Repressor Region Research Articles

Global coupling of R-loopdynamics with RNA polymerase II modulates gene expression and early development of Drosophila.

R-loops are involved in many biological processes in cells, yet the regulatory principles for R-loops in vivo and their impact on development remain to be explored. Here, we modified the CUT&Tag strategy to profile R-loops in Drosophila at multiple developmental stages. While high GC content promotes R-loop formation in mammalian cells, it is not required in Drosophila. In contrast, RNAPII abundance appears to be a universal inducing factor for R-loop formation, including active promoters and enhancers, and H3K27me3 decorated repressive regions and intergenic repeat sequences. Importantly, such a regulatory relationship is dynamically maintained throughout development, and development-related transcription factors may regulate RNAPII activation and R-loop dynamics. By ablating Spt6, we further showed the global R-loop induction coupled with RNAPII pausing. Importantly, depending on the gene length, genes underwent up- or down-regulation, both of which were largely reversed by rnh1 overexpression, suggesting that R-loops play a significant role in the divergent regulation of transcription by Spt6 ablation. DNA damage, defects in survival, and cuticle development were similarly alleviated by rnh1 overexpression. Altogether, our findings indicate that dynamic R-loop regulation is dictated by RNAPII pausing and transcription activity, and plays a feedback role in gene regulation, genome stability maintenance, and Drosophila development.

Discovery of a conserved translationally repressive upstream open reading frame within the iron-deficiency response regulator IDEF2

BackgroundIron (Fe) deficiency affects 30–50% of the world’s population. Genetic biofortification of staple crops is a promising strategy for improving human nutrition, but the number of effective precision breeding targets for Fe biofortification is small. Upstream open reading frames (uORFs) are cis-regulatory elements within the 5’ leader sequence (LS) of genes that generally repress translation of the main open reading frame (mORF).ResultsWe aligned publicly available rice (Oryza sativa L.) ribo-seq datasets and transcriptomes to identify putative uORFs within important Fe homeostasis genes. A dual luciferase assay (DLA) was used to determine whether these uORFs cause repression of mORF translation and pinpoint LS regions that can be mutated for mORF derepression. A translationally repressive uORF region was identified in two positive regulators of the Fe-deficiency response: IDEF1 and IDEF2. The IDEF2-uORF peptide was highly conserved among monocots and a mutation series in the 5’ LS of the wheat (Triticum aestivum L.) TaIDEF2-A1 gene demonstrated variable mORF derepression.ConclusionsTogether these results reveal a possible regulatory mechanism by which IDEF2 transcription factors modulate the Fe deficiency response in monocots, and highlight novel precision breeding targets to improve crop nutrition and abiotic stress tolerance.

Super-enhancer-driven LncRNA PPARα-seRNA exacerbates glucolipid metabolism and diabetic cardiomyopathy via recruiting KDM4B

ObjectiveAberrant glucolipid metabolism in the heart is a characteristic factor in diabetic cardiomyopathy (DbCM). Super-enhancers-driven noncoding RNAs (seRNAs) are emerging as powerful regulators in the progression of cardiac diseases. However, the functions of seRNAs in DbCM have not been fully elucidated. MethodsSuper enhancers and their associated seRNAs were screened and identified by H3K27ac ChIP-seq data in the Encyclopedia of DNA Elements (ENCODE) dataset. A dual-luciferase reporter assay was performed to analyze the function of super-enhancers on the transcription of peroxisome proliferator-activated receptor α-related seRNA (PPARα-seRNA). A DbCM mouse model was established using db/db leptin receptor-deficient mice. Adeno-associated virus serotype 9-seRNA (AAV9-seRNA) was injected via the tail vein to evaluate the role of seRNA in DbCM. The underlying mechanism was explored through RNA pull-down, RNA and chromatin immunoprecipitation, and chromatin isolation by RNA purification. ResultsPPARα-seRNA was regulated by super-enhancers and its levels were increased in response to high glucose and palmitic acid stimulation in cardiomyocytes. Functionally, PPARα-seRNA overexpression aggravated lipid deposition, reduced glucose uptake, and repressed energy production. In contrast, PPARα-seRNA knockdown ameliorated metabolic disorder in vitro. In vivo, overexpression of PPARα-seRNA exacerbated cardiac metabolic disorder and deteriorated cardiac dysfunction, myocardial fibrosis, and hypertrophy in DbCM. Mechanistically, PPARα-seRNA bound to the histone demethylase KDM4B (Lysine-specific demethylase 4B) and decreased H3K9me3 levels in the promoter region of PPARα, ultimately enhancing its transcription. ConclusionsOur study revealed the pivotal function of a super-enhancer-driven long noncoding RNA (lncRNA), PPARα-seRNA, in the deterioration of cardiac function and the exacerbation of metabolic abnormalities in diabetic cardiomyopathy, which recruited KDM4B to the promoter region of PPARα and repression of its transcription. This suggests a promising therapeutic strategy for the treatment of DbCM.

Abstract 2580: Targeting chromatin in ovarian cancer stem cells

Abstract The dynamic organization of chromatin, governed by epigenetic modifiers, critically regulates cell plasticity and determines cell fate in both normal and malignant stem cells. We hypothesized that chromatin architecture plays a role in regulating transcriptional heterogeneity in ovarian cancer stem cells (OCSCs) to maintain stemness and chemoresistance. PWS microscopy showed that ALDH+ OCSCs derived displayed higher nuclear fractal dimension D signals (P<0.05) than ALDH- non-OCSCs, indicating that chromatin in OCSCs possesses higher packing compared to non-OCSCs. Cut & TAG Sequencing showed that of the 3606-histone repressive H3K27m3 enriched regions (P<0.05, fold change >2), 3208 were detected in OCSCs while 398 peaks were present in non-OCSCs, consistent with findings supporting high D features in OCSCs. The active mark H3K4me3 was enriched at the promoter of stemness-associated genes, including SOX2, ALDH1A3, and FZD7 in OCSCs; and the repressive histone mark H3K27me3 was enriched at the promoter of the epithelial cell marker CDH1 in OCSCs. These results support that the chromatin of OCSCs contains open genomic regions, corresponding to stemness-associated genes and repressed regions associated with genes related to differentiation. RNA sequencing indicated that the coefficient of variance (COV) reflecting transcriptional heterogeneity in OCSCs was higher than in non-OCSCs at baseline. In response to platinum, genes with low baseline expression in OCSCs were most upregulated, supporting increased transcriptional heterogeneity in response to chemotherapy. Inhibitors of epigenetic modifiers (DNMTi, EZH2i, and Dot1Li) induced chromatin re-organization in OCSCs and promoted OCSCs transition toward differentiation, supporting that stemness-associated chromatin structure is targetable. We demonstrated that a Dot1L inhibitor (Dot1Li) targeted OCSCs by decreasing chromatin packing D, resulting in decreased transcriptional heterogeneity. In conclusion, OCSCs harbor highly packed chromatin, contributing to chemoresistance and cell plasticity. Epigenetic modifiers (such as Dot1Li) inhibit stemness by regulating chromatin organization and transcriptional malleability. Citation Format: Yinu Wang, Jane Frederick, Yaqi Zhang, Elizabeth Thomas Bartom, Greta Bauer, Edward Tanner, Vadim Backman, Daniela Matei. Targeting chromatin in ovarian cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2580.

Association between DNA Methylation in the Core Promoter Region of the CUT-like Homeobox 1 (CUX1) Gene and Lambskin Pattern in Hu Sheep.

CUT-like homeobox 1 (CUX1) has been proven to be a key regulator in sheep hair follicle development. In our previous study, CUX1 was identified as a differential expressed gene between Hu sheep lambskin with small wave patterns (SM) and straight wool patterns (ST); however, the exact molecular mechanism of CUX1 expression has been obscure. As DNA methylation can regulate the gene expression, the potential association between CUX1 core promotor region methylation and lambskin pattern in Hu sheep was explored in the present study. The results show that the core promoter region of CUX1 was present at (-1601-(-1) bp) upstream of the transcription start site. A repressive region (-1151-(-751) bp) was also detected, which had a strong inhibitory effect on CUX1 promoter activity. Bisulfite amplicon sequencing revealed that no significant difference was detected between the methylation levels of CUX1 core promoter region in SM tissues and ST tissues. Although the data demonstrated the differential expression of CUX1 between SM and ST probably has no association with DNA methylation, the identification of the core region and a potential repressive region of CUX1 promoter can enrich the role of CUX1 in Hu sheep hair follicle development.

Cumulative effects of weakly repressive regulatory regions in the 3’ UTR maintain PD-1 expression homeostasis in mammals

PD-1 has become a common target for cancer treatment. However, the molecular regulation of PD-1 expression homeostasis remains unclear. Here we report the PD-1 3’ UTR can dramatically repress gene expression via promoting mRNA decay. Deletion of the PD-1 3’ UTR inhibits T cell activity and promotes T-ALL cell proliferation. Interestingly, the robust repression is attributable to cumulative effects of many weak regulatory regions, which we show together are better able to maintain PD-1 expression homeostasis. We further identify several RNA binding proteins (RBPs) that modulate PD-1 expression via the 3’ UTR, including IGF2BP2, RBM38, SRSF7, and SRSF4. Moreover, despite rapid evolution, PD-1 3’ UTRs are functionally conserved and strongly repress gene expression through many common RBP binding sites. These findings reveal a previously unrecognized mechanism of maintaining PD-1 expression homeostasis and might represent a general model for how small regulatory effects play big roles in regulation of gene expression and biology.

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of β-hemoglobinopathies

Reactivation of fetal hemoglobin (HbF) is a commonly adapted strategy to ameliorate β-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits HbF tetramer production affecting the therapeutic benefits. Here, we evaluated deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified an 11-kb sequence, encompassing putative repressor region (PRR) to β-globin exon-1 (βE1), as the core deletion that ablates HbA and exhibits superior HbF production compared with HPFH or other well-established targets. PRR-βE1-edited hematopoietic stem and progenitor cells (HSPCs) retained their genome integrity and their engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice producing HbF positive cells invivo. Furthermore, PRR-βE1 gene editing is feasible without exvivo HSPC culture. Importantly, the editing induced therapeutically significant levels of HbF to reverse the phenotypes of both sickle cell disease and β-thalassemia major. These findings imply that PRR-βE1 gene editing of patient HSPCs could lead to improved therapeutic outcomes for β-hemoglobinopathy gene therapy.

DNA methylation dynamics and dysregulation delineated by high-throughput profiling in the mouse

SUMMARYWe have developed a mouse DNA methylation array that contains 296,070 probes representing the diversity of mouse DNA methylation biology. We present a mouse methylation atlas as a rich reference resource of 1,239 DNA samples encompassing distinct tissues, strains, ages, sexes, and pathologies. We describe applications for comparative epigenomics, genomic imprinting, epigenetic inhibitors, patient-derived xenograft assessment, backcross tracing, and epigenetic clocks. We dissect DNA methylation processes associated with differentiation, aging, and tumorigenesis. Notably, we find that tissue-specific methylation signatures localize to binding sites for transcription factors controlling the corresponding tissue development. Age-associated hypermethylation is enriched at regions of Polycomb repression, while hypomethylation is enhanced at regions bound by cohesin complex members. ApcMin/+ polyp-associated hypermethylation affects enhancers regulating intestinal differentiation, while hypomethylation targets AP-1 binding sites. This Infinium Mouse Methylation BeadChip (version MM285) is widely accessible to the research community and will accelerate high-sample-throughput studies in this important model organism.

Abstract 1462: Oncogenic activation of FOXR2 driven by somatic acquisition of a LINE-1 promoter in pediatric high-grade glioma

Abstract Long Interspersed Element-1 (LINE-1 or L1) is the only autonomously active mobile genetic element in the human genome. It has been shown to mobilize (i.e., retrotranspose) in cancers, causing de novo somatic L1 insertions. Somatic L1 retrotransposition can cause aberrant splicing of tumor suppressor genes by utilizing cryptic splice donor (SD) sites such as the nucleotide 97 SD in the L1 5’UTR or disrupting repressive regulatory regions of proto-oncogenes, inducing aberrant activation, leading to cancer. In contrast to these reported mechanisms, here we report the first evidence of a driver oncogene activation due to somatic acquisition of an L1 promoter in a pediatric high-grade glioma (HGG). While investigating activation of FOXR2 (forkhead box R2), a known oncogene in CNS embryonal tumors, in a recurrent pediatric diffuse HGG that showed a methylation profile of CNS FOXR2-activated tumors, we identified overexpression of a non-canonical FOXR2 isoform. Transcription of FOXR2 initiated from ~11kb upstream of exon 2 at a site harboring a somatic structural variation (SV) reminiscent of an L1 insertion. Targeted PCR amplification and subsequent PacBio sequencing revealed a 5’ inverted/deleted L1 insertion (~3kb sequence) containing an intact L1 5’UTR, presence of a 3’ transduction sequence, two poly-A tails, and target site duplications. The 3’ transduction sequence matches the sequence downstream an intact polymorphic full-length L1 element at 6p24.1, the likely source L1 element that initiated the FOXR2 somatic insertion. Strikingly, tumor RNA-seq data reveals FOXR2 transcription initiated from the intact sense pol-II promoter of the inserted L1 5’UTR, generating a fusion transcript joining the L1 5’UTR 97 SD to a canonical splice acceptor within exon 2 of FOXR2. Targeted bisulfite sequencing of the FOXR2 L1 5’UTR revealed a hypomethylated state, further supporting its role in FOXR2 transcription initiation. FOXR2 activation is likely a tumor initiating event as the following was observed in the primary tumor that occurred two years prior: a matching recurrent tumor methylation profile, the presence of L1/FOXR2 fusion transcripts, and overexpression of FOXR2. Additional driver mutations, clonal TP53 R175H mutation and PDGFRA amplification, identified in the recurrent tumor but absent in the primary tumor, support the somatic L1 insertion as a tumor initiating event occurring prior to later acquired alterations. Our data represent the first example of oncogenic activation by L1 “promoter donation” via somatic retrotransposition as a novel tumor initiating mechanism. Future large-scale research and development of new computational methods for detecting rearranged L1 insertions are required to determine the prevalence of L1 “promoter donation” in cancer or other diseases. Citation Format: Xiaolong Chen, Diane A. Flasch, Bensheng Ju, Heather L. Mulder, John Easton, Lu Wang, Suzanne J. Baker, Jason Chiang, Jinghui Zhang. Oncogenic activation of FOXR2 driven by somatic acquisition of a LINE-1 promoter in pediatric high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1462.

Histone Methylation Regulates Gene Expression in the Round Spermatids to Set the RNA Payloads of Sperm.

Gene expression during spermatogenesis undergoes significant changes due to a demanding sequence of mitosis, meiosis, and differentiation. We investigated the contribution of H3 histone modifications to gene regulation in the round spermatids. Round spermatids were purified from rat testes using centrifugal elutriation and Percoll density-gradient centrifugation. After enzymatic chromatin shearing, immuno-precipitation using antibodies against histone marks H3k4me3 and H3K9me3 was undertaken. The immunoprecipitated DNA fragments were subjected to massive parallel sequencing. Gene expression in round spermatids and sperm was analyzed by transcriptome sequencing using next-generation sequencing methods. ChIP-seq analysis showed significant peak enrichment in H3K4me3 marks in active chromatin regions and H3K9me3 peak enrichment in repressive regions. We found 53 genes which showed overlapping peak enrichment in both H3K4me3 and H3K9me3 marks. Some of the top H3K4me3-enriched genes were involved in sperm tail formation (Odf1, Odf3, Odf4, Oaz3, Ccdc42, Ccdc63,andCcdc181), chromatin condensation (Dync1h1, Dynll1,andKdm3a), and sperm functions such as acrosome reaction (AcrbpandFabp9), energy generation (Gapdhs), and signaling for motility (Tssk1b, Tssk2,andTssk4). Transcriptome sequencing in round spermatids found 64% transcripts of the H3K4me3-enriched genes at high levels and of about 25% of H3K9me3-enriched genes at very low levels. Transcriptome sequencing in sperm found that more than 99% of the ChIP-seq corresponding transcripts were also present in sperm. H3K4me3 enrichment in the round spermatids correlates significantly with gene expression and H3K9me3 correlates with gene silencing that contribute to sperm differentiation and setting the RNA payloads of sperm.

Troxerutin affects nephropathy signaling events in the kidney of type-1 diabetic male rats.

Objective:Nephropathy is known to be the leading cause of kidney failure in diabetic patients. Troxerutin, as a flavonoid component, could provide a novel protective strategy in the prevention of diabetic nephropathy. A large number of reports on the salutary effects of troxerutin inspired us to investigate its effect on the nephropathy signaling events (i.e., expression of TGF-β, miRNA192, and SIP1) in type-1 induced diabetic rats.Materials and Methods:50 male Wistar rats were divided into 5 groups including control group, sham group treated with troxerutin for 4 weeks, diabetic group induced by streptozotocin (STZ) injection, DI group including insulin-treated diabetic animals and DT group treated with troxerutin. Ultimately, rat kidneys were extracted, and the level of miR-192 (using qPCR), transforming growth factor-beta (TGF-β), and smad interacting protein 1 (SIP1) using an ELISA kit, was measured.Results:The level of TGF-β and miRNA192 significantly increased in the diabetic group. However, their expression levels decreased following the administration of troxerutin and insulin (p<0.05) compared to control group. SIP1 was down-regulated in the diabetic group, whereas a spike in the expression levels was observed after troxerutin administration compared to control and troxerutin groups (p<0.05). However, no significant difference was found in the effects of insulin and troxerutin on the level of miR-192, SIP1, and TGF- β. Conclusion:According to the previous literatures, during the progression of nephropathy, TGF-β represses SIP1 (the repression region in the collagen gene) by increasing the expression of miR-192. Ultimately, in this study, diabetes led to up-regulation of TGF-β while troxerutin proved to have a protective effect on the kidney by increasing SIP and lowering miR-192 levels.

Comprehensive characterization of the epigenetic landscape in Multiple Myeloma.

Background: Human multiple myeloma (MM) cell lines (HMCLs) have been widely used to understand the molecular processes that drive MM biology. Epigenetic modifications are involved in MM development, progression, and drug resistance. A comprehensive characterization of the epigenetic landscape of MM would advance our understanding of MM pathophysiology and may attempt to identify new therapeutic targets.Methods: We performed chromatin immunoprecipitation sequencing to analyze histone mark changes (H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3 and H3K36me3) on 16 HMCLs.Results: Differential analysis of histone modification profiles highlighted links between histone modifications and cytogenetic abnormalities or recurrent mutations. Using histone modifications associated to enhancer regions, we identified super-enhancers (SE) associated with genes involved in MM biology. We also identified promoters of genes enriched in H3K9me3 and H3K27me3 repressive marks associated to potential tumor suppressor functions. The prognostic value of genes associated with repressive domains and SE was used to build two distinct scores identifying high-risk MM patients in two independent cohorts (CoMMpass cohort; n = 674 and Montpellier cohort; n = 69). Finally, we explored H3K4me3 marks comparing drug-resistant and -sensitive HMCLs to identify regions involved in drug resistance. From these data, we developed epigenetic biomarkers based on the H3K4me3 modification predicting MM cell response to lenalidomide and histone deacetylase inhibitors (HDACi).Conclusions: The epigenetic landscape of MM cells represents a unique resource for future biological studies. Furthermore, risk-scores based on SE and repressive regions together with epigenetic biomarkers of drug response could represent new tools for precision medicine in MM.

Trendy Monsters: The Nazis, the Perpetrator Turn, and Popular Culture

Trendy Monsters: The Nazis, the Perpetrator Turn, and Popular Culture

Repression precedes independent evolutionary gains of a highly specific gene expression pattern.

SUMMARYHighly specific expression patterns can be caused by the overlapping activities of activator and repressor sequences in enhancers. However, few studies illuminate how these sequences evolve in the origin of new enhancers. Here, we show that expression of the bond gene in the semicircular wall epithelium (swe) of the Drosophila melanogaster male ejaculatory bulb (EB) is controlled by an enhancer consisting of an activator region that requires Abdominal-B driving expression in the entire EB and a repressor region that restricts this expression to the EB swe. Although this expression pattern is independently gained in the distantly related Scaptodrosophila lebanonensis and does not require Abdominal-B, we show that functionally similar repressor sequences are present in Scaptodrosophila and also in species that do not express bond in the EB. We suggest that during enhancer evolution, repressor sequences can precede the evolution of activator sequences and may lead to similar but independently evolved expression patterns.

Region-specific H3K9me3 gain in aged somatic tissues in Caenorhabditis elegans

Epigenetic alterations occur as organisms age, and lead to chromatin deterioration, loss of transcriptional silencing and genomic instability. Dysregulation of the epigenome has been associated with increased susceptibility to age-related disorders. In this study, we aimed to characterize the age-dependent changes of the epigenome and, in turn, to understand epigenetic processes that drive aging phenotypes. We focused on the aging-associated changes in the repressive histone marks H3K9me3 and H3K27me3 in C. elegans. We observed region-specific gain and loss of both histone marks, but the changes are more evident for H3K9me3. We further found alteration of heterochromatic boundaries in aged somatic tissues. Interestingly, we discovered that the most statistically significant changes reflected H3K9me3-marked regions that are formed during aging, and are absent in developing worms, which we termed "aging-specific repressive regions" (ASRRs). These ASRRs preferentially occur in genic regions that are marked by high levels of H3K9me2 and H3K36me2 in larval stages. Maintenance of high H3K9me2 levels in these regions have been shown to correlate with a longer lifespan. Next, we examined whether the changes in repressive histone marks lead to de-silencing of repetitive DNA elements, as reported for several other organisms. We observed increased expression of active repetitive DNA elements but not global re-activation of silent repeats in old worms, likely due to the distributed nature of repetitive elements in the C. elegans genome. Intriguingly, CELE45, a putative short interspersed nuclear element (SINE), was greatly overexpressed at old age and upon heat stress. SINEs have been suggested to regulate transcription in response to various cellular stresses in mammals. It is likely that CELE45 RNAs also play roles in stress response and aging in C. elegans. Taken together, our study revealed significant and specific age-dependent changes in repressive histone modifications and repetitive elements, providing important insights into aging biology.

The nuclear periphery is a scaffold for tissue-specific enhancers.

Nuclear architecture influences gene regulation and cell identity by controlling the three-dimensional organization of genes and their distal regulatory sequences, which may be far apart in linear space. The genome is functionally and spatially segregated in the eukaryotic nucleus with transcriptionally active regions in the nuclear interior separated from repressive regions, including those at the nuclear periphery. Here, we describe the identification of a novel type of nuclear peripheral chromatin domain that is enriched for tissue-specific transcriptional enhancers. Like other chromatin at the nuclear periphery, these regions are marked by H3K9me2. But unlike the nuclear peripheral Lamina-Associated Domains (LADs), these novel, enhancer-rich domains have limited Lamin B interaction. We therefore refer to them as H3K9me2-Only Domains (KODs). In mouse embryonic stem cells, KODs are found in Hi-C-defined A compartments and feature relatively accessible chromatin. KODs are characterized by low gene expression and enhancers located in these domains bear the histone marks of an inactive or poised state. These results indicate that KODs organize a subset of inactive, tissue-specific enhancers at the nuclear periphery. We hypothesize that KODs may play a role in facilitating and perhaps constraining the enhancer-promoter interactions underlying spatiotemporal regulation of gene expression programs in differentiation and development.

P300/CBP Sustains Polycomb Silencing by Non-Enzymatic Functions

Maintenance of appropriate cell states involves epigenetic mechanisms, including Polycomb group (PcG)-mediated transcriptional repression. While PcG proteins are known to induce chromatin compaction, how PcG proteins gain access to DNA in compact chromatin to achieve long-term silencing is poorly understood. Here we show that the p300/CBP co-activator is associated with repressive chromatin regions and required for PcG occupancy in Drosophila and mouse cells. CBP stabilizes RNA polymerase II (Pol II) at PcG bound sites and promotes Pol II pausing independently of its histone acetyltransferase activity. CBP and Pol II pausing are necessary for RNA-DNA hybrid (R-loop) formation and nucleosome depletion at Polycomb Response Elements (PREs), whereas transcription beyond the pause region is not. These results suggest that non-enzymatic activities of the CBP co-activator have been repurposed to support PcG-mediated silencing, revealing how chromatin regulator interplay maintains transcriptional states.

DNA methylation of GFI1 as a mediator of the association between prenatal smoking exposure and ADHD symptoms at 6\xa0years: the Hokkaido Study on Environment and Children\u2019s Health

BackgroundPrenatal smoking exposure has been associated with childhood attention-deficit/hyperactivity disorder (ADHD). However, the mechanism underlying this relationship remains unclear. We assessed whether DNA methylation differences may mediate the association between prenatal smoking exposure and ADHD symptoms at the age of 6 years.ResultsWe selected 1150 mother–infant pairs from the Hokkaido Study on the Environment and Children’s Health. Mothers were categorized into three groups according to plasma cotinine levels at the third trimester: non-smokers (≤ 0.21 ng/mL), passive smokers (0.21–11.48 ng/mL), and active smokers (≥ 11.49 ng/mL). The children’s ADHD symptoms were determined by the ADHD-Rating Scale at the age of 6 years. Maternal active smoking during pregnancy was significantly associated with an increased risk of ADHD symptoms (odds ratio, 1.89; 95% confidence interval, 1.14–3.15) compared to non-smoking after adjusting for covariates. DNA methylation of the growth factor-independent 1 transcriptional repressor (GFI1) region, as determined by bisulfite next-generation sequencing of cord blood samples, mediated 48.4% of the total effect of the association between maternal active smoking during pregnancy and ADHD symptoms. DNA methylation patterns of other genes (aryl-hydrocarbon receptor repressor [AHRR], cytochrome P450 family 1 subfamily A member 1 [CYP1A1], estrogen receptor 1 [ESR1], and myosin IG [MYO1G]) regions did not exert a statistically significant mediation effect.ConclusionsOur findings demonstrated that DNA methylation of GFI1 mediated the association between maternal active smoking during pregnancy and ADHD symptoms at the age of 6 years.

Epigenomic differences in the human and chimpanzee genomes are associated with structural variation.

Structural variation (SV), including insertions and deletions (indels), is a primary mechanism of genome evolution. However, the mechanism by which SV contributes to epigenome evolution is poorly understood. In this study, we characterized the association between lineage-specific indels and epigenome differences between human and chimpanzee to investigate how SVs might have shaped the epigenetic landscape. By intersecting medium-to-large human–chimpanzee indels (20 bp–50 kb) with putative promoters and enhancers in cranial neural crest cells (CNCCs) and repressed regions in induced pluripotent cells (iPSCs), we found that 12% of indels overlap putative regulatory and repressed regions (RRRs), and 15% of these indels are associated with lineage-biased RRRs. Indel-associated putative enhancer and repressive regions are approximately 1.3 times and approximately three times as likely to be lineage-biased, respectively, as those not associated with indels. We found a twofold enrichment of medium-sized indels (20–50 bp) in CpG island (CGI)–containing promoters than expected by chance. Lastly, from human-specific transposable element insertions, we identified putative regulatory elements, including NR2F1-bound putative CNCC enhancers derived from SVAs and putative iPSC promoters derived from LTR5s. Our results show that different types of indels are associated with specific epigenomic diversity between human and chimpanzee.

Comprehensive Characterization of the Epigenetic Landscape in Multiple Myeloma

Multiple myeloma (MM) is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow. Epigenetic modifications, including DNA methylation and histone post-translational changes, are involved in MM development and progression, but also in drug resistance. We have derived a large cohort of patient-derived HMCLs that remain dependent on the addition of exogenous MM bone marrow growth factors, reflecting primary tumor conditions. We have described their molecular diversity by analyzing the gene expression profile and mutational landscape and have showed that HMCL molecular diversity reflects part of the molecular heterogeneity of primary MM cells. However, the epigenetic landscape of HMCLs has never been described. A comprehensive characterization of the epigenetic landscape of HMCLs would advance our understanding of MM pathophysiology and may attempt to identify new therapeutic targets. In our study, we presented the epigenetic landscape of HMCLs. We performed chromatin immunoprecipitation sequencing (ChIP-seq) to analyze changes of the histone marks (H3K9me3 to follow heterochromatin, H3K4me1 and H3K27ac to follow enhancer activity, H3K4me3 and H3K36me3 to follow active transcription and H3K27me3 to follow Polycomb-silenced chromatin) on 16 HMCLs, representative of the molecular heterogeneity of MM. The differential analysis of histone modification profiles of HMCLs highlighted links between histone modifications and cytogenetic abnormalities or recurrent mutations. H3K4me3 and H3K27me3 profile analysis revealed specific clusters of HMCL related to 1q gain and t(4;14) translocation, respectively. These two cytogenetic abnormalities lead to deregulation of epigenetic player expression (e.g. SETDB1 and MMSET) and thus, could alter histone modification profile. Using histone modifications associated to enhancer regions (H3K4me1 and H3K27ac), we identified super-enhancers (SE) associated with genes involved in the biology of MM. 607 to 2510 predicted super-enhancers per HMCL were identified, including MAF, MYC, CCND1, CCND2, TRAF3 or NSD2. These super-enhancers differ from typical enhancers in both size and H3K4me1 and H3K27ac levels. The SE-associated genes identified in HMCLs with a prognostic value in two independent cohorts of newly diagnosed patients (CoMMpass cohort; N = 674 and Montpellier cohort; N = 69 with RNA-seq data) were used to build a score predicting MM patient outcome (Figure 1). Moreover, among the 28 genes that compose the risk-score (BSG, HK2, HNRNPC, HSPA9, IL10, ILF3, LDHB, MDH1, MYBPC2, NCL, NUDC, PARP1, PDIA6, PRPS1, RPL8, RPL13A, RPL27A, RPL35, SF3B2, SLC7A5, SLC25A39, SMARCA4, SPN, STC2, THY1, TNPO2, TPR), public datasets of RNAi and CRISPR/Cas9 screening revealed four genes (YWHAQ, IL10, HK2 and THY1) identified as significant essential myeloma genes, suggesting that they could represent potential therapeutic targets. We also identified promoters of genes characterized by a co-localization of H3K9me3 and H3K27me3 repressive marks in HMCLs. We evaluated the prognostic value of these genes in the CoMMpass and Montpellier cohorts, and selected genes associated with poor outcome when their expression is low in MM cells of patients (ARHGEF5, BIVM, DEF8, GRID2IP, HDAC9, HSPA1L, KDM4C, NLRP2, P4HA3, PAG1, PM20D1, RMND5A, SEMA6A, SFMBT2, THEMIS2, TPRKB, ZFP2 and ZNF5188B) underlining potential new tumor suppressor genes. These potential tumor suppressor genes associated with repressive histone marks were used to build a second risk score splitting MM patients in low- and high-risk groups in CoMMpass and Montpellier cohorts. Finally, we explored H3K4me3 marks comparing drug-resistant and -sensitive HMCLs (N = 16) to identify regions involved in drug resistance. From these data, we developed epigenetic biomarkers based on this H3K4me3 modification predicting lenalidomide and romidepsin HDCAi response. This study provides a comprehensive characterization of the MM epigenetic landscape representing unique resources for future biological studies and could help to identify novel critical epigenetic modifications involved in MM progression and drug resistance. Furthermore, risk-scores based on super enhancers and repressive regions together with epigenetic biomarkers of drug response could represent new tools for precision oncology in MM. Disclosures De Boussac: Diag2Tec: Current Employment. Bruyer:Diag2Tec: Current Employment. Vincent:janssen: Membership on an entity's Board of Directors or advisory committees, Other: Congress support; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Congress support; takeda: Membership on an entity's Board of Directors or advisory committees, Other: Congress support. Moreaux:Diag2Tec: Consultancy.