Proximal Gene Expression Research Articles

Sustained alterations in proximal tubule gene expression in primary culture associate with HNF4A loss

Primary cultures of proximal tubule cells are widely used to model the behavior of kidney epithelial cells in vitro. However, de-differentiation of primary cells upon culture has been observed and appreciated for decades, yet the mechanisms driving this phenomenon remain poorly understood. This confounds the interpretation of experiments using primary kidney epithelial cells and prevents their use to engineer functional kidney tissue ex vivo. In this report, we measure the dynamics of cell-state transformations in early primary culture of mouse proximal tubules to identify key pathways and processes that correlate with and may drive de-differentiation. Our data show that the loss of proximal-tubule-specific genes is rapid, uniform, and sustained even after confluent, polarized epithelial monolayers develop. This de-differentiation occurs uniformly across many common culture condition variations. Changes in early culture were strongly associated with the loss of HNF4A. Exogenous re-expression of HNF4A can promote expression of a subset of proximal tubule genes in a de-differentiated proximal tubule cell line. Using genetically labeled proximal tubule cells, we show that selective pressures very early in culture influence which cells grow to confluence. Together, these data indicate that the loss of in vivo function in proximal tubule cultures occurs very early and suggest that the sustained loss of HNF4A is a key regulatory event mediating this change.

A genetic-epigenetic interplay at 1q21.1 locus underlies CHD1L-mediated vulnerability to primary progressive multiple sclerosis

Multiple Sclerosis (MS) is a heterogeneous inflammatory and neurodegenerative disease with an unpredictable course towards progressive disability. Treating progressive MS is challenging due to limited insights into the underlying mechanisms. We examined the molecular changes associated with primary progressive MS (PPMS) using a cross-tissue (blood and post-mortem brain) and multilayered data (genetic, epigenetic, transcriptomic) from independent cohorts. In PPMS, we found hypermethylation of the 1q21.1 locus, controlled by PPMS-specific genetic variations and influencing the expression of proximal genes (CHD1L, PRKAB2) in the brain. Evidence from reporter assay and CRISPR/dCas9 experiments supports a causal link between methylation and expression and correlation network analysis further implicates these genes in PPMS brain processes. Knock-down of CHD1L in human iPSC-derived neurons and knock-out of chd1l in zebrafish led to developmental and functional deficits of neurons. Thus, several lines of evidence suggest a distinct genetic-epigenetic-transcriptional interplay in the 1q21.1 locus potentially contributing to PPMS pathogenesis.

Efficient activation of hundreds of LTR12C elements reveals cis-regulatory function determined by distinct epigenetic mechanisms.

Long terminal repeats (LTRs), which often contain promoter and enhancer sequences of intact endogenous retroviruses (ERVs), are known to be co-opted as cis-regulatory elements for fine-tuning host-coding gene expression. Since LTRs are mainly silenced by the deposition of repressive epigenetic marks, substantial activation of LTRs has been found in human cells after treatment with epigenetic inhibitors. Although the LTR12C family makes up the majority of ERVs activated by epigenetic inhibitors, how these epigenetically and transcriptionally activated LTR12C elements can regulate the host-coding gene expression remains unclear due to genome-wide alteration of transcriptional changes after epigenetic inhibitor treatments. Here, we specifically transactivated >600 LTR12C elements by using single guide RNA-based dCas9-SunTag-VP64, a site-specific targeting CRISPR activation (CRISPRa)system, with minimal off-target events. Interestingly, most of the transactivated LTR12C elements acquired the H3K27ac-marked enhancer feature, while only 20% were co-marked with promoter-associated H3K4me3 modifications. The enrichment of the H3K4me3 signal was intricately associated with downstream regions of LTR12C, such as internal regions of intact ERV9 or other types of retrotransposons. Here, we leverage an optimized CRISPRa system to identify two distinct epigenetic signatures that define LTR12C transcriptional activation, which modulate the expression of proximal protein-coding genes.

Abstract 1049: Long Noncoding RNA Regulation Of Inflammation In Atherosclerosis

Atherosclerosis is a chronic inflammatory disease driven by cholesterol deposition in the artery wall. Resolution of atherosclerotic inflammation and plaque regression are important clinical goals. As long noncoding RNAs (lncRNAs) are key regulators of gene expression, there is an intense interest in understanding their roles in atherosclerosis and their potential as therapeutic targets. To identify lncRNAs regulated during plaque progression and regression, we performed single cell RNA-sequencing of CD45 + immune cells isolated from aortic arch plaques of Ldlr –/– mice under conditions of plaque progression (16 wks Western diet; WD) or regression (16 wks WD + 4 wks apoB ASO). We screened for lncRNAs acting in cis to modify the expression of proximal protein-coding genes and identified Caslr ( C tsc a nti s ense l nc R NA) , a novel lncRNA transcribed from the opposite strand of Cathepsin C gene ( Ctsc ) encoding a pro-atherosclerotic lysosomal protease. Caslr–Ctsc pair is upregulated in macrophages of progressing plaques and in response to pro-inflammatory stimuli ( i.e . LPS, IL-6) in vitro , and conversely downregulated during atherosclerosis regression and in response to anti-inflammatory stimulus ( i.e. IL-4). LncRNA knockdown approaches combined with transcriptomic analyses and functional assays showed that Caslr regulates Ctsc expression and associated inflammatory responses, including NF-κB signaling and inflammasome-dependent IL-1β release. RNA purification strategies combined with mass-spectrometry revealed that Caslr interacts with spliceosome-associated proteins to coordinate splicing of Ctsc mRNA. Notably, Caslr shares functional homology with a human lncRNA, CASLR, proximal to the CTSC gene, which regulates CTSC expression and protease activity. Human CASLR and CTSC are highly expressed in the blood of hyperlipidemic patients and their expression is coordinately decreased upon aggressive cholesterol lowering by anti-PCSK9 treatment. Collectively, our data identify Caslr as an evolutionary conserved lncRNA acting in cis to drive the expression of cathepsin C with downstream consequences on inflammatory signaling in plaque macrophages, and suggest that targeting this lncRNA may be beneficial in treating atherosclerosis.

Whole-genome methylation profiling reveals regions associated with painful temporomandibular disorders and active recovery processes.

Temporomandibular disorders (TMDs), collectively representing one of the most common chronic pain conditions, have a substantial genetic component, but genetic variation alone has not fully explained the heritability of TMD risk. Reasoning that the unexplained heritability may be because of DNA methylation, an epigenetic phenomenon, we measured genome-wide DNA methylation using the Illumina MethylationEPIC platform with blood samples from participants in the Orofacial Pain: Prospective Evaluation and Risk Assessment (OPPERA) study. Associations with chronic TMD used methylation data from 496 chronic painful TMD cases and 452 TMD-free controls. Changes in methylation between enrollment and a 6-month follow-up visit were determined for a separate sample of 62 people with recent-onset painful TMD. More than 750,000 individual CpG sites were examined for association with chronic painful TMD. Six differentially methylated regions were significantly ( P < 5 × 10 -8 ) associated with chronic painful TMD, including loci near genes involved in the regulation of inflammatory and neuronal response. A majority of loci were similarly differentially methylated in acute TMD consistent with observed transience or persistence of symptoms at follow-up. Functional characterization of the identified regions found relationships between methylation at these loci and nearby genetic variation contributing to chronic painful TMD and with gene expression of proximal genes. These findings reveal epigenetic contributions to chronic painful TMD through methylation of the genes FMOD , PM20D1 , ZNF718 , ZFP57 , and RNF39 , following the development of acute painful TMD. Epigenetic regulation of these genes likely contributes to the trajectory of transcriptional events in affected tissues leading to resolution or chronicity of pain.

The pharmacoepigenomic landscape of cancer cell lines reveals the epigenetic component of drug sensitivity

Aberrant DNA methylation accompanies genetic alterations during oncogenesis and tumour homeostasis and contributes to the transcriptional deregulation of key signalling pathways in cancer. Despite increasing efforts in DNA methylation profiling of cancer patients, there is still a lack of epigenetic biomarkers to predict treatment efficacy. To address this, we analyse 721 cancer cell lines across 22 cancer types treated with 453 anti-cancer compounds. We systematically detect the predictive component of DNA methylation in the context of transcriptional and mutational patterns, i.e., in total 19 DNA methylation biomarkers across 17 drugs and five cancer types. DNA methylation constitutes drug sensitivity biomarkers by mediating the expression of proximal genes, thereby enhancing biological signals across multi-omics data modalities. Our method reproduces anticipated associations, and in addition, we find that the NEK9 promoter hypermethylation may confer sensitivity to the NEDD8-activating enzyme (NAE) inhibitor pevonedistat in melanoma through downregulation of NEK9. In summary, we envision that epigenomics will refine existing patient stratification, thus empowering the next generation of precision oncology.

Graph construction method impacts variation representation and analyses in a bovine super-pangenome

BackgroundSeveral models and algorithms have been proposed to build pangenomes from multiple input assemblies, but their impact on variant representation, and consequently downstream analyses, is largely unknown.ResultsWe create multi-species super-pangenomes using pggb, cactus, and minigraph with the Bos taurus taurus reference sequence and eleven haplotype-resolved assemblies from taurine and indicine cattle, bison, yak, and gaur. We recover 221 k nonredundant structural variations (SVs) from the pangenomes, of which 135 k (61%) are common to all three. SVs derived from assembly-based calling show high agreement with the consensus calls from the pangenomes (96%), but validate only a small proportion of variations private to each graph. Pggb and cactus, which also incorporate base-level variation, have approximately 95% exact matches with assembly-derived small variant calls, which significantly improves the edit rate when realigning assemblies compared to minigraph. We use the three pangenomes to investigate 9566 variable number tandem repeats (VNTRs), finding 63% have identical predicted repeat counts in the three graphs, while minigraph can over or underestimate the count given its approximate coordinate system. We examine a highly variable VNTR locus and show that repeat unit copy number impacts the expression of proximal genes and non-coding RNA.ConclusionsOur findings indicate good consensus between the three pangenome methods but also show their individual strengths and weaknesses that need to be considered when analysing different types of variants from multiple input assemblies.

Interactive Effect of Combined Intermittent and Sustained Hypoxia and High-Fat Diet on the Colonic Mucosal Microbiome and Host Gene Expression in Mice.

PurposeGut dysbiosis can cause cardiometabolic disease. Gut dysbiosis can be independently caused by high-fat diet (HFD) and intermittent hypoxia (IH; characterizing obstructive sleep apnea), but the interactive effect of combined intermittent and sustained hypoxia (IH+SH) (characterizing obesity hypoventilation syndrome) and HFD on gut dysbiosis is unclear. We aimed to investigate the interactive effect of a combination of IH and SH and HFD on proximal colonic microbiota and colonic gene expression pattern.MethodsMale mice (n=16) were randomly received four different combinations of diet (normal versus HFD) and oxygen conditions (normoxia versus IH+SH) for 4 weeks. Bacterial DNA and mucosal epithelial cell RNA from proximal colon were collected for analysis of adherent microbiome and host’s gene expression analysis.ResultsHFD during IH+SH (22.6 ± 5.73; SD) led to greater Firmicutes: Bacteroidetes ratio than HFD during normoxia (5.89 ± 1.19; p=0.029). HFD significantly decreased microbial diversity as compared to normal diet, but the addition of IH+SH to HFD mildly reversed such effects. When compared to HFD during normoxia, HFD with combination of IH+SH resulted in changes to host mucosal gene expression for apical junctional complexes and adhesion molecules. Specifically, when compared to HFD during normoxia, HFD during IH+SH led to upregulation of Claudin 2 and Syk (tight junction dysfunction and increased mucosal permeability), while the barrier promoting claudin 4 was downregulated.ConclusionHFD during combined IH and SH causes greater gut dysbiosis and potentially adverse changes in colonic epithelial transcriptome than HFD during normoxia. The latter changes are suggestive of impaired gut barrier function.

Proximal tubular epithelia-specific transcriptomics of diabetic mice treated with dapagliflozin

Based on recent clinical trials using sodium-glucose co-transporter 2 inhibitor (SGLT2i) demonstrating the significant improvement of outcomes of diabetic kidney disease (DKD), the paradigm shift from “glomerulocentric” to “tubule centric” pathophysiology in DKD progression has been highlighted. Several responsible mechanisms for renoprotective effects by SGLT2i have been proposed recently, but the changes in proximal tubule-specific gene expression by SGLT2i in diabetic mice have not been elucidated. We report the analysis of the proximal tubular-specific pathway, demonstrating the downregulation of oxidative phosphorylation in dapagliflozin-treated db/db mice, a type 2 diabetic model. After 8-week treatment of dapagliflozin for db/db mice having a proximal tubule-specific tdTomato reporter, tdTomato-positive cells were isolated by FACS. Pathway analysis of RNA sequencing of isolated tubular epithelia revealed that oxidative phosphorylation was downregulated in dapagliflozin-treated mice. However, depletion of renal tissue ATP content in db/db mice was ameliorated by dapagliflozin administration. Pimonidazole staining demonstrated renal cortical tissue hypoxia in db/db mice, which was improved by dapagliflozin administration. This study suggests that dapagliflozin can ameliorate the excessive oxygen and ATP consumption, and subsequent tissue hypoxia in the diabetic kidney, which may explain, in part, the responsible mechanisms of the renoprotective effects of dapagliflozin.

Genome-wide prediction of activating regulatory elements in rice by combining STARR-seq with FACS.

Self-transcribing active regulatory region sequencing (STARR-seq) is widely used to identify enhancers at the whole-genome level. However, whether STARR-seq works as efficiently in plants as in animal systems remains unclear. Here, we determined that the traditional STARR-seq method can be directly applied to rice (Oryza sativa) protoplasts to identify enhancers, though with limited efficiency. Intriguingly, we identified not only enhancers but also constitutive promoters with this technique. To increase the performance of STARR-seq in plants, we optimized two procedures. We coupled fluorescence activating cell sorting (FACS) with STARR-seq to alleviate the effect of background noise, and we minimized PCR cycles and retained duplicates during prediction, which significantly increased the positive rate for activating regulatory elements (AREs). Using this method, we determined that AREs are associated with AT-rich regions and are enriched for a motif that the AP2/ERF family can recognize. Based on GC content preferences, AREs are clustered into two groups corresponding to promoters and enhancers. Either AT- or GC-rich regions within AREs could boost transcription. Additionally, disruption of AREs resulted in abnormal expression of both proximal and distal genes, which suggests that STARR-seq-revealed elements function as enhancers invivo. In summary, our work provides a promising method to identify AREs in plants.

Alzheimer's genetic risk effects on cerebral blood flow across the lifespan are proximal to gene expression

Cerebrovascular dysregulation such as altered cerebral blood flow (CBF) can be observed in Alzheimer's disease (AD) and may precede symptom onset. Genome wide association studies show that AD has a polygenic aetiology, providing a tool for studying AD susceptibility across the lifespan. Here, we ascertain whether the AD genetic risk effects on CBF previously observed (Chandler et al., 2019) are also present in later life. Consistent with our prior observations, AD genetic risk score (AD-GRS) was associated with reduced CBF in the ADNI sample. The regional association between AD-GRS and CBF were also spatially similar. Furthermore, CBF was related to the regional mRNA transcript expression of AD risk genes proximal to AD-GRS risk loci. These observations suggest that AD risk alleles may reduce neurovascular process such as CBF, potentially via mechanisms such as regional expression of proximal AD risk genes as an antecedent AD pathophysiology. Our observations help establish processes that underpin AD genetic risk-related reductions in CBF as a therapeutic target prior to the onset of neurodegeneration.

Cis-Regulatory variation affecting gene expression contributes to the improvement of maize kernel size.

cis-Regulatory variations contribute to trait evolution and adaptation during crop domestication and improvement. As the most important harvested organ in maize (Zea mays L.), kernel size has undergone intensive selection for size. However, the associations between maize kernel size and cis-regulatory variations remain unclear. We chose two independent association populations to dissect the genetic architecture of maize kernel size together with transcriptomic and genotypic data. The resulting phenotypes reflected a strong influence of population structure on kernel size. Compared with genome-wide association studies (GWASs), which accounted for population structure and relatedness, GWAS based on a naïve or simple linear model revealed additional associated single-nucleotide polymorphisms significantly involved in the conserved pathways controlling seed size in plants. Regulation analyses through expression quantitative trait locus mapping revealed that cis-regulatory variations likely control kernel size by fine-tuning the expression of proximal genes, among which ZmKL1 (GRMZM2G098305) was transgenically validated. We also proved that the pyramiding of the favorable cis-regulatory variations has contributed to the improvement of maize kernel size. Collectively, our results demonstrate that cis-regulatory variations, together with their regulatory genes, provide excellent targets for future maize improvement.

Acetylated HOXB13 Regulated Super Enhancer Genes Define Therapeutic Vulnerabilities of Castration-Resistant Prostate Cancer.

Androgen receptor (AR) antagonism is exacerbated by HOXB13 in castration-resistant prostate cancers (CRPC). However, it is unclear when and how HOXB13 primes CRPCs for AR antagonism. By mass-spectrometry analysis of CRPC extract, we uncovered a novel lysine 13 (K13) acetylation in HOXB13 mediated by CBP/p300. To determine whether acetylated K13-HOXB13 is a clinical biomarker of CRPC development, we characterized its role in prostate cancer biology. We identified tumor-specific acK13-HOXB13 signal enriched super enhancer (SE)-regulated targets. We analyzed the effect of loss of HOXB13K13-acetylation on chromatin binding, SE proximal target gene expression, self-renewal, enzalutamide sensitivity, and CRPC tumor growth by employing isogenic parental and HOXB13K13A mutants. Finally, using primary human prostate organoids, we evaluated whether inhibiting an acK13-HOXB13 target, ACK1, with a selective inhibitor (R)-9b is superior to AR antagonists in inhibiting CRPC growth. acK13-HOXB13 promotes increased expression of lineage (AR, HOXB13), prostate cancer diagnostic (FOLH1), CRPC-promoting (ACK1), and angiogenesis (VEGFA, Angiopoietins) genes early in prostate cancer development by establishing tumor-specific SEs. acK13-HOXB13 recruitment to key SE-regulated targets is insensitive to enzalutamide. ACK1 expression is significantly reduced in the loss of function HOXB13K13A mutant CRPCs. Consequently, HOXB13K13A mutants display reduced self-renewal, increased sensitivity to enzalutamide, and impaired xenograft tumor growth. Primary human prostate tumor organoids expressing HOXB13 are significantly resistant to AR antagonists but sensitive to (R)-9b. In summary, acetylated HOXB13 is a biomarker of clinically significant prostate cancer. Importantly, PSMA-targeting agents and (R)-9b could be new therapeutic modalities to target HOXB13-ACK1 axis regulated prostate cancers.

Contribution and clinical relevance of germline variation to the cancer transcriptome

BackgroundSomatic alterations in the cancer genome, some of which are associated with changes in gene expression, have been characterized in multiple studies across diverse cancer types. However, less is known about germline variants that influence tumor biology by shaping the cancer transcriptome.MethodsWe performed expression quantitative trait loci (eQTL) analyses using multi-dimensional data from The Cancer Genome Atlas to explore the role of germline variation in mediating the cancer transcriptome. After accounting for associations between somatic alterations and gene expression, we determined the contribution of inherited variants to the cancer transcriptome relative to that of somatic variants. Finally, we performed an interaction analysis using estimates of tumor cellularity to identify cell type-restricted eQTLs.ResultsThe proportion of genes with at least one eQTL varied between cancer types, ranging between 0.8% in melanoma to 28.5% in thyroid cancer and was correlated more strongly with intratumor heterogeneity than with somatic alteration rates. Although contributions to variance in gene expression was low for most genes, some eQTLs accounted for more than 30% of expression of proximal genes. We identified cell type-restricted eQTLs in genes known to be cancer drivers including LPP and EZH2 that were associated with disease-specific mortality in TCGA but not associated with disease risk in published GWAS. Together, our results highlight the need to consider germline variation in interpreting cancer biology beyond risk prediction.

Epigenome-wide association study of kidney function identifies trans-ethnic and ethnic-specific loci

BackgroundDNA methylation (DNAm) is associated with gene regulation and estimated glomerular filtration rate (eGFR), a measure of kidney function. Decreased eGFR is more common among US Hispanics and African Americans. The causes for this are poorly understood. We aimed to identify trans-ethnic and ethnic-specific differentially methylated positions (DMPs) associated with eGFR using an agnostic, genome-wide approach.MethodsThe study included up to 5428 participants from multi-ethnic studies for discovery and 8109 participants for replication. We tested the associations between whole blood DNAm and eGFR using beta values from Illumina 450K or EPIC arrays. Ethnicity-stratified analyses were performed using linear mixed models adjusting for age, sex, smoking, and study-specific and technical variables. Summary results were meta-analyzed within and across ethnicities. Findings were assessed using integrative epigenomics methods and pathway analyses.ResultsWe identified 93 DMPs associated with eGFR at an FDR of 0.05 and replicated 13 and 1 DMPs across independent samples in trans-ethnic and African American meta-analyses, respectively. The study also validated 6 previously published DMPs. Identified DMPs showed significant overlap enrichment with DNase I hypersensitive sites in kidney tissue, sites associated with the expression of proximal genes, and transcription factor motifs and pathways associated with kidney tissue and kidney development.ConclusionsWe uncovered trans-ethnic and ethnic-specific DMPs associated with eGFR, including DMPs enriched in regulatory elements in kidney tissue and pathways related to kidney development. These findings shed light on epigenetic mechanisms associated with kidney function, bridging the gap between population-specific eGFR-associated DNAm and tissue-specific regulatory context.

TRIM28 Expression on Dendritic Cells Prevents Excessive T Cell Priming by Silencing Endogenous Retrovirus.

Acquired immune reaction is initiated by dendritic cells (DCs), which present Ags to a few naive Ag-specific T cells. Deregulation of gene expression in DCs may alter the outcome of the immune response toward immunodeficiency and/or autoimmune diseases. Expression of TRIM28, a nuclear protein that mediates gene silencing through heterochromatin, decreased in DCs from old mice, suggesting alteration of gene regulation. Mice specifically lacking TRIM28 in DCs show increased DC population in the spleen and enhanced T cell priming toward inflammatory effector T cells, leading to acceleration and exacerbation in experimental autoimmune encephalomyelitis. TRIM28-deficient DCs were found to ectopically transcribe endogenous retrovirus (ERV) elements. Combined genome-wide analysis revealed a strong colocalization among the decreased repressive histone mark H3K9me3-transcribed ERV elements and the derepressed host genes that were related to inflammation in TRIM28-deficient DCs. This suggests that TRIM28 occupancy of ERV elements critically represses expression of proximal inflammatory genes on the genome. We propose that gene silencing through repressive histone modification by TRIM28 plays a role in maintaining the integrity of precise gene regulation in DCs, which prevents aberrant T cell priming to inflammatory effector T cells.

The Middle Part of the Plucked Hair Follicle Outer Root Sheath Is Identified as an Area Rich in Lineage-Specific Stem Cell Markers.

Hair follicle outer root sheath (ORS) is a putative source of stem cells with therapeutic capacity. ORS contains several multipotent stem cell populations, primarily in the distal compartment of the bulge region. However, the bulge is routinely obtained using invasive isolation methods, which require human scalp tissue ex vivo. Non-invasive sampling has been standardized by means of the plucking procedure, enabling to reproducibly obtain the mid-ORS part. The mid-ORS shows potential for giving rise to multiple stem cell populations in vitro. To demonstrate the phenotypic features of distal, middle, and proximal ORS parts, gene and protein expression profiles were studied in physically separated portions. The mid-part of the ORS showed a comparable or higher NGFR, nestin/NES, CD34, CD73, CD44, CD133, CK5, PAX3, MITF, and PMEL expression on both protein and gene levels, when compared to the distal ORS part. Distinct subpopulations of cells exhibiting small and round morphology were characterized with flow cytometry as simultaneously expressing CD73/CD271, CD49f/CD105, nestin, and not CK10. Potentially, these distinct subpopulations can give rise to cultured neuroectodermal and mesenchymal stem cell populations in vitro. In conclusion, the mid part of the ORS holds the potential for yielding multiple stem cells, in particular mesenchymal stem cells.

Chromatin Accessibility Identifies Regulatory Elements Predictive of Oncogene Expression in Multiple Myeloma

Chromatin Accessibility Identifies Regulatory Elements Predictive of Oncogene Expression in Multiple Myeloma

Identification of epigenetic variation associated with synchronous pod maturity in mungbean (Vigna radiata L.)

Cytosine methylation in genomic DNA affects gene expression, potentially causing phenotypic variation. Mungbean, an agronomically and nutritionally important legume species, is characterized by nonsynchronous pod maturity, resulting in multiple harvest which costs extra time and labor. To elucidate the epigenetic influences on synchronous pod maturity (SPM) in mungbean, we determined the genome-wide DNA methylation profiles of eight mungbean recombinant inbred lines (RILs) and their parental genotypes, and compared DNA methylation profiles between high SPM and low SPM RILs, thus revealing differentially methylated regions (DMRs). A total of 3, 18, and 28 pure DMRs, defined as regions showing no significant correlation between nucleotide sequence variation and methylation level, were identified in CpG, CHG, and CHH contexts, respectively. These DMRs were proximal to 20 genes. Among the 544 single nucleotide polymorphisms identified near the 20 genes, only one caused critical change in gene expression by early termination. Analysis of these genome-wide DNA methylation profiles suggests that epigenetic changes can influence the expression of proximal genes, regardless of nucleotide sequence variation, and that SPM is mediated through gibberellin-mediated hormone signaling pathways. These results provide insights into how epialleles contribute to phenotypic variation and improve SPM in mungbean cultivars.

Genetic and epigenetic profiling indicates the proximal tubule origin of renal cancers in end-stage renal disease.

End‐stage renal disease (ESRD) patients on dialysis therapy have a higher incidence of renal cell carcinomas (RCCs), which consist of 2 major histopathological types: clear‐cell RCCs (ESRD‐ccRCCs) and acquired cystic disease (ACD)‐associated RCCs. However, their genetic and epigenetic alterations are still poorly understood. Here, we investigated somatic mutations, copy number alterations (CNAs), and DNA methylation profiles in 9 ESRD‐ccRCCs and 7 ACD‐associated RCCs to identify their molecular alterations and cellular origins. Targeted sequencing of 409 cancer‐related genes, including VHL, PBRM1, SETD2, BAP1, KDM5C, MET, KMT2C (MLL3), and TP53, showed ESRD‐ccRCCs harbored frequent VHL mutations, while ACD‐associated RCCs did not. CNA analysis showed that ESRD‐ccRCCs had a frequent loss of chromosome 3p while ACD‐associated RCCs had a gain of chromosome 16. Beadarray methylation analysis showed that ESRD‐ccRCCs had methylation profiles similar to those of sporadic ccRCCs, while ACD‐associated RCCs had profiles similar to those of papillary RCCs. Expression analysis of genes whose expression levels are characteristic to individual segments of a nephron showed that ESRD‐ccRCCs and ACD‐associated RCCs had high expression of proximal tubule cell marker genes, while chromophobe RCCs had high expression of distal tubule cell/collecting duct cell marker genes. In conclusion, ESRD‐ccRCCs and ACD‐associated RCCs had mutation and methylation profiles similar to those of sporadic ccRCCs and papillary RCCs, respectively, and these 2 histopathological types of RCCs were indicated to have originated from proximal tubule cells of the nephron.