regulation. Also, our understanding of the networks controlling plant gene expression in response to environmental changes, and examples of these regulatory pathways intersecting, remains limited. Here, to investigate the role of smRNAs in stress responses we examined smRNA transcriptomes of Brachypodium distachyon plants subjected to various abiotic stresses. We found that exposure to different abiotic stresses specifically induced a group of novel, endogenous small interfering RNAs (stress-induced, UTR-derived siRNAs, or sutr-siRNAs) that originate from the 3' UTRs of a subset of coding genes. Our bioinformatics analyses predicted that sutr-siRNAs have potential regulatory functions and that over 90% of sutr-siRNAs target intronic regions of many mRNAs in trans. Importantly, a subgroup of these sutr-siRNAs target the important intron regulatory regions, such as branch point sequences, that could affect splicing. Our study indicates that in Brachypodium, sutr-siRNAs may affect splicing by masking or changing accessibility of specific cis-elements through base-pairing interactions to mediate gene expression in response to stresses. We hypothesize that this mode of regulation of gene expression may also serve as a general mechanism for regulation of gene expression in plants and potentially in other eukaryotes.

The role of the Arabidopsis Exosome in siRNA-independent silencing of heterochromatic loci.

The exosome functions throughout eukaryotic RNA metabolism and has a prominent role in gene silencing in yeast. In Arabidopsis, exosome regulates expression of a "hidden" transcriptome layer from centromeric, pericentromeric, and other heterochromatic loci that are also controlled by small (sm)RNA-based de novo DNA methylation (RdDM). However, the relationship between exosome and smRNAs in gene silencing in Arabidopsis remains unexplored. To investigate whether exosome interacts with RdDM, we profiled Arabidopsis smRNAs by deep sequencing in exosome and RdDM mutants and also analyzed RdDM-controlled loci. We found that exosome loss had a very minor effect on global smRNA populations, suggesting that, in contrast to fission yeast, in Arabidopsis the exosome does not control the spurious entry of RNAs into smRNA pathways. Exosome defects resulted in decreased histone H3K9 dimethylation at RdDM-controlled loci, without affecting smRNAs or DNA methylation. Exosome also exhibits a strong genetic interaction with RNA Pol V, but not Pol IV, and physically associates with transcripts produced from the scaffold RNAs generating region. We also show that two Arabidopsis rrp6 homologues act in gene silencing. Our data suggest that Arabidopsis exosome may act in parallel with RdDM in gene silencing, by epigenetic effects on chromatin structure, not through siRNAs or DNA methylation.