Changes In Promoter Methylation Research Articles

Exposure to a Titanium Dioxide Product Alters DNA Methylation in Human Cells.

The safety of titanium dioxide (TiO2), widely used in foods and personal care products, has been of ongoing concern. Significant toxicity of TiO2 has been reported, suggesting a risk to human health. To evaluate its potential epigenotoxicity, the effect of exposure to a TiO2 product to which humans could be exposed on DNA methylation, a primary epigenetic mechanism, was investigated using two human cell lines (Caco-2 (colorectal) and HepG2 (liver)) relevant to human exposure. Global methylation was determined by enzyme-linked immunosorbent assay-based immunochemical analysis. Gene promoter methylation was evaluated using EpiTect Methyl II Signature PCR System Array technology. Expression of DNA methyltransferases, MBD2, and URHF1 was quantified by qRT-PCR. A decrease in global DNA methylation was observed in both cell lines. Across the cell lines, seven genes (BNIP3, DNAJC15, GADD45G, GDF15, INSIG1, SCARA3, and TP53) were identified in which promoters were methylated. Changes in promoter methylation were associated with gene expression. Results also revealed aberrant expression of regulatory genes, DNA methyltransferases, MBD2, and UHRF1. Findings from the study clearly demonstrate the impact of TiO2 exposure on DNA methylation in two cell types, supporting the potential involvement of this epigenetic mechanism in its biological responses. Hence, epigenetic studies are critical for complete assessment of potential risk from exposure.

Integrated multi-omics reveals contrasting epigenetic patterns in leaf and root morphogenesis in Aegilops speltoides

Aegilops speltoides, the closest ancestor of the wheat B subgenome, has been well studied genomically. However, the epigenetic landscape of Ae. speltoides and the effects of epigenetics on its growth and development remain poorly understood. Here, we present a comprehensive multi-omics atlas of leaves and roots in Ae. speltoides, encompassing transcriptome, DNA methylation, histone modifications, and small RNA profiling. Divergent DNA methylation levels were detected between leaves and roots, and were associated with differences in accumulated 24-nt siRNAs. DNA methylation changes in promoters and gene bodies showed strong connections with altered expression between leaves and roots. Transcriptional regulatory networks (TRN) reconstructed between leaves and roots were driven by tissue-specific TF families. DNA methylation and histone modification act together as switches that shape root and leaf morphogenesis by modulating the binding of tissue-specific TFs to their target genes. The TRNs in leaves and roots reshaped during wheat polyploidization were associated with alterations in epigenetic modifications. Collectively, these results not only shed light on the critical contribution of epigenetic regulation in the morphogenesis of leaves and roots in Ae. speltoides but also provide new insights for future investigations into the complex interplay of genetic and epigenetic factors in the developmental biology of common wheat.

STUB1-mediated K63-linked ubiquitination of UHRF1 promotes the progression of cholangiocarcinoma by maintaining DNA hypermethylation of PLA2G2A

BackgroundCholangiocarcinoma (CCA) is a highly malignant tumor characterized by a lack of effective targeted therapeutic strategies. The protein UHRF1 plays a pivotal role in the preservation of DNA methylation and works synergistically with DNMT1. Posttranscriptional modifications (PTMs), such as ubiquitination, play indispensable roles in facilitating this process. Nevertheless, the specific PTMs that regulate UHRF1 in CCA remain unidentified.MethodsWe confirmed the interaction between STUB1 and UHRF1 through mass spectrometry analysis. Furthermore, we investigated the underlying mechanisms of the STUB1-UHRF1/DNMT1 axis via co-IP experiments, denaturing IP ubiquitination experiments, nuclear‒cytoplasmic separation and immunofluorescence experiments. The downstream PLA2G2A gene, regulated by the STUB1-UHRF1/DNMT1 axis, was identified via RNA-seq. The negative regulatory mechanism of PLA2G2A was explored via bisulfite sequencing PCR (BSP) experiments to assess changes in promoter methylation. The roles of PLA2G2A and STUB1 in the proliferation, invasion, and migration of CCA cells were assessed using the CCK-8 assay, colony formation assay, Transwell assay, wound healing assay and xenograft mouse model. We evaluated the effects of STUB1/UHRF1 on cholangiocarcinoma by utilizing a primary CCA mouse model.ResultsThis study revealed that STUB1 interacts with UHRF1, resulting in an increase in the K63-linked ubiquitination of UHRF1. Consequently, this facilitates the nuclear translocation of UHRF1 and enhances its binding affinity with DNMT1. The STUB1-UHRF1/DNMT1 axis led to increased DNA methylation of the PLA2G2A promoter, subsequently repressing its expression. Increased STUB1 expression in CCA was inversely correlated with tumor progression and overall survival. Conversely, PLA2G2A functions as a tumor suppressor in CCA by inhibiting cell proliferation, invasion and migration.ConclusionsThese findings suggest that the STUB1-mediated ubiquitination of UHRF1 plays a pivotal role in tumor progression by epigenetically silencing PLA2G2A, underscoring the potential of STUB1 as both a prognostic biomarker and therapeutic target for CCA.Graphical

Promoter Methylation Changes in DNA Damage-Response Genes in Ovarian Cancer and Their Correlation with Prognosis

Background: Ovarian cancer has a poor prognosis, and DNA damage-response (DDR) genes are associated with both its occurrence and prognosis. However, previous studies have mostly focused on genetic mutations, with no clear conclusions on epigenetic factors such as DNA methylation. Methods: In this study, we comprehensively investigated the relationship between promoter methylation of DDR genes and ovarian cancer prognosis. We performed combined multidata analysis of the promoter methylation, expression, homologous recombination defieiency (HRD) score, and drug sensitivity of 377 DDR genes in ovarian cancer by utilizing The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. We then validated abnormal promoter methylation and its relationship with overall survival in clinical samples. Results: Our analysis identified 52 methylation-driven DDR genes that exhibited abnormal expression due to abnormal promoter methylation. These genes are mostly related to BRCA1-related DNA damage repair and cell cycle regulatory pathways. Further studies revealed six of these genes, BRCA1, PTTG1, TTK, AURKA, CDC6, and E2F1, to be significantly associated with HRD scores. Among them, E2F1, PTTG1, and CDC6 are associated with drug sensitivity. Finally, we verified in 81 ovarian cancer samples that methylation of the promoter of these three genes was significantly associated with patient survival. Conclusions: Our study identified a large number of methylation-driven aberrantly expressed DDR genes in ovarian cancer, some of which affect disease prognosis. Levels of methylation of these gene promoters may serve as potential prognostic markers.

THU157 Epigenetic Regulation Of Genes Involved In The Signaling Cascade Of The GH Axis - IGF1 And Insulin. Implication In Children Postnatal Growth

Abstract Disclosure: L. Zoff: None. F. Garcia: None. G. Aschettino: None. G. Veneruzzo: None. M.C. Mattone: None. N. Perez Garrido: None. M. Juanes: None. N.I. Saraco: None. C. Alonso: None. G. Guercio: None. A. Belgorosky: None. M.S. Baquedano: None. Growth is influenced by genetic, nutritional, environmental, and hormonal factors, but it proceeds in a predictable pattern characterized by a constant growth deceleration between childhood and adolescence. This growth deceleration is coordinated in tissues and organs in order to maintain body proportions. Growth hormone (GH)/ insulin-like growth factor type 1 (IGF1) axis and insulin are crucial stimulators of growth, cell proliferation and metabolism. However, their levels do not change with age in a pattern that would explain the prepubertal decline in growth rate. DNA methylation represents a fundamental epigenetic mark that is associated with transcriptional repression during development. We hypothesize that the postnatal growth pattern could be orchestrated by epigenetic mechanisms. In order to analyze age related physiological changes, we evaluated promoter methylation in the GHR, IGF1R and INSR genes in peripheral blood from 40 healthy children of both sexes (females (F) n=21 and males (M) n=19), between 3 and 15 years old by using targeted deep-amplicon bisulfite sequencing on a MiSeq system. Sequencing libraries of 4 promoter CpG rich regions for GHR, 3 for IGF1R and 5 for INSR (104, 103 and 141 CpG sites, respectively) were analyzed for mean methylation values of all CpG sites using amplikyzer2 software. The results would suggest a sexual dimorphism in the epigenetic regulation of GH, IGF1 and insulin receptors gene expression. A significant negative correlation between GHR methylation and age was observed in both sexes (Multiple Spearman correlation, p<0.05), but in different GHR promoter regions. Specifically, a decrease in themethylation levels with age were detected in CpG-8, CpG+183 and CpG+243 in F; and in CpG+343, CpG+346, CpG+385, CpG+391, CpG+407 and CpG+487 in M. In contrast, in the IGF1R promoter region, a positive correlation was observed exclusively in F, between mean methylation levels and age in CpG-51, CpG-43, CpG-41, CpG-23, CpG-17, CpG-15, CpG-13, CpG-11, and CpG-9, p<0.05. Male-specific positive correlation between INSR promotermethylation values and age were found in CpG-768, CpG+10, CpG+31, CpG+87, CpG+95, CpG+163, CpG+199, CpG+206, CpG+238, CpG+700, CpG+815 and CpG+900, p<0.05.Taking together, our findings are in agreement with the hypothesis that age-associated DNA promoter methylation changes could be involved in the physiological growth pattern. Finally, the observed sexual dimorphism suggests age- and sex-dependent regulatory mechanisms for normal growth. Presentation: Thursday, June 15, 2023

Abstract 3155: Gene expression profiles reveal distinct regulatory activities of transcription factors GATA1 and TAL1 upon AML relapse

Abstract The purpose of this study is to identify key regulatory pathways that potentially drive abnormal gene expression program in relapsed Acute Myeloid Leukemia (AML) patients, by integrative computational analyses on multi-omics molecular profiles. Relapsed AML remains a clinical challenge. Epigenetic heterogeneity may contribute to transcriptional dysregulation and disease progression in AML. However, what specific transcriptional programs and potential regulatory mechanisms contribute to disease relapse are not yet well understood. To characterize the global transcriptional landscapes in relapsed AML, we integrated genomics data from two cohorts of matched diagnosis and relapse patient specimens. We identified 5,416 differentially expressed genes (DEGs) between diagnosis and relapse in Cohort I. Unsupervised clustering yielded three distinct DEG groups: group A, B and C genes that were predominantly (88%) down-regulated, divergently regulated, or predominantly (65%) up-regulated, respectively, upon relapse. The expression pattern of all DEGs separated the patients into two clusters, most robustly by Group B genes. Interestingly, the majority of DEGs did not associate with changes in gene promoter methylation. Similar patterns were observed in Cohort II. We used Binding Analysis for Regulation of Transcription (BART) to identify transcriptional regulators (TRs) that potentially regulated the DEGs not associated with DNA methylation changes, and assessed the differential expression of identified TRs during disease progression. PU.1 was identified as a potential TR for Group A genes and was down-regulated upon relapse. GATA1 and TAL1 were identified as regulating Group B genes and were up-regulated in patient cluster1 and down-regulated in cluster2, consistent with the expression pattern of Group B genes. RBBP5 was a top predicted TR for Group C genes and was up-regulated upon relapse. We next validated the potential functionality of those predicted factors. In NSG mice transplanted with a human AML specimen, TAL1 and GATA1 were downregulated in AML cells collected four weeks after chemotherapy treatment, and were inferred as TRs for the down-regulated genes, similar to the patient data. PU.1 was inferred as regulating the up-regulated genes. Furthermore, we found that the level of differential expression of TAL1, GATA1, and PU.1 in each patient specimen associated with the correlation of DEG profiles between the patient specimen and TR perturbation in human-derived hematopoietic cell lines. Our results support the possibility that in some AML patients, TRs with roles in hematopoiesis and leukemia might contribute to disease relapse. Further mechanistic studies deciphering the molecular and phenotypic events facilitated by these TRs will yield significant insight into disease biology and possible therapeutic targeting approaches in relapsed AML. Citation Format: Zhenjia Wang, Yaseswini Neelamraju, Cem Meydan, Nicholas Dunham, Jorge Gandara, Tak Lee, Subhash Prajapati, Franck Rapaport, Caroline Sheridan, Paul Zumbo, Michael Becker, Lars Bullinger, Martin Carroll, Richard D’Andrea, Richard Dillon, Ross Levine, Christopher E. Mason, Ari Melnick, Donna Neuberg, Stefan Bekiranov, Chongzhi Zang, Francine E. Garrett-Bakelman. Gene expression profiles reveal distinct regulatory activities of transcription factors GATA1 and TAL1 upon AML relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3155.

Identification of Epigenetically Regulated Genes Distinguishing Intracranial from Extracranial Melanoma Metastases

Despite remarkable advances in treating patients with metastatic melanoma, the management of melanoma brain metastases remains challenging. Recent evidence suggests that epigenetic reprogramming is an important mechanism for the adaptation of melanoma cells to the brain environment. In this study, the methylomes and transcriptomes of a cohort of matched melanoma metastases were evaluated by integrated omics data analysis. The identified 38 candidate genes displayed distinct promoter methylation and corresponding gene expression changes in intracranial compared with extracranial metastases. The 11 most promising genes were validated on protein level in both tumor and surrounding normal tissue using immunohistochemistry. In accordance with the underlying promoter methylation and gene expression changes, a significantly different protein expression was confirmed for STK10, PDXK, WDR24, CSSP1, NMB, RASL11B, phosphorylated PRKCZ, PRKCZ, and phosphorylated GRB10 in the intracranial metastases. The observed changes imply a distinct intracranial phenotype with increased protein kinase B phosphorylation and a higher frequency of proliferating cells. Knockdown of PRKCZ or GRB10 altered the expression of phosphorylated protein kinase B and decreased the viability of a brain-specific melanoma cell line. In summary, epigenetically regulated cancer-relevant alterations were identified that provide insights into the molecular mechanisms that discriminate brain metastases from other organ metastases, which could be exploited by targeting the affected signaling pathways.

Neonatal inflammation increases hippocampal KCC2 expression through methylation-mediated TGF-β1 downregulation leading to impaired hippocampal cognitive function and synaptic plasticity in adult mice

The mechanisms by which neonatal inflammation leads to cognitive deficits in adulthood remain poorly understood. Inhibitory GABAergic synaptic transmission plays a vital role in controlling learning, memory and synaptic plasticity. Since early-life inflammation has been reported to adversely affect the GABAergic synaptic transmission, the aim of this study was to investigate whether and how neonatal inflammation affects GABAergic synaptic transmission resulting in cognitive impairment. Neonatal mice received a daily subcutaneous injection of lipopolysaccharide (LPS, 50 μg/kg) or saline on postnatal days 3–5. It was found that blocking GABAergic synaptic transmission reversed the deficit in hippocampus-dependent memory or the induction failure of long-term potentiation in the dorsal CA1 in adult LPS mice. An increase of mIPSCs amplitude was further detected in adult LPS mice indicative of postsynaptic potentiation of GABAergic transmission. Additionally, neonatal LPS resulted in the increased expression and function of K+–Cl−-cotransporter 2 (KCC2) and the decreased expression of transforming growth factor-beta 1 (TGF-β1) in the dorsal CA1 during adulthood. The local TGF-β1 overexpression improved KCC2 expression and function, synaptic plasticity and memory of adult LPS mice. Adult LPS mice show hypermethylation of TGFb1 promoter and negatively correlate with reduced TGF-β1 transcripts. 5-Aza-deoxycytidine restored the changes in TGFb1 promoter methylation and TGF-β1 expression. Altogether, the results suggest that hypermethylation-induced reduction of TGF-β1 leads to enhanced GABAergic synaptic inhibition through increased KCC2 expression, which is a underlying mechanism of neonatal inflammation-induced hippocampus-dependent memory impairment in adult mice.

Hypomethylation in MTNR1B: a novel epigenetic marker for atherosclerosis profiling using stenosis radiophenotype and blood inflammatory cells

BackgroundChanges in gene-specific promoter methylation may result from aging and environmental influences. Atherosclerosis is associated with aging and environmental effects. Thus, promoter methylation profiling may be used as an epigenetic tool to evaluate the impact of aging and the environment on atherosclerosis development. However, gene-specific methylation changes are currently inadequate epigenetic markers for predicting atherosclerosis and cardiovascular disease pathogenesis.ResultsWe profiled and validated changes in gene-specific promoter methylation associated with atherosclerosis using stenosis radiophenotypes of cranial vessels and blood inflammatory cells rather than direct sampling of atherosclerotic plaques. First, we profiled gene-specific promoter methylation changes using digital restriction enzyme analysis of methylation (DREAM) sequencing in peripheral blood mononuclear cells from eight samples each of cranial vessels with and without severe-stenosis radiophenotypes. Using DREAM sequencing profiling, 11 tags were detected in the promoter regions of the ACVR1C, ADCK5, EFNA2, ENOSF1, GLS2, KNDC1, MTNR1B, PACSIN3, PAX8-AS1, TLDC1, and ZNF7 genes. Using methylation evaluation, we found that EFNA2, ENOSF1, GLS2, KNDC1, MTNR1B, PAX8-AS1, and TLDC1 showed > 5% promoter methylation in non-plaque intima, atherosclerotic vascular tissues, and buffy coats. Using logistic regression analysis, we identified hypomethylation of MTNR1B as an independent variable for the stenosis radiophenotype prediction model by combining it with traditional atherosclerosis risk factors including age, hypertension history, and increases in creatinine, lipoprotein (a), and homocysteine. We performed fivefold cross-validation of the prediction model using 384 patients with ischemic stroke (50 [13%] no-stenosis and 334 [87%] > 1 stenosis radiophenotype). For the cross-validation, the training dataset included 70% of the dataset. The prediction model showed an accuracy of 0.887, specificity to predict stenosis radiophenotype of 0.940, sensitivity to predict no-stenosis radiophenotype of 0.533, and area under receiver operating characteristic curve of 0.877 to predict stenosis radiophenotype from the test dataset including 30% of the dataset.ConclusionsWe identified and validated MTNR1B hypomethylation as an epigenetic marker to predict cranial vessel atherosclerosis using stenosis radiophenotypes and blood inflammatory cells rather than direct atherosclerotic plaque sampling.

Single-base resolution methylome of different ecotype from Pyrus betulaefolia reveals epigenomic changes in response to salt stress

• Drawing the cytosine methylation map across the whole genome of Pyrus betulaefolia roots. • The changes of methylomes in two ecotypes after salt exposed dependent on their salt tolerance abilities. • DNA demethylation induced higher expression levels of some Ca 2+ sensor genes in P. betulaefolia roots. • The higher K + /Na + ratio in the roots of salt-tolerance ecotype is relationship with Ca 2+ sensor genes transcription causing by hypomethylation. The root is the first organ to feel salt stress in woody fruit trees. Understanding the potential epigenetic mechanism induced by salt stress in roots of Pyrus betulaefolia is important for pear breeding using diverse genetic resources. With the help of whole-genome bisulfite sequencing, cytosine methylation was mapped at single-base resolution across the whole genome of P. betulaefolia roots. The P. betulaefolia root genome revealed nearly 11.40%, 39.93%, 22.73%, and 4.17% methylation across all sequenced C sites and in the CG, CHG, and CHH sequence contexts, respectively. Then, the changes in the methylome of the roots relating to salt stress were comparatively analysed in two ecotypes with different salt-stress tolerances. After a global methylome and transcriptome combined analysis, gene body methylation demonstrated an obvious negative correlation with transcription levels, whereas promoter-methylated genes presented lower expression levels than promoter-unmethylated genes. Furthermore, the methylation profiles of Ca 2+ sensor genes in roots of P. betulaefolia . were described, and some were demethylated under salt-stress conditions. In detail, the up-regulation of Ca 2+ sensors and their downstream genes, PbCDPK20.1, PbCDPK20.2, PbAKT1 , and PbHAK25 , resulted from salt-induced promoter methylation changes . Finally, K + accumulation was promoted, and a higher K + /Na + ratio was maintained in roots under salt-stress compared with normal conditions. Additionally, the application of the DNA methylation inhibitor 5-azacytidine in vitro induced DNA demethylation and promoted K + enrichment, whereas the accelerator methyl trifluoromethanesulfonate produced the opposite effects. In conclusion, our result revealed the relationship of DNA methylation and gene expression in P. betulaefolia during salt stress, which will increase our understanding of the epigenetic mechanisms of woody trees.

Cri du chat syndrome patients have DNA methylation changes in genes linked to symptoms of the disease

BackgroundCri du chat (also called 5p deletion, or monosomy 5p) syndrome is a genetic disease caused by deletions of various lengths in the short (p) arm of chromosome 5. Genetic analysis and phenotyping have been used to suggest dose-sensitive genes in this region that may cause symptoms when a gene copy is lost, but the heterogeneity of symptoms for patients with similar deletions complicates the picture. The epigenetics of the syndrome has only recently been looked at with DNA methylation measurements of blood from a single patient, suggesting epigenetic changes in these patients. Here, we conduct the deepest epigenetic analysis of the syndrome to date with DNA methylation analysis of eight Cri du chat patients with sibling- and age-matched controls.ResultsThe genome-wide patterns of DNA methylation in the blood of Cri du chat patients reveal distinct changes compared to controls. In the p-arm of chromosome 5 where patients are hemizygous, we find stronger changes in methylation of CpG sites than what is seen in the rest of the genome, but this effect is less pronounced in gene regulatory sequences. Gene set enrichment analysis using patient DNA methylation changes in gene promoters revealed enrichment of genes controlling embryonic development and genes linked to symptoms which are among the most common symptoms of Cri du chat syndrome: developmental delay and microcephaly. Importantly, this relative enrichment is not driven by changes in the methylation of genes on chromosome 5. CpG sites linked to these symptoms where Cri du chat patients have strong DNA methylation changes are enriched for binding of the polycomb EZH2 complex, H3K27me3, and H3K4me2, indicating changes to bivalent promoters, known to be central to embryonic developmental processes.ConclusionsFinding DNA methylation changes in the blood of Cri du chat patients linked to the most common symptoms of the syndrome is suggestive of epigenetic changes early in embryonic development that may be contributing to the development of symptoms. However, with the present data we cannot conclude about the sequence of events between DNA methylation changes and other cellular functions—the observed differences could be directly driving epigenetic changes, a result of other epigenetic changes, or they could be a reflection of other gene regulatory changes such as changed gene expression levels. We do not know which gene(s) on the p-arm of chromosome 5 that causes epigenetic changes when hemizygous, but an important contribution from this work is making the pool of possible causative genes smaller.

Promoter methylation changes in the placenta involved in the relationship between prenatal depression and small for gestational age

BackgroundRecent studies suggest that the incidence of small for gestational age (SGA) birth related to maternal depression, but the mechanism is unclear. The aim of this study was to explore the changes of promoter methylation in the placenta which may be involved in the relationship between prenatal depression and SGA.MethodsThree hundred forty-five pregnant women were enrolled in this prospective cohort study. Perinatal emotion and sleep quality in the second and third trimesters were assessed using self-rating depression scale, self-rating anxiety scale, and Pittsburgh sleep quality index. According to the exposure (depressed emotion of mother) and outcome (SGA), the placentas were divided into four groups. Methylation of the promoter regions of the placental CRH, HSD11β2, SLA16A10, DIO3, and MTNR1B genes was determined using next generation sequencing based on bisulfite sequencing PCR.ResultsThere were 97 (28.1%) and 95 (27.5%) pregnant women who had depression in the second trimester and third trimester, respectively. Thirty-five pregnant women had an SGA birth. The incidence of SGA births in this prospective cohort was 10.1%. The risk factors of SGA birth were low BMI of pregnancy women (RR = 0.71, 95%CI = 0.54 ~ 0.92), hypertensive disorder complicating pregnancy (HDCP, RR = 4.7, 95%CI = 1.18 ~ 18.72), and maternal depression in the second trimester (RR = 3.71, 95%CI = 1.31 ~ 12.16). We found that the CRH and HSD11β2 methylation levels were higher in the depression group than those in the non-depression group. Methylation levels of DIO3 were higher in SGA group than that in the non-SGA group. Higher methylation levels of CRH correlated with higher methylation levels of DIO3 in the placenta.ConclusionsMaternal depression in the second trimester may lead to the changes of methylation levels in the promoter region of CRH and HSD11β2 gene, while the changes of methylation of DIO3 in subsequent could related to SGA. This study suggests that maternal depressed emotion during pregnancy may result in SGA due to the epigenetic changes of placenta.

Renal functional, transcriptome, and methylome adaptations in pregnant Sprague Dawley and Brown Norway rats.

Pregnancy induces maternal renal adaptations that include increased glomerular filtration rate and renal blood flow which can be compromised in obstetrical complications such as preeclampsia. Brown Norway (BN) rat pregnancies are characterized by placental insufficiency, maternal hypertension, and proteinuria. We hypothesized that BN pregnancies would show renal functional, anatomical, or molecular features of preeclampsia. We used the Sprague-Dawley (CD) rat as a model of normal pregnancy. Pregnancy increased the glomerular filtration rate by 50% in CD rats and 12.2% in BN rats compared to non-pregnancy, and induced proteinuria only in BN rats. BN pregnancies showed a decrease in maternal plasma calcitriol levels, which correlated with renal downregulation of 1-alpha hydroxylase and upregulation of 24-hydroxylase. RNA sequencing revealed that pregnancy induced 297 differentially expressed genes (DEGs) in CD rats and 174 DEGs in BN rats, indicating a 70% increased response to pregnancy in CD compared to BN rats. Pregnancy induced activation of innate immune pathways such as ‘Role of Pattern Recognition Receptors’, and ‘Interferon signaling’ with interferon regulatory factor 7 as a common upregulated upstream factor in both rat strains. Comparison of rat strain transcriptomic profiles revealed 475 DEGs at non-pregnancy and 569 DEGs at pregnancy with 205 DEGs shared at non-pregnancy (36%), indicating that pregnancy interacted with rat strain in regulating 64% of the DEGs. Pathway analysis revealed that pregnancy induced a switch in renal transcriptomics in BN rats from ‘inhibition of renal damage’ to ‘acute phase reaction’, ‘recruitment of immune cells’ and ‘inhibition of 1,25-(OH)2-vitamin D synthesis’. Key upstream regulators included peroxisome-proliferator-activated receptor alpha (PPARA), platelet-derived growth factor B dimer (PDGF-BB), and NF-kB p65 (RELA). DNA methylome profiling by reduced representation bisulfite sequencing studies revealed that the DEGs did not correlate with changes in promoter methylation. In sum, BN rat kidneys respond to pregnancy-specific signals with an increase in pro-inflammatory gene networks and alteration of metabolic pathways including vitamin D deficiency in association with mild proteinuria and blunted GFR increase. However, the lack of glomerular endotheliosis and mild hypertension/proteinuria in pregnant BN rats limits the relevance of this rat strain for preeclampsia research.

Promoter Methylation Changes in KRT17: A Novel Epigenetic Marker for Wool Production in Angora Rabbit.

Wool production is an important economic trait of Angora rabbits. Exploring molecular markers related to wool production is one of the essentials of Angora rabbits’ breeding. KRT17 (Keratin 17) is an important gene of hair follicle development, which must be explored for genetic/epigenetic variation to assess its effect on wool production. Based on the effective wool production data of 217 Angora rabbits, the high and low yield groups were screened with 1.5 standard deviations of the population mean. The full-length sequence of KRT17 was obtained by rapid amplification of cDNA ends technology, and the polymorphism was analyzed in the promoter, exon, and intron regions by direct sequencing. KRT17, SP1 over-expression plasmids, and siRNA were constructed and transfected into dermal papilla cells. The mRNA expressions of relevant genes were analyzed by RT-qPCR. The methylation level of the KRT17 promoter was determined by Bisulfite Sequencing PCR. Dual-luciferase system, site-directed mutagenesis, and electrophoretic mobility shift assays were used to analyze the binding relationship between SP1 and the promoter of KRT17. The structure map of KRT17 was drawn, and no SNPs were found in the promoter, exon, and intron, indicating a relatively conserved structure of KRT17. Expression of KRT17 was significantly higher in cutaneous tissues than in other tissues and was significantly upregulated in the high-yield group compared to the low-yield group (p < 0.05). Furthermore, the overall high methylation levels of KRT17 CpG I and CpG III showed significant association with low wool yield; the methylation levels of 5 CpG locus (CpG I site 4 and CpG III site 2–5) were significantly different between the high and low yield groups (p < 0.05). The methylation levels of 3 CpG locus (CpG I site 4 and CpG III site 4, 14) showed a significant correlation with KRT17 expression (p < 0.05). Overall, CpG III site 4 significantly affects wool production and KRT17 expressions (p < 0.05). This site promotes SP1 binding to the KRT17 promoter region (CGCTACGCCC) to positively regulate the KRT17 expression. KRT17 CpG III site 4 can be used as candidate epigenetic markers for the breeding of high wool-producing Angora rabbits.

Abstract A045: Multiple roles for plasticity in metastasis and therapy resistance in long-term survivors of metastatic colorectal cancer

Abstract Long-term survivors (LS) of metastatic colorectal cancer (mCRC) who experience multiple recurrences with resectable oligometastatic disease provide an opportunity to explore co-evolution of the tumor and immune microenvironment. We profiled 16 LS of mCRC with a median follow-up of 9.3 years and median of 3 biopsies/resections per patient (range 2-7). We performed multi-omic profiling of 56 primary and 176 metastatic samples from formalin-fixed paraffin-embedded tissue using low pass whole genome sequencing, 3’ RNA sequencing and DNA methylation arrays. A machine learning cell classifier was used to quantify immune cell and fibroblast infiltration from hematoxylin and eosin staining. Copy number profiling showed that the fraction of genome altered remained relatively stable across time and tissue type but that already-altered segments underwent progressive fragmentation. Inter-timepoint divergence of copy number alterations was significantly higher than intra-timepoint divergence, and intra-timepoint divergence was lower for liver and lung metastases than for primary tumors. Chemotherapy treatment did not significantly affect either divergence type. Differential expression and gene set enrichment analysis (GSEA) revealed common pathways dysregulated in metastases compared to primaries, including reductions in E2F (important in G1/S checkpoint) and G2M checkpoint, suggestive of the onset of senescence in metastases. Tumors underwent progressive hypomethylation over time and analysis of genes with concordant changes in promoter methylation and expression revealed dysregulation in pathways related to endocytosis, cell adhesion and migration. This suggests an important role for phenotypic plasticity in driving the metastatic phenotype. There were transient increases in the proportion of macrophages, lymphocytes and neutrophils in tumors that had undergone neoadjuvant chemotherapy in the 6 months prior to resection and slight increases in M1 macrophage activity (by GSEA) in tumors that were previously therapy naïve. There were concordant transient increases in pathways associated with immune response (MYC V1 and MTORC1), as well as xenobiotic metabolism. The latter is a known mechanism of drug resistance to both the platinum- and fluoropyridine-based therapies used in CRC. There were also more sustained increases post-chemotherapy in inflammatory and immune pathways associated with the adaptive immune response and tissue injury and repair. These findings were corroborated by concordant changes in promoter methylation. These data suggest that there is a threshold level of aneuploidy required to facilitate CRC metastasis but the migratory phenotype and adaptation to the metastatic niche are driven by plasticity. Chemotherapy induces differing short-term and long-term anti-tumor immune responses in the local microenvironment. We see evidence that mCRCs are able to mount plastic pro-survival mechanisms in response to chemotherapy. Citation Format: Alison May Berner, Calum Gabbutt, Salpie Nowinski, Jacob Househam, Nick Trahearn, George D. Cresswell, Vinaya Srirangam Nadhamuni, Christopher Kimberley, Matteo Fassan, Ann-Marie Baker, Andrea Sottoriva, Christina Thirlwell, John Bridgewater, Trevor Graham. Multiple roles for plasticity in metastasis and therapy resistance in long-term survivors of metastatic colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A045.

Skeletal Muscle LINE‐1 Gene Regulation and Inflammatory Response to Chronic, Voluntary Endurance Training in Rodents

ObjectiveWe sought to determine the effects of chronic, voluntary wheel running on Long Interspersed Nuclear Element‐1 (LINE‐1) mRNA expression and promoter methylation, as well as markers of the cGAS‐STING inflammatory pathway, in skeletal muscle.HypothesesLINE‐1 mRNA expression will increase with age, and exercise will mitigate this increase. This difference in expression will be driven through changes in promoter methylation. The cGAS‐STING inflammatory pathway will be upregulated with age and downregulated with rats that exercise‐trained.MethodsPlantaris and soleus skeletal muscles were collected from female Lewis rats that belonged to one of three groups: a young control group (CTL; n=10; 6 mos old), an aged sedentary group (SED; n=12; 15 mos old), and an aged exercise group with access to a running wheel for 9 months (EX; n=12; 15 mos old). RNA, DNA, and protein were isolated from the tissues for analysis. Analysis included RT‐qPCR for LINE‐1 mRNA expression, methylated DNA Immunoprecipitation (MeDIP) to assess the methylation status of the LINE‐1 promoter via RT‐qPCR, and Western blotting for markers of the cGAS‐STING pathway and 4Hydroxynonenal (4HNE). Two primer sets were used for RT‐qPCR: L1‐3 probed for the most active form of LINE‐1 and L1‐Tot probed for all full‐length LINE‐1 elements.ResultsThere was no significant difference in plantaris LINE‐1 mRNA expression or for L1‐3 or L1‐Tot. Plantaris LINE‐1 promoter methylation was undetectable in most samples, so the results are not presented herein. Soleus L1‐3 mRNA was significantly higher in EX compared to CTL (p=0.036) and there was a trend between SED and EX (p=0.053). L1‐Tot also was trending (p=0.059), where EX was higher than CTL (p=0.025). Methylation of the LINE‐1 promoter was significantly different for both L1‐3 and L1‐Tot (p=0.021 and p=0.028, respectively). In both instances, LINE‐1 DNA in SED and EX were significantly more methylated compared to CTL. There were no differences in cGAS protein expression, p‐/pan STING protein expression, or 4HNE expression.ConclusionsContrary to the original hypothesis, LINE‐1 mRNA expression and DNA methylation were higher in the exercise group. Additionally, chronic exercise did not affect the cGAS‐STING inflammatory pathway, highlighting this pathway may be more relevant in extreme age and disease states. Previous skeletal muscle research has focused on using mixed muscle types to look at LINE‐1 expression and regulation, so the differences seen between type I (soleus) and type II (plantaris) fibers is a novel finding that warrants further investigation to understand the underlying mechanisms.

Association of Aberrant Promoter Methylation Changes in the Suppressor of Cytokine Signaling 3 (SOCS3) Gene with Susceptibility to Crohn's Disease.

Background:Growing evidence supports that changes in the methylation state of Inflammatory Bowel Disease (IBD)-associated genes could significantly alter levels of gene expression, potentially contributing to disease onset and progression. We supposed that alterations in DNA methylation status at promoter region within the suppressor of cytokine signaling 3 (SOCS3) gene in intestinal tissues may be involved in the susceptibility to Crohn's Disease (CD).Methods:DNA methylation status in the promoter region of the human SOCS3 gene of intestinal tissues from 15 patients with CD and 15 age- and sex-matched healthy controls were profiled using the real-time Quantitative Multiplex Methylation Specific PCR (QM-MSP) assay.Results:Based on methylation assay data profiling, we found that patients with CD showed a higher degree of methylation of the SOCS3 gene promoter region than did the healthy controls (unmethylated DNA in CD vs. healthy controls; 0.00048±0.0011 vs. 0.07±0.142, p<0.000).Conclusion:The data presented here demonstrate that aberrant methylation of the CpG islands within promoter regions of SOCS3 gene in colonic mucosa of CD was associated with mucosal inflammatory status, providing insights into the involvement of methylation could contribute to the initiation of the inflammatory process and development of CD.

DNA methylation variation along the cancer epigenome and the identification of novel epigenetic driver events.

While large-scale studies applying various statistical approaches have identified hundreds of mutated driver genes across various cancer types, the contribution of epigenetic changes to cancer remains more enigmatic. This is partly due to the fact that certain regions of the cancer genome, due to their genomic and epigenomic properties, are more prone to dysregulated DNA methylation than others. Thus, it has been difficult to distinguish which promoter methylation changes are really driving carcinogenesis from those that are mostly just a reflection of their genomic location. By developing a novel method that corrects for epigenetic covariates, we reveal a small, concise set of potential epigenetic driver events. Interestingly, those changes suggest different modes of epigenetic carcinogenesis: first, we observe recurrent inactivation of known cancer genes across tumour types suggesting a higher convergence on common tumour suppressor pathways than previously anticipated. Second, in prostate cancer, a cancer type with few recurrently mutated genes, we demonstrate how the epigenome primes tumours towards higher tolerance of other aberrations.

Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System.

Simple SummaryDNA methylation is an important modification of the genome that is implicated in the pathogenesis of numerous human diseases, including cancer. DNA methylation changes can alter the expression of critical genes, predisposing to disease progression. Existing techniques that can modify DNA methylation to investigate disease etiology are severely limited with regard to specificity, which means that establishing a causal link between DNA methylation changes and disease progression is difficult. The advent of CRISPR-based technologies has provided a powerful tool for more specific editing of DNA methylation. Here, we describe a comprehensive protocol for the design and application of a CRISPR-dCas9-based tool for editing DNA methylation at a target locus in human melanoma cell lines alongside protocols for downstream techniques used to evaluate subsequent methylation and gene expression changes in methylation-edited cells. Furthermore, we demonstrate highly efficacious methylation and demethylation of the EBF3 promoter across a panel of melanoma cell lines.DNA methylation is a key epigenetic modification implicated in the pathogenesis of numerous human diseases, including cancer development and metastasis. Gene promoter methylation changes are widely associated with transcriptional deregulation and disease progression. The advent of CRISPR-based technologies has provided a powerful toolkit for locus-specific manipulation of the epigenome. Here, we describe a comprehensive global workflow for the design and application of a dCas9-SunTag-based tool for editing the DNA methylation locus in human melanoma cells alongside protocols for downstream techniques used to evaluate subsequent methylation and gene expression changes in methylation-edited cells. Using transient system delivery, we demonstrate both highly efficacious methylation and demethylation of the EBF3 promoter, which is a putative epigenetic driver of melanoma metastasis, achieving up to a 304.00% gain of methylation and 99.99% relative demethylation, respectively. Furthermore, we employ a novel, targeted screening approach to confirm the minimal off-target activity and high on-target specificity of our designed guide RNA within our target locus.

Transgenerational inheritance of promoter methylation changes in extrauterine growth restriction-induced pulmonary arterial pressure disorders.

BackgroundThis study aimed to investigate the influence of extrauterine growth restriction (EUGR) on pulmonary arterial pressure (PAP) and the transgenerational inheritance of promoter methylation changes in pulmonary vascular endothelial cells (PVECs) of 2 consecutive generations under EUGR stress.MethodsAfter modeling, PAP values of F1 and F2 pups were investigated at 9-week-old. The methyl-DNA immune precipitation chip was used to analyze DNA methylation profiling. Differential enrichment peaks (DEPs) and regions of interest (ROIs) were identified, based on which Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and reactome pathway enrichments were analyzed.ResultsThe F1 male rats in the EUGR group had significantly increased PAP levels compared to the control group; however, this increase was not observed in female rats. Interestingly, in F2 female rats, the EUGR group had decreased PAP. In the X chromosome of the F1 males, there were 16 differential ROI genes in the F1 generation, while in F2 females, there were 86 differential ROI genes. Similarly, there were 105 DEPs in the F1 generation and 38 DEPs in the F2 generation. In combination with the 5 common ROIs and 14 common DEPs, 18 genes were regarded as the key candidate genes associated with hereditable PAP variation in the EUGR model. Enrichment analysis showed that synaptic and neurotransmitter relative pathways might be involved in the process of EUGR-induced PAH development. Among common DEPs, Smad1 and Serpine1 were also found in 102 PAH-associated genes in the MalaCards database.ConclusionsTogether, there is a transgenerational inheritance of promoter methylation changes in the X chromosome in EUGR-induced PAP disorders, which involves the participation of synaptic and neurotransmitter relative pathways. Also, attenuated methylation of Smad1 and Serpine1 in the promoter region may be a partial driver of PAH in later life.