Differential Allelic Expression Research Articles

Identification of candidate causal variants and target genes at 41 breast cancer risk loci through differential allelic expression analysis

Understanding breast cancer genetic risk relies on identifying causal variants and candidate target genes in risk loci identified by genome-wide association studies (GWAS), which remains challenging. Since most loci fall in active gene regulatory regions, we developed a novel approach facilitated by pinpointing the variants with greater regulatory potential in the disease’s tissue of origin. Through genome-wide differential allelic expression (DAE) analysis, using microarray data from 64 normal breast tissue samples, we mapped the variants associated with DAE (daeQTLs). Then, we intersected these with GWAS data to reveal candidate risk regulatory variants and analysed their cis-acting regulatory potential. Finally, we validated our approach by extensive functional analysis of the 5q14.1 breast cancer risk locus. We observed widespread gene expression regulation by cis-acting variants in breast tissue, with 65% of coding and noncoding expressed genes displaying DAE (daeGenes). We identified over 54 K daeQTLs for 6761 (26%) daeGenes, including 385 daeGenes harbouring variants previously associated with BC risk. We found 1431 daeQTLs mapped to 93 different loci in strong linkage disequilibrium with risk-associated variants (risk-daeQTLs), suggesting a link between risk-causing variants and cis-regulation. There were 122 risk-daeQTL with stronger cis-acting potential in active regulatory regions with protein binding evidence. These variants mapped to 41 risk loci, of which 29 had no previous report of target genes and were candidates for regulating the expression levels of 65 genes. As validation, we identified and functionally characterised five candidate causal variants at the 5q14.1 risk locus targeting the ATG10 and ATP6AP1L genes, likely acting via modulation of alternative transcription and transcription factor binding. Our study demonstrates the power of DAE analysis and daeQTL mapping to identify causal regulatory variants and target genes at breast cancer risk loci, including those with complex regulatory landscapes. It additionally provides a genome-wide resource of variants associated with DAE for future functional studies.

Genetic regulation of B cell receptor signaling pathway: Insights from expression quantitative trait locus analysis using a mixed model

The B cell receptor (BCR) signaling pathway regulates non-immune cellular response through various pathways like MAPK, NF-kB, and PI3K-Akt. This study aimed to identify expression quantitative trait loci (eQTL) and their regulatory functions on BCR signaling pathway genes. A mixed model was employed to analyze eQTL using RNA expression levels in lymphoblastoid from 376 Europeans in the GEUVADIS dataset. In total, 266 SNPs, including 115 cis-acting SNPs, were found for association with transcription of 13 genes (P < 5 × 10−8), revealing 19 independent signals for five genes through linkage disequilibrium analysis. Functional analysis, aligning them with DNase sensitive sites, transcription factor binding sites, histone modification, promoters/enhancers, CpG islands, and ChIA-PET, identified regulatory variants targeting SYK, VAV2, and PLCG2. Notably, rs2562397 was validated as a SYK promoter variant, and rs694505, rs636667, and rs4889409 were confirmed as enhancer variants for VAV2 and PLCG2. Their allelic differences in gene expression were also confirmed using ENCODE ChIP-seq and Sei neural network prediction. Persistent differential expression of these genes by alleles might impact the adaptive immune system, vascular development, and/or relevant diseases that have been previously associated with other variants of the genes. Comprehensive genetic architecture studies of the BCR signaling pathway, along with experiments demonstrating related mechanisms, will greatly contribute to understanding the underlying mechanisms of relevant disease development and implementing precision medicine.

Study on Allele Specific Expression of Long-Term Residents in High Altitude Areas.

In diploid organisms, half of the chromosomes in each cell come from the father and half from the mother. Through previous studies, it was found that the paternal chromosome and the maternal chromosome can be regulated and expressed independently, leading to the emergence of allele specific expression (ASE). In this study, we analyzed the differential expression of alleles in the high-altitude population and the normal population based on the RNA sequencing data. Through gene cluster analysis and protein interaction network analysis, we found some changes occurred at the gene level, and some negative effects. During the study, we realized that the calmodulin homology domain may have a certain correlation with long-term survival at high altitude. The plateau environment is characterized by hypoxia, low air pressure, strong ultraviolet radiation, and low temperature. Accordingly, the genetic changes in the process of adaptation are mainly reflected in these characteristics. High altitude generation living is also highly related to cancer, immune disease, cardiovascular disease, neurological disease, endocrine disease, and other diseases. Therefore, the medical system in high altitude areas should pay more attention to these diseases.

Phased genomics reveals hidden somatic mutations and provides insight into fruit development in sweet orange.

Although revisiting the discoveries and implications of genetic variations using phased genomics is critical, such efforts are still lacking. Somatic mutations represent a crucial source of genetic diversity for breeding and are especially remarkable in heterozygous perennial and asexual crops. In this study, we focused on a diploid sweet orange (Citrus sinensis) and constructed a haplotype-resolved genome using high fidelity (HiFi) reads, which revealed 10.6% new sequences. Based on the phased genome, we elucidate significant genetic admixtures and haplotype differences. We developed a somatic detection strategy that reveals hidden somatic mutations overlooked in a single reference genome. We generated a phased somatic variation map by combining high-depth whole-genome sequencing (WGS) data from 87 sweet orange somatic varieties. Notably, we found twice as many somatic mutations relative to a single reference genome. Using these hidden somatic mutations, we separated sweet oranges into seven major clades and provide insight into unprecedented genetic mosaicism and strong positive selection. Furthermore, these phased genomics data indicate that genomic heterozygous variations contribute to allele-specific expression during fruit development. By integrating allelic expression differences and somatic mutations, we identified a somatic mutation that induces increases in fruit size. Applications of phased genomics will lead to powerful approaches for discovering genetic variations and uncovering their effects in highly heterozygous plants. Our data provide insight into the hidden somatic mutation landscape in the sweet orange genome, which will facilitate citrus breeding.

Expression analysis of NF1-mutated alleles in a rare compound heterozygous spinal NF1 patient by digital PCR.

Neurofibromatosis type 1 (NF1) is a heterogeneous neurocutaneous disorder. Spinal neurofibromatosis (SNF) is a distinct clinical entity of NF1, characterized by bilateral neurofibromas involving all spinal nerve roots. Although both forms are caused by intragenic heterozygous variants of NF1, missense variants have been associated with SNF, according to a dominant inheritance model causing haploinsufficiency. Most patients carry pathogenic variants in one of theNF1 alleles; nevertheless, patients with both NF1-mutated copies have been described. Interestingly, all NF1 variants carried by the known SNF compound heterozygotes were missense/splicing variants or in-frame insertion-deletions. To investigate whether there is a differential expression of NF1 variant alleles in an NF1 compound heterozygous SNF patient possibly contributing to clinical phenotype. We performed an allele-specific expression study, by chip-based digital PCR, in an SNF family carrying two NF1 missense variants. We evaluated the expression levels of the two NF1-mutated alleles both carried by the compound heterozygous SNF patient and his relatives. Both alleles were expressed at comparable levels in the patient and hyper-expressed compared to the wild-type alleles of healthy controls. Here we provide new insights into expression studies of NF1-mutated transcripts suggesting that a novel pathogenetic mechanism, caused by gain-of-function variants, could be associated with SNF. Further studies should be performed in larger cohorts, opening new perspectives in the NF1 pathogenesis comprehension.

Single-cell allele-specific expression analysis reveals dynamic and cell-type-specific regulatory effects

Differential allele-specific expression (ASE) is a powerful tool to study context-specific cis-regulation of gene expression. Such effects can reflect the interaction between genetic or epigenetic factors and a measured context or condition. Single-cell RNA sequencing (scRNA-seq) allows the measurement of ASE at individual-cell resolution, but there is a lack of statistical methods to analyze such data. We present Differential Allelic Expression using Single-Cell data (DAESC), a powerful method for differential ASE analysis using scRNA-seq from multiple individuals, with statistical behavior confirmed through simulation. DAESC accounts for non-independence between cells from the same individual and incorporates implicit haplotype phasing. Application to data from 105 induced pluripotent stem cell (iPSC) lines identifies 657 genes dynamically regulated during endoderm differentiation, with enrichment for changes in chromatin state. Application to a type-2 diabetes dataset identifies several differentially regulated genes between patients and controls in pancreatic endocrine cells. DAESC is a powerful method for single-cell ASE analysis and can uncover novel insights on gene regulation.

Allelic chromatin structure precedes imprinted expression of Kcnk9 during neurogenesis.

Differences in chromatin state inherited from the parental gametes influence the regulation of maternal and paternal alleles in offspring. This phenomenon, known as genomic imprinting, results in genes preferentially transcribed from one parental allele. While local epigenetic factors such as DNA methylation are known to be important for the establishment of imprinted gene expression, less is known about the mechanisms by which differentially methylated regions (DMRs) lead to differences in allelic expression across broad stretches of chromatin. Allele-specific higher-order chromatin structure has been observed at multiple imprinted loci, consistent with the observation of allelic binding of the chromatin-organizing factor CTCF at multiple DMRs. However, whether allelic chromatin structure impacts allelic gene expression is not known for most imprinted loci. Here we characterize the mechanisms underlying brain-specific imprinted expression of the Peg13-Kcnk9 locus, an imprinted region associated with intellectual disability. We performed region capture Hi-C on mouse brains from reciprocal hybrid crosses and found imprinted higher-order chromatin structure caused by the allelic binding of CTCF to the Peg13 DMR. Using an in vitro neuron differentiation system, we showed that imprinted chromatin structure precedes imprinted expression at the locus. Additionally, activation of a distal enhancer induced imprinted expression of Kcnk9 in an allelic chromatin structure-dependent manner. This work provides a high-resolution map of imprinted chromatin structure and demonstrates that chromatin state established in early development can promote imprinted expression upon differentiation.

Genetic analysis and allele-specific expression of SMAD7 3′UTR variants in human colorectal cancer reveal a novel somatic variant exhibiting allelic imbalance

BackgroundConsidering the impact of SMAD7 deregulation in colorectal cancer (CRC) progression and the significance of single nucleotide variant (SNV)-mediated disruptions of microRNA (miRNA)-dependent regulation for cancer susceptibility, our study aimed to analyze genetic variation in the SMAD7 3′ untranslated region ( 3′UTR) in CRC, measure differences in allelic mRNA expression, and evaluate its interference with miRNA-mediated post-transcriptional regulation. Patients and MethodsThis study included 80 patients with different CRC stages and six human colon cancer cell lines of various histological origins. SMAD7 3′UTR was analyzed by direct sequencing, followed by the relative quantification of differential allelic expression of detected variants by allele-specific qRT-PCR. In silico tools were employed for predictions of regulatory consequences of detected variants. ResultsA total of four different SNVs in one cell line and nine patients were found, among which were a novel somatic point variant and three already known germline variants (rs16950113, rs1050799536, and rs1043778717). All evaluated SNVs exhibited variable extents of allelic imbalance in expression. In silico analysis predicted significant effects of SNVs on miRNA binding efficiency, with each SNV disrupting existing and creating new target sites for one or more miRNAs. ConclusionImbalance observed in the expression of SNV alleles altering miRNA binding suggests that all investigated SNVs are potential contributing factors impacting SMAD7 expression regulation in CRC that further studies should investigate.

Spatial and temporal regulation of parent-of-origin allelic expression in the endosperm.

Genomic imprinting promotes differential expression of parental alleles in the endosperm of flowering plants and is regulated by epigenetic modification such as DNA methylation and histone tail modifications in chromatin. After fertilization, the endosperm develops through a syncytial stage before it cellularizes and becomes a nutrient source for the growing embryo. Regional compartmentalization has been shown both in early and late endosperm development, and different transcriptional domains suggest divergent spatial and temporal regional functions. The analysis of the role of parent-of-origin allelic expression in the endosperm as a whole and the investigation of domain-specific functions have been hampered by the inaccessibility of the tissue for high-throughput transcriptome analyses and contamination from surrounding tissue. Here, we used fluorescence-activated nuclear sorting (FANS) of nuclear targeted GFP fluorescent genetic markers to capture parental-specific allelic expression from different developmental stages and specific endosperm domains. This approach allowed us to successfully identify differential genomic imprinting with temporal and spatial resolution. We used a systematic approach to report temporal regulation of imprinted genes in the endosperm, as well as region-specific imprinting in endosperm domains. Analysis of our data identified loci that are spatially differentially imprinted in one domain of the endosperm, while biparentally expressed in other domains. These findings suggest that the regulation of genomic imprinting is dynamic and challenge the canonical mechanisms for genomic imprinting.

Conserved noncoding sequences and de novo Mutator insertion alleles are imprinted in maize.

Genomic imprinting is an epigenetic phenomenon in which differential allele expression occurs in a parent-of-origin-dependent manner. Imprinting in plants is tightly linked to transposable elements (TEs), and it has been hypothesized that genomic imprinting may be a consequence of demethylation of TEs. Here, we performed high-throughput sequencing of ribonucleic acids from four maize (Zea mays) endosperms that segregated newly silenced Mutator (Mu) transposons and identified 110 paternally expressed imprinted genes (PEGs) and 139 maternally expressed imprinted genes (MEGs). Additionally, two potentially novel paternally suppressed MEGs are associated with de novo Mu insertions. In addition, we find evidence for parent-of-origin effects on expression of 407 conserved noncoding sequences (CNSs) in maize endosperm. The imprinted CNSs are largely localized within genic regions and near genes, but the imprinting status of the CNSs are largely independent of their associated genes. Both imprinted CNSs and PEGs have been subject to relaxed selection. However, our data suggest that although MEGs were already subject to a higher mutation rate prior to their being imprinted, imprinting may be the cause of the relaxed selection of PEGs. In addition, although DNA methylation is lower in the maternal alleles of both the maternally and paternally expressed CNSs (mat and pat CNSs), the difference between the two alleles in H3K27me3 levels was only observed in pat CNSs. Together, our findings point to the importance of both transposons and CNSs in genomic imprinting in maize.

Single-cell variations in the expression of codominant alleles A and B on RBC of AB blood group individuals

A key question in biology is whether all cells of a given 'cell type' within an individual have more or less the same phenotype, especially in relation to nonimprinted autosomal loci. Some studies have shown differential allelic expression of autosomal genes to confer phenotypic variability at the individual cell level. Here, we report the amount of A and B histo-blood group antigens, products of classic examples of codominant alleles, in individual red blood cells (RBCs). Using immunofluorescence with Cy3-tagged and FITC-tagged antibodies, we quantified the levels of these antigens in 2512 RBCs from 24 individuals in the AB blood group. When these data were fit to a normal distribution, we could detect four groups: showing normal distribution for both antigens, either antigen, and neither antigen. Surprisingly, very few samples showed a significant positive correlation between the amounts of A and B antigens on individual RBC; in fact, the ratio of antigen A to antigen B in the entire set of samples spanned over five orders of magnitude. This variability in the amount of antigens A and/or B, combined with a lack of correlation between the amounts of these two antigens, resulted in unique staining patterns of RBC, generating widespread mosaicism in the RBC population of AB blood group individuals.

Sex-biased and parental allele-specific gene regulation by KDM6A

BackgroundKDM6A is a demethylase encoded by a gene with female-biased expression due to escape from X inactivation. Its main role is to facilitate gene expression through removal of the repressive H3K27me3 mark, with evidence of some additional histone demethylase-independent functions. KDM6A mutations have been implicated in congenital disorders such as Kabuki Syndrome, as well as in sex differences in cancer.MethodsKdm6a was knocked out using CRISPR/Cas9 gene editing in F1 male and female mouse embryonic stem cells (ES) derived from reciprocal crosses between C57BL6 x Mus castaneus. Diploid and allelic RNA-seq analyses were done to compare gene expression between wild-type and Kdm6a knockout (KO) clones. The effects of Kdm6a KO on sex-biased gene expression were investigated by comparing gene expression between male and female ES cells. Changes in H3K27me3 enrichment and chromatin accessibility at promoter regions of genes with expression changes were characterized by ChIP-seq and ATAC-seq followed by diploid and allelic analyses.ResultsWe report that Kdm6a KO in male and female embryonic stem (ES) cells derived from F1 hybrid mice cause extensive gene dysregulation, disruption of sex biases, and specific parental allele effects. Among the dysregulated genes are candidate genes that may explain abnormal developmental features of Kabuki syndrome caused by KDM6A mutations in human. Strikingly, Kdm6a knockouts result in a decrease in sex-biased expression and in preferential downregulation of the maternal alleles of a number of genes. Most promoters of dysregulated genes show concordant epigenetic changes including gain of H3K27me3 and loss of chromatin accessibility, but there was less concordance when considering allelic changes.ConclusionsOur study reveals new sex-related roles of KDM6A in the regulation of developmental genes, the maintenance of sex-biased gene expression, and the differential expression of parental alleles.

Favoring Expression of Yak Alleles in Interspecies F1 Hybrids of Cattle and Yak Under High-Altitude Environments.

Both cis- and trans-regulation could cause differential expression between the parental alleles in diploid species that might have broad biological implications. Due to the relatively distant genetic divergence between cattle and yak, as well as their differential adaptation to high-altitude environments, we investigated genome-wide allelic differential expression (ADE) in their F1 hybrids using Nanopore long-read RNA-seq technology. From adult F1 hybrids raised in high-altitude, ten lung and liver tissues were individually sequenced for producing 31.6 M full-length transcript sequences. Mapping against autosomal homologous regions between cattle and yak, we detected 17,744 and 14,542 protein-encoding genes expressed in lung and liver tissues, respectively. According to the parental assignments of transcript sequences, a total of 3,381 genes were detected to show ADE in at least one sample. There were 186 genes showing ubiquitous ADE in all the studied animals, and among them 135 and 37 genes had consistent higher expression of yak and cattle alleles, respectively. Functional analyses revealed that the genes with favoring expression of yak alleles have been involved in the biological progresses related with hypoxia adaptation and immune response. In contrast, the genes with favoring expression of cattle alleles have been enriched into different biological progresses, such as secretion of endocrine hormones and lipid metabolism. Our results would support unequal contribution of parental genes to environmental adaptation in the F1 hybrids of cattle and yak.

Abstract 1213: Uncovering HLA loss of heterozygosity and allelic imbalance in cancer for the improvement of personalized neoTCR immunotherapy with PACT-ESCAPE

Abstract Introduction: PACT Pharma is a clinical-stage adoptive cell therapy company that develops personalized neoTCR-T cells using a state-of-the-art approach to discover and validate predicted neoepitopes and their cognate T cell receptors (clinical trial NCT03970382). An important mechanism of resistance to adoptive cell therapy targeting tumor-specific neoantigens is the genetic loss of human leukocyte antigen (HLA) alleles or alterations in HLA expression in tumor cells. Therapies targeting neoantigens presented by HLA alleles that have either been deleted, mutated or are strongly downregulated are likely to lack efficacy due to the absence or reduction of epitope presentation. Surveying neoepitope presentation escape mechanisms is key for personalized immunotherapy. Methods: PACT Pharma has developed PACT-ESCAPE, a method that integrates DNA and RNA sequencing data to quantify allelic imbalance at HLA loci, including the most extreme case of loss of heterozygosity (LOH). Allelic imbalance is first determined at the DNA level by determining the patient’s maternal and paternal haplotypes across chromosome 6p to infer their copy number states. Twenty-six HLA Class I and II loci from chromosome 6 are then genotyped at high resolution, enabling accurate assignment of copy number states to the patient’s alleles, including any alleles that have been deleted in the tumor. Unlike other currently available methods, PACT-ESCAPE also measures the relative RNA expression of HLA alleles to provide an orthogonal estimate of allelic imbalance across chromosome 6 and support loss of heterozygosity calls made at the DNA level. Presentation machinery genes are also surveyed for loss of function mutations. To benchmark PACT-ESCAPE, we compared loss of heterozygosity calls at HLA-A, HLA-B, and HLA-C between our method and a previously published DNA-based HLA LOH classifier for 17 samples for which we had matched tumor/normal whole exome sequencing. Results: We found 100% concordance between PACT-ESCAPE and the benchmark, with four samples showing LOH at HLA-A, HLA-B, and HLA-C at the DNA level. Next, we evaluated RNA sequencing from tumor samples and confirmed that the relative expression of the lost allele compared to the kept allele was lower when LOH was called. High levels of allelic imbalance in gene expression, however, were not unique to samples with LOH, suggesting that differential expression of HLA alleles might also be an important contributor to immune escape in cancer. Conclusions: Evaluation of allelic imbalance in DNA copy number and RNA expression in PACT-ESCAPE provides new insights into immune escape in cancer. Adoptive cell therapy targeting neoantigens will benefit by enabling the accurate selection of targets most likely to be presented by the tumor. Citation Format: Chad C. Smith, Yan Ma, Katie Campbell, Zheng Pan, Eric Stawiski. Uncovering HLA loss of heterozygosity and allelic imbalance in cancer for the improvement of personalized neoTCR immunotherapy with PACT-ESCAPE [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1213.

Abstract 3818: Infrequent chromosomal loss and recurrent gains lead to imbalanced expression of HLA genes in melanoma

Abstract Introduction: Loss-of-heterozygosity (LOH) events in chromosome 6p, comprising the human leukocyte antigen (HLA) genes, have been reported in about 10% of cutaneous melanoma (compared to 20-40% of squamous cell carcinomas), while copy number gains in this region have been observed in over 50% of melanoma. Recent studies focused in HLA allelic loss have been restricted to DNA-based approaches, and have not been validated orthogonally at the RNA or protein levels. Here, using clinical melanoma biopsies and patient-derived melanoma cell lines, we show that genetic alterations in HLA genes results in imbalanced allele expression, subsequently skewing antigen presentation by melanoma cells. Methods: Whole exome and RNA sequencing (WES, RNAseq) analyses were performed on 760 melanoma biopsies and 60 patient-derived melanoma cell lines. Patient-matched normal WES was used to perform HLA haplotyping across Class I and II HLA genes. Tumor WES was analyzed for copy number alterations in chromosome 6, identifying which alleles were lost or gained. Differential expression of HLA alleles was quantified in tumor RNAseq, correlating the allelic imbalance at the DNA and RNA levels. Melanoma cell lines heterozygous for HLA-A*02, A*03, and A*24 were analyzed by flow cytometry for surface-level HLA protein expression using allele-specific antibodies to quantify allelic densities and compare the imbalance of HLA-A alleles at the DNA, RNA, and protein levels. Results: Across 760 melanoma biopsies, copy number alterations in chromosome 6p were identified in 76% of tumors; 12% had LOH, and 54% had copy number gains that resulted in imbalanced copies of alleles. In paired tumor WES and RNAseq (N=682), genetic imbalance was correlated with imbalanced expression of HLA alleles in the classical Class I HLA genes (Spearman rho=0.64-0.7; p=2.2e-16); this association was strengthened in tumors with high tumor cellularity, and was not associated with the total expression of the HLA genes.These patterns were explored in a 60 patient-derived melanoma cell lines with matched tumor WES and RNAseq, confirming that alleles gained at the genetic level were also expressed at higher levels than alleles that were not gained or lost. In 10 cell lines heterozygous for either HLA-A*02, A*03, or A*24, allelic imbalance at the DNA and RNA level resulted in correlative imbalanced surface presentation of alleles at the protein level. Conclusions: Evaluation of paired tumor WES and RNAseq revealed orthogonal validation of HLA allelic imbalance, and analysis in cell lines suggested that these patterns were likely tumor intrinsic. Experimental validation of these findings at the protein level suggests that antigen presentation density can be modulated by chromosomal gains, and not just allelic loss, in HLA genes. This knowledge is important for the design of cancer vaccines or T cell therapies targeting neoantigens presented by HLA class I complexes. Citation Format: Katie M. Campbell, Justin Saco, Egmidio Medina, Meelad Amouzgar, Shannon M. Pfeiffer, Cynthia R. Gonzalez, Gabriela Steiner, Ameya Champhekar, Cristina Puig Saus, Jesse Zaretsky, Gabriel Abril Rodriguez, Agustin Vega-Crespo, Ignacio Baselga Carretero, Mito Tariveranmoshabad, Anusha Kalbasi, Christine Spencer, Zachary L. Skidmore, Malachi Griffith, Obi L. Griffith, Daniel K. Wells, Antoni Ribas. Infrequent chromosomal loss and recurrent gains lead to imbalanced expression of HLA genes in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3818.

SARS-CoV-2-Encoded MiRNAs Inhibit Host Type I Interferon Pathway and Mediate Allelic Differential Expression of Susceptible Gene.

Infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the rapid spread of coronavirus disease 2019 (COVID-19), has generated a public health crisis worldwide. The molecular mechanisms of SARS-CoV-2 infection and virus–host interactions are still unclear. In this study, we identified four unique microRNA-like small RNAs encoded by SARS-CoV-2. SCV2-miR-ORF1ab-1-3p and SCV2-miR-ORF1ab-2-5p play an important role in evasion of type I interferon response through targeting several genes in type I interferon signaling pathway. Particularly worth mentioning is that highly expressed SCV2-miR-ORF1ab-2-5p inhibits some key genes in the host innate immune response, such as IRF7, IRF9, STAT2, OAS1, and OAS2. SCV2-miR-ORF1ab-2-5p has also been found to mediate allelic differential expression of COVID-19-susceptible gene OAS1. In conclusion, these results suggest that SARS-CoV-2 uses its miRNAs to evade the type I interferon response and links the functional viral sequence to the susceptible genetic background of the host.

Significance of differential allelic expression in phenotypic plasticity and evolutionary potential of microbial eukaryotes

BackgroundDifferential allelic expression (DAE) plays a key role in the regulation of many biological processes, and it may also play a role in adaptive evolution. Recently, environment‐dependent DAE has been observed in species of marine phytoplankton, and most remarkably, alleles that showed the highest level of DAE also showed the fastest rate of evolution.MethodsTo better understand the role of DAE in adaptive evolution and phenotypic plasticity, we developed a 2‐D cellular automata model “DAEsy‐World” that builds on the classical Daisyworld model.ResultsSimulations show that DAE delineates the evolution of alternative alleles of a gene, enabling the two alleles to adapt to different environmental conditions and sub‐functionalize. With DAE, the build‐up of genetic polymorphisms within genes is driven by positive selection rather than strict neutral evolution, and this can enhance phenotypic plasticity. Moreover, in sexually reproducing organisms, DAE also increased the standing genetic variation, augmenting a species’ adaptive evolutionary potential and ability to respond to fluctuating and/or changing conditions ( cf. genetic assimilation). We furthermore show that DAE is likely to evolve in fluctuating environmental conditions.ConclusionsDAE increases the adaptive evolutionary potential of both sexual and asexually reproducing organisms, and it may affect the pattern of nucleotide substitutions of genes.

Differential Allelic Expression among Long Non-Coding RNAs.

Long non-coding RNAs (lncRNA) comprise a diverse group of non-protein-coding RNAs >200 bp in length that are involved in various normal cellular processes and disease states, and can affect coding gene expression through mechanisms in cis or in trans. Since the discovery of the first functional lncRNAs transcribed by RNA Polymerase II, H19 and Xist, many others have been identified and noted for their unusual transcriptional pattern, whereby expression from one chromosome homolog is strongly favored over the other, also known as mono-allelic or differential allelic expression. lncRNAs with differential allelic expression have been observed to play critical roles in developmental gene regulation, chromosome structure, and disease. Here, we will focus on known examples of differential allelic expression of lncRNAs and highlight recent research describing functional lncRNAs expressed from both imprinted and random mono-allelic expression domains.

Imprinting of canine IGF2 and H19.

Genomic imprinting occurs in therian mammals and is a phenomenon whereby the two alleles of a gene are differentially expressed, based on the sex of the parent from whom the alleles were inherited. The allelic differences in expression are the consequence of different epigenetic modifications that are established in the sperm or oocyte during gametogenesis and transmitted at fertilization to offspring. A small minority of genes is regulated in this way but they have important biological functions, and aberrant regulation of imprinted genes contributes to disease aetiology in humans and other animals. The factors driving the evolution of imprinted genes are also of considerable interest, as these genes appear to forego the benefits of diploidy. To broaden the phylogenetic analysis of genomic imprinting, we began a study of imprinted genes in the domestic dog, Canis familiaris. In this report, we show that canine IGF2 and H19 are imprinted, with parent-of origin-dependent monoallelic expression patterns in neonatal umbilical cord. We identify a putative imprint control region associated with the genes, and provide evidence for differential methylation of this region in a somatic tissue (umbilical cord) and for its hypermethylation in the male germline. Canis familiaris is fast becoming a highly informative system for elucidating disease processes and evolution, and the study of imprinted genes in this species may help in understanding how these genes contribute to the generation of morphological and behavioral diversity.

Genome-wide analysis of R2R3-MYB transcription factors family in the autopolyploid Saccharum spontaneum: an exploration of dominance expression and stress response

BackgroundSugarcane (Saccharum) is the most critical sugar crop worldwide. As one of the most enriched transcription factor families in plants, MYB genes display a great potential to contribute to sugarcane improvement by trait modification. We have identified the sugarcane MYB gene family at a whole-genome level through systematic evolution analyses and expression profiling. R2R3-MYB is a large subfamily involved in many plant-specific processes.ResultsA total of 202 R2R3-MYB genes (356 alleles) were identified in the polyploid Saccharum spontaneum genomic sequence and classified into 15 subgroups by phylogenetic analysis. The sugarcane MYB family had more members by a comparative analysis in sorghum and significant advantages among most plants, especially grasses. Collinearity analysis revealed that 70% of the SsR2R3-MYB genes had experienced duplication events, logically suggesting the contributors to the MYB gene family expansion. Functional characterization was performed to identify 56 SsR2R3-MYB genes involved in various plant bioprocesses with expression profiling analysis on 60 RNA-seq databases. We identified 22 MYB genes specifically expressed in the stem, of which RT-qPCR validated MYB43, MYB53, MYB65, MYB78, and MYB99. Allelic expression dominance analysis implied the differential expression of alleles might be responsible for the high expression of MYB in the stem. MYB169, MYB181, MYB192 were identified as candidate C4 photosynthetic regulators by C4 expression pattern and robust circadian oscillations. Furthermore, stress expression analysis showed that MYB36, MYB48, MYB54, MYB61 actively responded to drought treatment; 19 and 10 MYB genes were involved in response to the sugarcane pokkah boeng and mosaic disease, respectively.ConclusionsThis is the first report on genome-wide analysis of the MYB gene family in sugarcane. SsMYBs probably played an essential role in stem development and the adaptation of various stress conditions. The results will provide detailed insights and rich resources to understand the functional diversity of MYB transcription factors and facilitate the breeding of essential traits in sugarcane.