WITHDRAWN: Short tandem repeats and methylation in the promoter region affect expression of cystathionine beta-synthase gene in the laying hen

Short tandem repeats and methylation in the promoter region affect expression of cystathionine beta-synthase gene in the laying hen

Short tandem repeats (STRs) contribute significantly to genetic diversity in humans, including disease-causing variation. Although the effect of STR variation on gene expression has been extensively assessed, their impact on epigenetics has been poorly studied and limited to specific genomic regions. Here, we investigated the hypothesis that some STRs act as independent regulators of local DNA methylation in the human genome and modify risk of common human traits. To address these questions, we first analyzed two independent data sets comprising PCR-free whole-genome sequencing (WGS) and genome-wide DNA methylation levels derived from whole-blood samples in 245 (discovery cohort) and 484 individuals (replication cohort). Using genotypes for 131,635 polymorphic STRs derived from WGS using HipSTR, we identified 11,870 STRs that associated with DNA methylation levels (mSTRs) of 11,774 CpGs (Bonferroni P < 0.001) in our discovery cohort, with 90% successfully replicating in our second cohort. Subsequently, through fine-mapping using CAVIAR we defined 585 of these mSTRs as the likely causal variants underlying the observed associations (fm-mSTRs) and linked a fraction of these to previously reported genome-wide association study signals, providing insights into the mechanisms underlying complex human traits. Furthermore, by integrating gene expression data, we observed that 12.5% of the tested fm-mSTRs also modulate expression levels of nearby genes, reinforcing their regulatory potential. Overall, our findings expand the catalog of functional sequence variants that affect genome regulation, highlighting the importance of incorporating STRs in future genetic association analysis and epigenetics data for the interpretation of trait-associated variants.

Over recent years several examples of randomly switching methyltransferases, associated with Type III restriction‐modification (R‐M) systems, have been described in pathogenic bacteria. In every case examined, changes in simple DNA sequence repeats result in variable methyltransferase expression and result in global changes in gene expression, and differentiation of the bacterial cell into distinct phenotypes. These epigenetic regulatory systems are called phasevarions, phase‐variable regulons, and are widespread in bacteria, with 17.4% of Type III R‐M system containing simple DNA sequence repeats. A distinct, recombination‐driven random switching system has also been described in Streptococci in Type I R‐M systems that also regulate gene expression. Here, we interrogate the most extensive and well‐curated database of R‐M systems, REBASE, by searching for all possible simple DNA sequence repeats in the hsdRMS genes that encode Type I R‐M systems. We report that 7.9% of hsdS, 2% of hsdM, and of 4.3% of hsdR genes contain simple sequence repeats that are capable of mediating phase variation. Phase variation of both hsdM and hsdS genes will lead to differential methyltransferase expression or specificity, and thereby the potential to control phasevarions. These data suggest that in addition to well characterized phasevarions controlled by Type III mod genes, and the previously described Streptococcal Type I R‐M systems that switch via recombination, approximately 10% of all Type I R‐M systems surveyed herein have independently evolved the ability to randomly switch expression via simple DNA sequence repeats.

DNA methylation inhibits transcription driven by the collagen alpha2(I) promoter and the 5' end of the gene in transient transfection and in vitro transcription assays. DNA-binding proteins in a unique family of ubiquitously expressed proteins, methylated DNA-binding protein (MDBP)/regulatory factor for X box (RFX), form specific complexes with a sequence overlapping the transcription start site of the collagen alpha2(I) gene. Complex formation increased when the CpG site at +7 base pairs from the transcription start site was methylated. The identity of the protein was demonstrated by co-migration and cross-competition for a characteristic slowly migrating doublet complex formed on MDBP/RFX recognition sequences and the collagen sequences by band shift assays. A RFX1-specific antibody supershifted the collagen DNA-protein complexes. Furthermore, in vitro translated RFX1 protein formed a specific complex with the collagen sequence that was also supershifted with the RFX1 antibody. MDBP/RFX displayed a higher affinity binding to the collagen sequence if the CpG at +7 was mutated in a manner similar to TpG. This same mutation within reporter constructs inhibited transcription in transfection and in vitro transcription assay. These results support the hypothesis that DNA methylation-induced inactivation of collagen alpha2(I) gene transcription is mediated, in part, by increased binding of MDBP/RFX to the first exon in response to methylation in this region.

Forensic genetics

Through sos3 (salt overly sensitive 3) suppressor screening, two allelic suppressor mutants that are weak alleles of the strong sos3 suppressor sos3hkt1-1 were recovered. Molecular characterization identified T-DNA insertions in the distal promoter region of the Arabidopsis thaliana HKT1 (AtHKT1, At4g10310) in these two weak sos3 suppressors, which results in physical separation of a tandem repeat from the proximal region of the AtHKT1 promoter. The tandem repeat is approximately 3.9 kb upstream of the ATG start codon and functions as an enhancer element to promote reporter gene expression. A putative small RNA target region about 2.6 kb upstream of the ATG start codon is heavily methylated. CHG and CHH methylation but not CG methylation is significantly reduced in the small RNA biogenesis mutant rdr2, indicating that non-CG methylation in this region is mediated by small RNAs. Analysis of AtHKT1 expression in rdr2 suggests that non-CG methylation in the putative small RNA target region represses AtHKT1 expression in shoots. The DNA methylation-deficient mutant met1-3 has nearly complete loss of total cytosine methylation in the putative small RNA target region and is hypersensitive to salt stress. The putative small RNA target region and the tandem repeat are essential for maintaining AtHKT1 expression patterns crucial for salt tolerance.

Polymorphic Short Tandem Repeats (STR) emerged as a separate class of genetic mutation, which together with Single Nucleotide Polymorphisms (SNPs) and Copy Number Variations (CNVs) can explain variability in response to pharmacotherapy. STR draws interest in pharmacogenomics research because of their prevalence in the human genome, and their putative functional role as regulators of gene expression. Depending on the search algorithm, there are approximately 700,000–1,000,000 STR loci with 2-6 bp long motifs in the human reference genome. STR is non-randomly distributed across Untranslated Regions (UTRs), protein-coding sequences, and introns, and is overrepresented in the promoter regions of the human genes. The functional role of STR has been demonstrated by effects on gene expression, splicing, protein sequence, and association with pathogenic effects. An intrinsic property of STR is the high rate of mutation by expansion or contraction in the number of repeat units. Variation in the length of STR plays an important role in modulating gene expression, and STR is likely to be general regulatory elements which attenuate expression of multiple genes. Elucidating the effects of STR on gene expression may in part explain variability in drug response, something that cannot be achieved by focusing analysis exclusively on SNPs or CNV. This review summarizes the role of polymorphic STR in clinical manifestations including response to pharmacotherapy.

Systematic evaluation of the early access applied biosystems precision ID Globalfiler mixture ID and Globalfiler NGS STR panels for the ion S5 system

Short tandem repeat (STR) replacements in UTRs and introns suggest an important role for certain STRs in gene expression and disease

The tea aphid, Aphis aurantii Boyer de Fonscolombe (Hemiptera: Aphididae), is found in regions where the tea plant grows, and has become one of the most important pests in tea gardens in the tropics and subtropics (Han et al., 2012; Deng et al., 2019). This species is also known as the black citrus aphid and destroys citrus orchards (Wang and Tsai, 2001). Ap. aurantii is a polyphagous aphid with over 190 genera hosts, including many other economically important plants in addition to tea and citrus, such as coffee, cacao, loquat, litchi, mango, and camellia (Carver, 1978; Deng et al., 2019). This aphid directly damages trees by sucking the phloem sap out of the shoot tip or from new fresh leaves and injecting its saliva, which causes phytotoxicity and stunting in the plant (Guidolin and Consoli, 2018). Moreover, this aphid secretes honeydew when sap-feeding, and sooty molds frequently grow on the honeydew, which hinders photosynthetic activity (Sevim et al., 2012). Aphids are wide-spread pests that feed on a wide range of fruits and vegetables, most of them are vectors of plant viruses (Huang and Qiao, 2014; Hulle et al., 2020). Except the tea aphid, there have been previous functional studies on genes associated with the development (Ding et al., 2017; Ye et al., 2019), reproduction (Shang et al., 2018; Ullah et al., 2019b), wing development (Shang et al., 2020b), response to the stress (Gao et al., 2018; Jing et al., 2018), and pest control (Mohammed et al., 2018; Ullah et al., 2019a) of other aphids pests (e.g., Ap. citricidus, Ap. gossypii, Acyrthosiphon pisum). Moreover, functional studies of the new insecticide targets in aphids were also conducted in these aphids (Ye et al., 2019; Shang et al., 2020a). However, most previously published papers have focused on the ecology (Alizadeh Kafeshani et al., 2018; Guidolin and Consoli, 2018; Li et al., 2019), control (Aslam et al., 2015; Gholamzadeh-Chitgar and Pourmoradi, 2017), and mitochondrial genome (Wang et al., 2019) in tea aphid Ap. aurantii. There have been few functional studies that focused on the development and reproduction of Ap. aurantii, because there are limited reference sequences. Quantitative and qualitative transcriptome analyses can reveal the integrated biochemical and physiological processes at a molecular level that are associated with specific aspects of the organism, such as identification of the critical genes during the different developmental stages in insects (Morandin et al., 2018; Liu et al., 2020). For instance, RNA-Seq was used to elucidate the underlying molecular mechanisms of metamorphic development of Henosepilachna vigintioctopunctata (Zhang et al., 2018). Using available insect genomes, comparative transcriptome analysis was conducted to analyze gene expression during all developmental stages of Zeugodacus cucurbitae (Wei et al., 2020), Bactrocera dorsalis (Liu et al., 2020). In addition, gene expression has been studied by RNA-Seq in multiple tissues to identify tissue-specific genes involved in female fertility in B. dorsalis [e.g., vitellogenin and vitelline membrane protein in female (Wei et al., 2018, 2019)]. In aphids, RNA-Seq was used to analyze the gene expression between dispersing and non-dispersing morphs (Shang et al., 2016, 2020b). Thus, RNA-Seq technology allows us to determine the gene expression to underlie certain biological functions of critical genes and identify potential targets of new environmentally friendly insecticides for pest control. Simple sequence repeats (SSRs), also known as microsatellites, are short, tandemly arranged, repeating motifs (1–6 bp), which are widely distributed throughout the genomes of eukaryotic organisms (Temnykh et al., 2001). SSRs are co-dominant, hypervariable, neutral, and reproducible molecular markers; therefore, they have become the most widely used molecular markers in population genetic and conservation studies to evaluate the level of genetic variation in a species (King, 2012). Transcriptomic sequencing is also a highly efficient approach to identify SSRs in insects with no accessible genome. This method was extensively used to identify and analyze the SSRs in Liposcelis entomophila (Wei et al., 2013) and H. vigintioctopunctata (Zhang et al., 2018). SSRs were also identified in specific tissues in B. dorsalis (Wei et al., 2015). In this study, RNA-Seq was conducted on samples from six stages of Ap. aurantii with four biological replicates. A comprehensive transcriptome was sequenced, the transcripts were de novo assembled, and the gene's functional annotation was performed. Gene expression during development and SSRs were analyzed. These results will be valuable for the future functional studies of genes involved in Ap. aurantii development, reproduction, and wing differentiation.

BackgroundLate‐Onset Alzheimer’s Disease (LOAD) is characterized by genetic heterogeneity and there is no single model explaining the genetic mode of inheritance. Short tandem repeats (STRs), which are hyper‐mutable sequences in the human genome could explain some of the missing heritability in LOAD. STRs are involved in several neuro‐degenerative disorders. We systematically evaluated the impact of 31 disease‐associated STRs on neuropathological LOAD features.MethodFrom whole‐genome sequencing (WGS) data in for 1,134 unrelated individuals of European ancestry from Religious Orders Study (ROS) and Rush Memory and Aging project (MAP) cohorts, we identified known pathogenic STRs in 31 loci using ExpansionHunter. WGS was generated from DNA extracted from blood and brain tissues. We tested the association of STRs with a) neuropathological LOAD status, b) beta‐amyloid levels, c) neurofibrillary tangle (NFT) burden, d) global measure of pathology based on the scaled scores of 5 brain regions and e) estimated slope of global cognition using longitudinal measurements. Regression models adjusting for age, sex and first three principal components. Subsequently, we examined if STRs influenced gene expression in dorsolateral prefrontal cortex (DLPFC), posterior cingulate cortex (PCC) and the anterior cingulate (AC) and tested if the association of STRs with neuropathological traits were mediated by altered gene expression.ResultTGC repeat in ATXN1 was associated with cognitive decline (b = ‐0.007, p = 0.014) and risk of clinical AD (b = 0.126, p = 0.03). Variation in CAG repeats in ATN1 was associated with cognition (b = 0.004, p = 0.022) and risk of pathological AD (b = ‐0.069, p = 0.035). Longer Repeats at ATXN1 increased gene expression in DLPFC (b = 0.012, p = 0.049) and PCC (b = 0.019, p = 0.006) and repeats in ATN1 altered DLPFC (b = 0.01, p = 0.016) and PCC (b = 0.01, p = 0.026) expression. Mediation analysis determined that the effect of the CAG repeats in ATN1 on tau was mediated by gene expression in PCC (p = 0.004).ConclusionWe demonstrate that disease causing STRs influence the underlying gene expression in brain and are associated with neuropathological and cognitive endophenotypes of AD. This suggests that STRs could explain some of the missing heritability in LOAD.

Genome-wide profiling of polymorphic short tandem repeats and their influence on gene expression and trait variation in diverse rice populations.

Rapid genotyping of mutant mice using dried blood spots for polymerase chain reaction (PCR) analysis

Background: Short tandem repeats (STRs) are highly variable elements that play a pivotal role in multiple genetic diseases and the regulation of gene expression. Long-read sequencing (LRS) offers a potential solution to genome-wide STR analysis. However, characterizing STRs in human genomes using LRS on a large population scale has not been reported.Methods: We conducted the large LRS-based STR analysis in 193 unrelated samples of the Chinese population and performed genome-wide profiling of STR variation in the human genome. The repeat dynamic index (RDI) was introduced to evaluate the variability of STR. We sourced the expression data from the Genotype-Tissue Expression to explore the tissue specificity of highly variable STRs related genes across tissues. Enrichment analyses were also conducted to identify potential functional roles of the high variable STRs.Results: This study reports the large-scale analysis of human STR variation by LRS and offers a reference STR database based on the LRS dataset. We found that the disease-associated STRs (dSTRs) and STRs associated with the expression of nearby genes (eSTRs) were highly variable in the general population. Moreover, tissue-specific expression analysis showed that those highly variable STRs related genes presented the highest expression level in brain tissues, and enrichment pathways analysis found those STRs are involved in synaptic function-related pathways.Conclusion: Our study profiled the genome-wide landscape of STR using LRS and highlighted the highly variable STRs in the human genome, which provide a valuable resource for studying the role of STRs in human disease and complex traits.

7593 Background: DAB2IP loss promotes primary tumor growth by activating Ras and drives metastasis through NFkB, serving as a signaling scaffold to coordinately regulate these pathways. DAB2IP is frequently methylated in lung cancer, and methylation in the m2a region is a key regulatory factor for DAB2IP expression in prostate cancer. We examined DAB2IP methylation in cell lines and in serum from erlotinib-treated NSCLC p with EGFR mutations. Methods: In human lung, breast and colorectal cancer cell lines, we analyzed DAB2IP promoter methylation in regions m2a and m2b by methylation-specific PCR (MSP) and bisulfite genomic sequencing. In circulating serum DNA from 152 erlotinib-treated NSCLC p with EGFR mutations, we analyzed methylation in the m2a and m2b promoter regions of DAB2IP by MSP. Methylation status was correlated with clinical outcome. Results: Methylation was detected in the m2a region of 42 (27.63%) p, and in the m2b region in 51 (33.55%) p. There were no major differences in clinical characteristics (age, gender, smoking history, EGFR mutation type, metastatic sites) between p with methylation in the m2a region and p with methylation in the m2b region. Overall progression-free survival (PFS) was 15 months (m), and median survival (MS) 28 m for all 152 p. For the 41 p with bone metastases (mets), PFS was 14 m for 30 p without methylation in the m2a region vs 8 m for 11 p with methylation in the m2a region (P=0.01), and MS was 23 m vs 10 m, respectively (P=0.19). For the 57 p with distant mets but no lung mets, PFS was 18 m for 36 p without methylation in the m2a region vs 10 m for 21 p with methylation in the m2a region (P=0.01), and MS was 24 m vs 16 m, respectively (P=0.03). No differences in either PFS or MS were observed according to the methylation status of the m2b region. Conclusions: Methylation in the m2a region of DAB2IP in serum DNA correlates with PFS and MS to erlotinib in NSCLC p with EGFR mutations with non-lung mets. Surveillance of DAB2IP methylation status in circulating DNA could be a useful tool to predict outcome to erlotinib in EGFR-mutated NSCLC p with non-lung mets.