Genic Targets Research Articles

Differential Expression of Iron Deficiency Responsive Rice Genes under Low Phosphorus and Iron Toxicity Conditions and Association of OsIRO3 with Yield in Acidic Soils

With the hypothesis that iron (Fe) deficiency responsive genes may play a role in Fe toxicity conditions, we studied five such genes OsNAS1, OsNAS3, OsIRO2, OsIRO3 and OsYSL16 across six contrasting rice genotypes for expression under high Fe and low phosphorus (P) conditions, and sequence polymorphism. Genotypes Sahbhagi Dhan, Chakhao Poirieton and Shasharang were high yielders with no bronzing symptom visible under Fe toxic field conditions, and BAM350 and BAM811 were low yielders but did not show bronzing symptoms. Hydroponic screening revealed that the number of crown roots and root length can be traits for consideration for identifying Fe toxicity tolerance in rice genotypes. Fe contents in rice roots and shoots of a high-yielding genotype KMR3 showing leaf bronzing were significantly high. In response to 24 h high Fe stress, the expression levels of OsNAS3 were up-regulated in all genotypes except KMR3. In response to 48 h high Fe stress, the expression levels of OsNAS1 were 3-fold higher in tolerant Shasharang, whereas in KMR3, it was significantly down-regulated. Even in response to 7 d excess Fe stress, the transcript abundances of OsIRO2 and OsNAS3 were contrasting in genotypes Shasharang and KMR3. This suggested that the reported Fe deficiency genes had a role in Fe toxicity and that in genotype KMR3 under excess Fe stress, there was disruption of metal homeostasis. Under the 48 h low P conditions, OsIRO2 and OsYSL16 were significantly up-regulated in Fe tolerant genotype Shasharang and in low P tolerant genotype Chakhao Poirieton, respectively. In silico sequence analysis across 3 024 rice genotypes revealed polymorphism for 4 genes. Sequencing across OsIRO3 and OsNAS3 revealed nucleotide polymorphism between tolerant and susceptible genotypes for Fe toxicity. Non-synonymous single nucleotide polymorphisms and insertion/deletions (InDels) differing in tolerant and susceptible genotypes were identified. A marker targeting 25-bp InDel in OsIRO3, when run on a diverse panel of 43 rice genotypes and a biparental population, was associated with superior performance for yield under acidic lowland field conditions. This study highlights the potential of one of the vital genes involved in Fe homeostasis as a genic target for improving rice yield in acidic soils.

Duplex sequencing identifies genomic features that determine susceptibility to benzo(a)pyrene-induced in vivo mutations

Exposure to environmental mutagens increases the risk of cancer and genetic disorders. We used Duplex Sequencing (DS), a high-accuracy error-corrected sequencing technology, to analyze mutation induction across twenty 2.4 kb intergenic and genic targets in the bone marrow of MutaMouse males exposed to benzo(a)pyrene (BaP), a widespread environmental pollutant. DS revealed a linear dose-related induction of mutations across all targets with low intra-group variability. Heterochromatic and intergenic regions exhibited the highest mutation frequencies (MF). C:G > A:T transversions at CCA, CCC and GCC trinucleotides were enriched in BaP-exposed mice consistent with the known etiology of BaP mutagenesis. However, GC-content had no effect on mutation susceptibility. A positive correlation was observed between DS and the “gold-standard” transgenic rodent gene mutation assay. Overall, we demonstrate that DS is a promising approach to study in vivo mutagenesis and yields critical insight into the genomic features governing mutation susceptibility, spectrum, and variability across the genome.

Transposable elements contribute to the spatiotemporal microRNA landscape in human brain development.

Transposable elements (TEs) contribute to the evolution of gene regulatory networks and are dynamically expressed throughout human brain development and disease. One gene regulatory mechanism influenced by TEs is the miRNA system of post-transcriptional control. miRNA sequences frequently overlap TE loci and this miRNA expression landscape is crucial for control of gene expression in adult brain and different cellular contexts. Despite this, a thorough investigation of the spatiotemporal expression of TE-embedded miRNAs in human brain development is lacking. Here, we identify a spatiotemporally dynamic TE-embedded miRNA expression landscape between childhood and adolescent stages of human brain development. These miRNAs sometimes arise from two apposed TEs of the same subfamily, such as for L2 or MIR elements, but in the majority of cases stem from solo TEs. They give rise to in silico predicted high-confidence pre-miRNA hairpin structures, likely represent functional miRNAs, and have predicted genic targets associated with neurogenesis. TE-embedded miRNA expression is distinct in the cerebellum when compared to other brain regions, as has previously been described for gene and TE expression. Furthermore, we detect expression of previously nonannotated TE-embedded miRNAs throughout human brain development, suggestive of a previously undetected miRNA control network. Together, as with non-TE-embedded miRNAs, TE-embedded sequences give rise to spatiotemporally dynamic miRNA expression networks, the implications of which for human brain development constitute extensive avenues of future experimental research. To facilitate interactive exploration of these spatiotemporal miRNA expression dynamics, we provide the “Brain miRTExplorer” web application freely accessible for the community.

A large chromosomal inversion shapes gene expression in seaweed flies (Coelopa frigida).

Inversions often underlie complex adaptive traits, but the genic targets inside them are largely unknown. Gene expression profiling provides a powerful way to link inversions with their phenotypic consequences. We examined the effects of the Cf‐Inv(1) inversion in the seaweed fly Coelopa frigida on gene expression variation across sexes and life stages. Our analyses revealed that Cf‐Inv(1) shapes global expression patterns, most likely via linked variation, but the extent of this effect is variable, with much stronger effects in adults than larvae. Furthermore, within adults, both common as well as sex‐specific patterns were found. The vast majority of these differentially expressed genes mapped to Cf‐Inv(1). However, genes that were differentially expressed in a single context (i.e., in males, females, or larvae) were more likely to be located outside of Cf‐Inv(1). By combining our findings with genomic scans for environmentally associated SNPs, we were able to pinpoint candidate variants in the inversion that may underlie mechanistic pathways that determine phenotypes. Together the results of this study, combined with previous findings, support the notion that the polymorphic Cf‐Inv(1) inversion in this species is a major factor shaping both coding and regulatory variation resulting in highly complex adaptive effects.

Detection of Neanderthal Adaptively Introgressed Genetic Variants That Modulate Reporter Gene Expression in Human Immune Cells.

Although some variation introgressed from Neanderthals has undergone selective sweeps, little is known about its functional significance. We used a Massively Parallel Reporter Assay (MPRA) to assay 5,353 high-frequency introgressed variants for their ability to modulate the gene expression within 170 bp of endogenous sequence. We identified 2,548 variants in active putative cis-regulatory elements (CREs) and 292 expression-modulating variants (emVars). These emVars are predicted to alter the binding motifs of important immune transcription factors, are enriched for associations with neutrophil and white blood cell count, and are associated with the expression of genes that function in innate immune pathways including inflammatory response and antiviral defense. We combined the MPRA data with other data sets to identify strong candidates to be driver variants of positive selection including an emVar that may contribute to protection against severe COVID-19 response. We endogenously deleted two CREs containing expression-modulation variants linked to immune function, rs11624425 and rs80317430, identifying their primary genic targets as ELMSAN1, and PAN2 and STAT2, respectively, three genes differentially expressed during influenza infection. Overall, we present the first database of experimentally identified expression-modulating Neanderthal-introgressed alleles contributing to potential immune response in modern humans.

Familial analysis reveals rare risk variants for migraine in regulatory regions

The most recent genome-wide association study of migraine increased the total number of known migraine risk loci to 38. Still, most of the heritability of migraine remains unexplained, and it has been suggested that rare gene dysregulatory variants play an important role in migraine etiology. Addressing the missing heritability of migraine, we aim to fine-map signals from the known migraine risk loci to regulatory mechanisms and associate these to downstream genic targets. We analyzed a large cohort of whole-genome sequenced patients from extended migraine pedigrees (1040 individuals from 155 families). We test for association between rare variants segregating in regulatory regions with migraine. The findings were replicated in an independent case-control cohort (2027 migraineurs, 1650 controls). We report an increased burden of rare variants in one CpG island and three polycomb group response elements near four migraine risk loci. We found that the association is independent of the common risk variants in the loci. The regulatory regions are suggested to affect different genes than those originally tagged by the index SNPs of the migraine loci. Families with familial clustering of migraine have an increased burden of rare variants in regulatory regions near known migraine risk loci, with effects that are independent of the variants in the loci. The possible regulatory targets suggest different genes than those originally tagged by the index SNPs of the migraine loci.

Distinct genomic signals of lifespan and life history evolution in response to postponed reproduction and larval diet in Drosophila.

Reproduction and diet are two major factors controlling the physiology of aging and life history, but how they interact to affect the evolution of longevity is unknown. Moreover, although studies of large‐effect mutants suggest an important role of nutrient sensing pathways in regulating aging, the genetic basis of evolutionary changes in lifespan remains poorly understood. To address these questions, we analyzed the genomes of experimentally evolved Drosophila melanogaster populations subjected to a factorial combination of two selection regimes: reproductive age (early versus postponed), and diet during the larval stage (“low,” “control,” “high”), resulting in six treatment combinations with four replicate populations each. Selection on reproductive age consistently affected lifespan, with flies from the postponed reproduction regime having evolved a longer lifespan. In contrast, larval diet affected lifespan only in early‐reproducing populations: flies adapted to the “low” diet lived longer than those adapted to control diet. Here, we find genomic evidence for strong independent evolutionary responses to either selection regime, as well as loci that diverged in response to both regimes, thus representing genomic interactions between the two. Overall, we find that the genomic basis of longevity is largely independent of dietary adaptation. Differentiated loci were not enriched for “canonical” longevity genes, suggesting that naturally occurring genic targets of selection for longevity differ qualitatively from variants found in mutant screens. Comparing our candidate loci to those from other “evolve and resequence” studies of longevity demonstrated significant overlap among independent experiments. This suggests that the evolution of longevity, despite its presumed complex and polygenic nature, might be to some extent convergent and predictable.

Marker-trait association for low-light intensity tolerance in rice genotypes from Eastern India.

Light intensity is a crucial environmental factor that affects photosynthesis and ultimately, grain yield in rice. However, no gene or marker directly associated with improved performance under low-light intensity under field conditions has been identified till date. With an aim of identifying genes and markers associated with improved performance (measured in terms of better yields) under low-light intensity, an integrated field screening, in silico and wet lab validation analysis was performed. Field-based screening of a diverse set of 110 genotypes led to the identification of two physiological and three morphological parameters critical for low-light tolerance in rice. In silico analysis using information available in public databases led to the identification of a set of 90 potential candidate genes which were narrowed to thirteen genic targets for possible marker-trait association. Marker-trait association on the panel of 48 diverse rice genotypes varying in their response to low-light intensity led to the identification of six markers [HvSSR02-44 (biological yield), HvSSR02-52 (spikelet fertility), HvSSR02-54 (grain yield), HvSSR06-56 (spikelet fertility), HvSSR06-69 (spikelet fertility; biological yield), HvSSR09-45 (spikelet fertility)] lying on chromosomes 2, 6 and 9 showing significant association (R2 > 0.1) for traits like grain yield/plant, biological yield and spikelet fertility under low light. Eight rice genes [including member of BBX (B-box) family] lying within 10kb distance of these identified markers already reported for their role in response to stress or change in plant architecture in rice were also identified. The eight rice genotypes, five traits, eight genes and six markers identified in the current study will help in devising strategies to increase yield under low light intensity and pave way for future application in marker-assisted breeding.

Putative bovine topological association domains and CTCF binding motifs can reduce the search space for causative regulatory variants of complex traits

BackgroundTopological association domains (TADs) are chromosomal domains characterised by frequent internal DNA-DNA interactions. The transcription factor CTCF binds to conserved DNA sequence patterns called CTCF binding motifs to either prohibit or facilitate chromosomal interactions. TADs and CTCF binding motifs control gene expression, but they are not yet well defined in the bovine genome. In this paper, we sought to improve the annotation of bovine TADs and CTCF binding motifs, and assess whether the new annotation can reduce the search space for cis-regulatory variants.ResultsWe used genomic synteny to map TADs and CTCF binding motifs from humans, mice, dogs and macaques to the bovine genome. We found that our mapped TADs exhibited the same hallmark properties of those sourced from experimental data, such as housekeeping genes, transfer RNA genes, CTCF binding motifs, short interspersed elements, H3K4me3 and H3K27ac. We showed that runs of genes with the same pattern of allele-specific expression (ASE) (either favouring paternal or maternal allele) were often located in the same TAD or between the same conserved CTCF binding motifs. Analyses of variance showed that when averaged across all bovine tissues tested, TADs explained 14% of ASE variation (standard deviation, SD: 0.056), while CTCF explained 27% (SD: 0.078). Furthermore, we showed that the quantitative trait loci (QTLs) associated with gene expression variation (eQTLs) or ASE variation (aseQTLs), which were identified from mRNA transcripts from 141 lactating cows’ white blood and milk cells, were highly enriched at putative bovine CTCF binding motifs. The linearly-furthermost, and most-significant aseQTL and eQTL for each genic target were located within the same TAD as the gene more often than expected (Chi-Squared test P-value < 0.001).ConclusionsOur results suggest that genomic synteny can be used to functionally annotate conserved transcriptional components, and provides a tool to reduce the search space for causative regulatory variants in the bovine genome.

TERRA RNA Antagonizes ATRX and Protects Telomeres

Through an integration of genomic and proteomic approaches to advance understanding of long noncoding RNAs, we investigate the function of the telomeric transcript, TERRA. By identifying thousands ofTERRA target sites in the mouse genome, we demonstrate that TERRA can bind both in cis to telomeres and in trans to genic targets. We then define a large network of interacting proteins, including epigenetic factors, telomeric proteins, and the RNA helicase, ATRX. TERRA and ATRX share hundreds of target genes and are functionally antagonistic at these loci: whereas TERRA activates, ATRX represses gene expression. At telomeres, TERRA competes with telomeric DNA for ATRX binding, suppresses ATRX localization, and ensures telomeric stability. Depleting TERRA increases telomerase activity and induces telomeric pathologies, including formation of telomere-induced DNA damage foci and loss or duplication of telomeric sequences. We conclude that TERRA functions as an epigenomic modulator in trans and as an essential regulator of telomeres in cis.

Genomics of clinal variation in Drosophila: disentangling the interactions of selection and demography.

Clines in phenotypes and genotype frequencies across environmental gradients are commonly taken as evidence for spatially varying selection. Classical examples include the latitudinal clines in various species of Drosophila, which often occur in parallel fashion on multiple continents. Today, genomewide analysis of such clinal systems provides a fantastic opportunity for unravelling the genetics of adaptation, yet major challenges remain. A well-known but often neglected problem is that demographic processes can also generate clinality, independent of or coincident with selection. A closely related issue is how to identify true genic targets of clinal selection. In this issue of Molecular Ecology, three studies illustrate these challenges and how they might be met. Bergland etal. report evidence suggesting that the well-known parallel latitudinal clines in North American and Australian D.melanogaster are confounded by admixture from Africa and Europe, highlighting the importance of distinguishing demographic from adaptive clines. In a companion study, Machado etal. provide the first genomic comparison of latitudinal differentiation in D.melanogaster and its sister species D.simulans. While D.simulans is less clinal than D.melanogaster, a significant fraction of clinal genes is shared between both species, suggesting the existence of convergent adaptation to clinaly varying selection pressures. Finally, by drawing on several independent sources of evidence, Božičević etal. identify a functional network of eight clinal genes that are likely involved in cold adaptation. Together, these studies remind us that clinality does not necessarily imply selection and that separating adaptive signal from demographic noise requires great effort and care.

PiRNA-directed cleavage of meiotic transcripts regulates spermatogenesis.

MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.

Expression of pituitary tumor transforming gene in patients with acute lymphoblastic leukemia

The aim of this study was to explore the expression of pituitary tumor-transforming gene (PTTG) in acute lymphoblastic leukemia (ALL) and its relationship with the pathogenesis of ALL, as well as study the difference of the PTTG expression in ALL patients with Ph1 chromosome and without Ph1 chromosome. The mRNA expressions of PTTG in bone marrow from 28 patients with ALL and 28 normal controls were quantitatively detected by real-time quantitative polymerase chain reaction (real-time PCR). The results indicated that the expression of PTTG mRNA was significantly higher in ALL patients (1.9428E5 ± 1.8372E5) than that in normal controls (4.5766E3 ± 1.1817E3) (P < 0.05). The expression of PTTG mRNA was higher in Ph1 chromosome positive patients. The initial expression of PTTG mRNA was lower in patients achieved complete remission than that in patients with non-complete remission. It is concluded that the overexpression of PTTG may be related to the progression and genesis of ALL. Overexpression of PTTG may be intimately related to the progression and genesis of Ph1 chromosome positive ALL. It provides a new ideas to research the pathogenesis and genic target treatment of ALL.

Stearoyl-acyl carrier protein desaturases are associated with floral isolation in sexually deceptive orchids

The orchids Ophrys sphegodes and O. exaltata are reproductively isolated from each other by the attraction of two different, highly specific pollinator species. For pollinator attraction, flowers chemically mimic the pollinators' sex pheromones, the key components of which are alkenes with different double-bond positions. This study identifies genes likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs. The expression of two isoforms, SAD1 and SAD2, is flower-specific and broadly parallels alkene production during flower development. SAD2 shows a significant association with alkene production, and in vitro assays show that O. sphegodes SAD2 has activity both as an 18:0-ACP Δ(9) and a 16:0-ACP Δ(4) desaturase. Downstream metabolism of the SAD2 reaction products would give rise to alkenes with double-bonds at position 9 or position 12, matching double-bond positions observed in alkenes in the odor bouquet of O. sphegodes. SAD1 and SAD2 show evidence of purifying selection before, and positive or relaxed purifying selection after gene duplication. By contributing to the production of species-specific alkene bouquets, SAD2 is suggested to contribute to differential pollinator attraction and reproductive isolation among these species. Taken together, these data are consistent with the hypothesis that SAD2 is a florally expressed barrier gene of large phenotypic effect and, possibly, a genic target of pollinator-mediated selection.

Mutagenic selectivity of carcinogenic nitroso compounds: II. N,N-Dimethylnitrosamine

The genetic properties of the hepatocarcinogen N,N-dimethylnitrosamine (DMN) were examined in Drosophila for the assessment of the role of dose, cellular metabolism and genic target in its mutagenicity. Genetic activity was assayed with respect to the induction of the non-specific X-chromosome recessives (lethals and visibles) relative to the effects on specific genic sites, especially rDNA, which yields bobbed (bb) mutations. Dose dependence followed a quadratic course for all mutational classes and germ cell types, which indicated that DMN induced at least some multiple-hit mutagenic events. The genetic activity of DMN was favoured by cellular metabolism for all mutational classes, as was indicated by the progressive increase in mutation yield during spermatogenesis — from the metabolically inert mature sperm to the actively metabolizing spermatocytes and spermatogonia. The role of DNA methylation in the mutagenicity of DMN was deduced from quantitative assays of its genetic activity relative to the methylating nitrosamide — N-methyl- N-nitrosourethane (MNUr) — over the same dose range (1–10 m M) and on identical cell types and genic targets. In the metabolically inert cells (mature sperm), the two compounds were equally active with respect to the non-specific effects (X-recessives), but MNUr was considerably more effective on rDNA ( bb's). Conversely, in the metabolically active cells (spermatocytes and spermatogonia), DMN exerted a much higher non-specific mutagenicity than MNUr, but the two compounds were equally effective on rDNA. These results could not be entirely interpreted by the methylation hypothesis and indicated that a DMN aldehydic metabolite, structurally analogous to MNUr, might be responsible for the induction of the rDNA mutations. The rDNA selectivity index of DMN was significantly lower than for MNUr, which paralleled their relative carcinogenic versatilities. However, DMN was comparatively more effective on the tRNA genes, a feature which might be associated with its oncogenic specificity.