Enhancer Region Research Articles

Disruption of the FOXM1 Regulatory Region Inhibits Tumor Progression in Ovarian Cancer by CRISPR-Cas9.

Ovarian cancer is the seventh most common lethal tumor among women in the world. FOXM1 is a transcription factor implicated in the initiation and progression of ovarian cancer by regulating key oncogenic genes. The role of regulatory regions in regulating the expression of FOXM1 in ovarian cancer is not completely clarified. Treatment with bromodomain and extraterminal (BET) inhibitors JQ-1 and I-BET were explored in ovarian cancer cell lines (OVCAR3, A2780, or SKOV3) to evaluate FOXM1 expression and biological behavior by qPCR, CCK8 assay, colony formation assay, wound-healing, and transwell assays. The regulatory regions (enhancer sequence spanning promoter or exon 1) of FOXM1 were deleted using CRISPR-Cas9 in the OVCAR3 cell line. FOXM1 expression and tumor biological behavior were further assessed in FOXM1 regulatory regions deleted OVCAR3 cell line. The mouse xenograft model was assessed at the indicated time points following subcutaneous injection of enhancer-deleted cells. Treatment with the JQ-1 and I-BET reduced the expression of FOXM1, decreasing cell proliferation, migration, and invasion in a panel of ovarian cancer cell lines including OVCAR3, A2780, and SKOV3 cells. By mining the published ChIP-sequencing data (H3K27Ac) from 12 ovarian cancer cell lines, we identified a potential enhancer and promoter region. Deletion of the spanning enhancer and promoter region of FOXM1 reduced mRNA and protein expression. Similarly, cell proliferation, migration, invasion, and tumorigenesis in both cells and mouse xenograft models were significantly attenuated. Our study demonstrates that JQ-1 and I-BET can regulate the expression of the FOXM1 gene-relating network. These data also indicate that disruption of the span enhancer and promoter region activity of FOXM1 has a vital role in the anti-ovarian cancer effect, hiding a potential opportunity for the evaluation of this non-coding DNA deletion disrupts the FOXM1 transcriptional network in ovarian cancer development.

P0008 A multi-omics approach across multiple chronic inflammatory diseases identifies a distinct type-1-interferon endotype of Inflammatory Bowel Disease

Abstract Background IBD shares many clinical and immunological features with other chronic inflammatory diseases (CIDs) like rheumatoid arthritis (RA) and psoriasis (Pso). Extraintestinal manifestations, particularly in the joints and skin, often overlap with these CIDs. We hypothesize that a systems medicine approach involving omics-driven phenotyping of multiple CIDs can yield a novel, therapy-relevant molecular taxonomy of disease. In this study, we aim to identify IBD-specific molecular signatures and those shared with other CIDs, as well as novel endotypes of IBD. Methods We recruited a cross-sectional cohort of 650 patients with CIDs (CD, UC, RA, Pso, psoriatic arthritis [PsA], and systemic lupus erythematosus [SLE]) along with 202 healthy controls (HC) at the University Hospital Kiel. Active or inactive disease was defined by clinical activity scores (i.e., CD: CDAI <150; UC: partial Mayo ≤2). Blood samples were collected and subjected to RNA sequencing and methylation profiling. Detailed clinical phenotypes and 5 years-follow-up data were obtained by chart review. We conducted differential gene expression and methylation analyses comparing each CID with HCs to identify shared molecular signatures. Core (activity-independent) signatures were defined by the overlap of differential expression in both active vs. HC and inactive disease vs. HC. Biclustering methods (UnPaSt) were used to uncover distinct patient endotypes based on expression profiles. Results IBD shared the most differential signatures with RA, indicating common pathways in their pathogenesis. Core signatures upregulated across all CIDs correlated with DNA methylation changes, reflecting the molecular mechanism of chronicity, and were enriched in pathways related to cytokine signaling, neutrophil chemotaxis, and humoral immune response. While methylation signatures in enhancer regions were shared among most CIDs, disease-specific methylation was mainly located in CpG islands. One disease-overarching subgroup of 71 patients with active disease, identified by biclustering, exhibited upregulation of interleukin-27 and type-1 interferon signaling, including 16 out of 121 active IBD patients with a skewed sex ratio toward females and higher rates of therapy escalations within 5 years (Log-rank test: p=0.0089). Conclusion Our study highlights shared and disease-specific immune dysregulation pathways contributing to the pathogenesis of IBD and other CIDs. We show the clear presence of core signatures of disease regardless of disease activity, which could represent insights into actionable targets for causative treatments and better disease control of CIDs. With our approach, we identified a distinct molecular endotype of IBD with worse prognosis, warranting further validation studies. References This project has received funding from the DFG Cluster of Excellence 2167 "Precision medicine in chronic inflammation", the BMBF (e:Med Network iTREAT 01ZX2202A), the European Union/Horizon2020 (SYSCID, No 733100) and the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 831434 (3TR) and No 853995 (ImmUniverse).The JU receives support from the European Union's Horizon 2020 research and innovation programme and EFPIA. The content provided in this publication reflects the author's views only. Neither the Innovative Medicines Initiative (IMI JU) nor the European Commission are responsible for any use that may be made of the information it contains.

The Histone Demethylase LSD1/ZNF217/CoREST Complex is a Major Restriction Factor of Epstein-Barr Virus Lytic Reactivation.

Epstein-Barr virus (EBV) contributes to ~1.5% of human cancers, including lymphomas, gastric and nasopharyngeal carcinomas. In most of these, nearly 80 viral lytic genes are silenced by incompletely understood epigenetic mechanisms, precluding use of antiviral agents such as ganciclovir to treat the 200,000 EBV-associated cancers/year. To identify host factors critical for EBV latency, we performed a human genome-wide CRISPR-Cas9 screen in Burkitt B-cells. Top hits included the lysine-specific histone demethylase LSD1 and its co-repressors ZNF217 and CoREST. LSD1 removes histone 3 lysine 4 (H3K4) and histone 3 lysine 9 (H3K9) methylation marks to downmodulate chromatin activation. LSD1, ZNF217 or CoREST knockout triggered EBV reactivation, as did a LSD1 small molecule antagonist, whose effects were additive with histone deacetylase inhibition. LSD1 blockade reactivated EBV in Burkitt lymphoma, gastric carcinoma and nasopharyngeal carcinoma models, sensitized cells to ganciclovir cytotoxicity and induced EBV reactivation in murine xenografts. ZNF217 and LSD1 co-occupied the EBV immediate early gene BZLF1 promoter, which drives B-cell lytic cycle, as well as to the oriLyt enhancer regions recently implicated in EBV reactivation. LSD1 depletion increased levels of activating histone 3 lysine 4 (H3K4) methylation but not repressive histone lysine 9 methylation marks at BZLF1 and oriLyt and induced their interaction by long-range DNA looping. An orthogonal CRISPR screen highlighted a key H3K4 methyltransferase KMT2D role in driving EBV reactivation. Our results highlight H3K4 methylation as a major EBV lytic switch regulator and suggest novel therapeutic approaches.

Standardisation of the radiological definition of supramaximal resection in glioblastoma.

Glioblastoma remains the most common and lethal primary malignant brain tumour, with high rates of recurrence and progression despite gross-total resection of the contrast-enhancing region based on T1-weighted MRI. There has been growing interest in exploring "supramaximal" resections that extend beyond contrast-enhancing borders, with initial retrospective data suggesting survival benefit, but there is currently no consensus definition. In this systematic review, we explore the evolution of supramaximal resection in glioblastoma, dissect the incongruencies in the literature regarding its definition, qualitatively appraise each definition and discuss the results of various studies that have explored its impacts on patient outcomes. MEDLINE, EMBASE, SCOPUS, Cochrane Registry of Clinical Trials and Pub-MED were systematically searched for studies of glioblastoma patients who had undergone supramaximal resection. After screening and applying eligibility criteria, 25 studies were included in the final review. Definitions were grouped according to radiological modality and visualisation adjuncts and included various extents of resection of hyperintensity visualised using T2-Fluid-Attenuated Inversion Recovery, various volumetric resections of the non-contrast enhancing region, removal of methionine-uptake areas on PET, complete removal of 5-aminolevulinic acid fluorescent tissue and lobectomies. Our systematic review identified a general trend suggesting a survival benefit from supramaximal resection compared to gross-total resection but, more importantly, demonstrated the limitations of these studies due to selection bias and substantial methodological heterogeneity. Ultimately, our findings demonstrate the need for an applicable, standardised and specific definition for supramaximal resection so that prospective studies can determine prognostically significant clinical data to guide the surgical management of glioblastoma.

Lower expression of BIN1's neuronal isoform in vulnerable excitatory neurons increases risk in Alzheimer's disease

Background: Neurons in Alzheimer's disease (AD) experience elevated DNA damage, with DNA repair sites enriched at enhancer regions of genes essential for neuronal survival. Excitatory neurons in the cortical superficial layers expressing CUX2 and RORB (Cux2+/Rorb+), are selectively vulnerable in AD, but their relationship to single nucleotide polymorphisms (SNPs) in AD genome-wide association studies (GWAS) is unclear. Objective: This study aimed to identify and characterize functional AD-GWAS SNPs using single-nucleus RNA sequencing data, focusing on selectively vulnerable neurons and DNA repair hotspots. Methods: Filters were applied to identify candidate SNPs based on overlap with repair hotspots, RNA expression, transcription factor binding, AD association, and epigenetic significance. In vitro assays and analyses of large datasets from bulk RNA-seq (n = 1894), proteomics (n = 400), and single-nucleus RNA-seq (n = 424, 1.6 M cells) were conducted. Results: BIN1 SNP, rs78710909, met multiple criteria - located in an AD-GWAS locus, repair hotspot, and promoter region. rs78710909C exhibits 1.52× higher AD risk and 5.4× differential transcription factor binding. In vitro, rs78710909C shows greater enhancer activity and weaker p53 but stronger E2F1 binding. BIN1's neuronal isoform is neuroprotective, but its AD expression is lower (p < 0.01). Moreover, only in AD and Cux2+/Rorb + neurons, rs78710909C is associated with a lower average BIN1 neuronal isoform ratio (p < 0.01). The genes upregulated in neurons with lower neuronal isoform ratio were associated with the hallmarks of AD pathology. Conclusions: In a disease-relevant mechanism, the BIN1 SNP rs78710909C is associated with a lower ratio of BIN1's neuronal isoform which increases the vulnerability of specific excitatory neurons in AD patients.

Biologically-targeted discovery-replication scan identifies G×G interaction in relation to risk of Barrett's esophagus and esophageal adenocarcinoma.

Inherited genetics represents an important contributor to risk of esophageal adenocarcinoma (EAC), and its precursor Barrett's esophagus (BE). Genome-wide association studies have identified ∼30 susceptibility variants for BE/EAC, yet genetic interactions remain unexamined. To address challenges in large-scale G×G scans, we combined knowledge-guided filtering and machine learning approaches, focusing on genes with (A) known/plausible links to BE/EAC pathogenesis (n=493) or (B) prior evidence of biological interactions (n=4,196). ∼75 x 106 SNP×SNP interactions were screened via hierarchical group lasso (glinternet) using BEACON GWAS data. The top ∼2000 interactions retained in each scan were prioritized using P values from single logistic models. Identical scans were repeated among males only (78%), with two independent GWAS datasets used for replication. In overall and male-specific primary replications, 11 of 187 and 20 of 191 interactions satisfied P<0.05, respectively. The strongest evidence for secondary replication was for rs17744726×rs3217992 among males, with consistent directionality across all cohorts (Pmeta=2.19×10-8); rs3217992 "T" was associated with reduced risk only in individuals homozygous for rs17744726 "G". Rs3217992 maps to the CDKN2B 3'UTR and reportedly disrupts microRNA-mediated repression. Rs17744726 maps to an intronic enhancer region in BLK. Through in-silico prioritization and experimental validation, we identified a nearby proxy variant (rs4841556) as a functional modulator of enhancer activity. Enhancer-gene mapping and eQTLs implicated BLK and FAM167A as targets. The first systematic G×G investigation in BE/EAC, this study uncovers differential risk associations for CDKN2B variation by BLK genotype, suggesting novel biological dependency between two risk loci encoding key mediators of tumor suppression and inflammation.

Genetic predisposition to Behcet's disease mediated by a IL10RA enhancer polymorphism.

Several studies suggested the genetic association between IL10RA variants and susceptibility to Behcet's disease (BD). However, the precise mechanism of the association is still unknown. The purpose of this study was to investigate the mechanism underlying the genetic associations between IL10RA polymorphisms and the risk of BD. To analyse the genetic susceptibility to BD mediated by IL10RA causal polymorphisms, we performed a study on data from our previous genome-wide association studies (GWAS), the bioinformatic analysis of post-annotation of GWAS and relevant mechanism verification experiments, including chromatin immunoprecipitation, luciferase gene-reporter assay, electrophoretic mobility shift assays, and enzyme-linked immunosorbent assay. Among 125 single nucleotide polymorphisms (SNPs) with P<1×10-5 identified in our previous GWAS study on BD, rs4936415 (G/C) was predicted with the highest conserved score as an expression quantitative-trait-locus SNP for IL10RA in whole blood. There were H3K27ac and H3K4Me1 enhancer-specific enrichments around SNP rs4936415. Luciferase gene-reporter assays revealed that the rs4936415G-allele construct showed a higher enhancer activity as compared to the empty and the C-allele construct. NF-κB1 was identified to bind the C-allele rather than the G-allele, although the enhancer SNP (rs4936415) region was found to control transcription factor binding sites. Interaction of C-allele and NF-κB1 gene construct resulted in an increased enhancer activity. BD patients showed a significantly lower serum level of the IL-10Rα. This study identified a single functional causal SNP, rs4936415, in the IL10RA super-enhancer, conferring BD susceptibility. The protective G-allele of non-coding rs4936415 located inside an enhancer region of IL10RA promoted the enhancer activity and increased the expression of IL10RA.The risk C-allele is able to specifically bind NF-κB1 and, in turn, promotes enhancer activity of IL10RA. This subsequently leads to an increased expression of IL10RA. Low expression of IL-10RA suggests a relative deficiency of NF-κB1 in BD.

Brd4 modulates metabolic endotoxemia-induced inflammation by regulating colonic macrophage infiltration in high-fat diet-fed mice

High-fat diet (HFD) induces low-grade chronic inflammation, contributing to obesity and insulin resistance. However, the precise mechanisms triggering obesity-associated metabolic inflammation remain elusive. In this study, we identified epigenetic factor Brd4 as a key player in this process by regulating the expression of Ccr2/Ccr5 in colonic macrophage. Upon 4-week HFD, myeloid-lineage-specific Brd4 deletion (Brd4-CKO) mice showed reduced colonic inflammation and macrophage infiltration with decreased expression of Ccr2 and Ccr5. Mechanistically, Brd4 was recruited by NF-κB to the enhancer regions of Ccr2 and Ccr5, promoting enhancer RNA expression, which facilitated Ccr2/Ccr5 expression and macrophage migration. Furthermore, decreased infiltration of Ccr2/Ccr5-positive colonic macrophages in Brd4-CKO mice altered gut microbiota composition and reduced intestinal permeability, thereby lowering metabolic endotoxemia. Finally, Brd4-CKO mice subjected to a 4-week LPS infusion exhibited restored susceptibility to HFD-induced obesity and insulin resistance. This study identifies Brd4 as a critical initiator of colonic macrophage-mediated inflammation and metabolic endotoxemia upon HFD, suggesting Brd4 as a potential target for mitigating HFD-induced inflammation, obesity, and its metabolic complications.

Efficacy of glutathione inhibitor eprenetapopt against the vulnerability of glutathione metabolism in SMARCA4-, SMARCB1- and PBRM1-deficient cancer cells

Mutation of genes related to the SWI/SNF chromatin remodeling complex is detected in 20% of all cancers. The SWI/SNF chromatin remodeling complex comprises about 15 subunits and is classified into three subcomplexes: cBAF, PBAF, and ncBAF. Previously, we showed that ovarian clear cell carcinoma cells deficient in ARID1A, a subunit of the cBAF complex, are synthetic lethal with several genes required for glutathione (GSH) synthesis and are therefore sensitive to the GSH inhibitor eprenetapopt (APR-246). However, we do not know whether cancer cells deficient in SWI/SNF components other than ARID1A are selectively sensitive to treatment with eprenetapopt. Here, we show that SMARCA4-, SMARCB1-, and PBRM1-deficient cells are more sensitive to eprenetapopt than SWI/SNF-proficient cells. We found that deficiency of SMARCA4, SMARCB1, or PBRM1 attenuates transcription of the SLC7A11 gene (which supplies cysteine as a raw metabolic material for GSH synthesis) by the failure of recruitment of cBAF and PBAF to the promotor and enhancer regions of the SLC7A11 locus, thereby reducing basal levels of GSH. In addition, eprenetapopt decreased the amount of intracellular GSH and increased the intracellular amount of reactive oxygen species (ROS), followed by induction of apoptosis. Taken together, eprenetapopt could be a promising selective agent for SWI/SNF-deficient cancer cells derived from SMARCA4-deficient lung cancers, SMARCB1-deficient rhabdoid tumors, and PBRM1-deficient kidney cancers.

Archaeological heritage in the Shabè region and challenges of enhancement: the example of the “Etoo” fortified site

The conservation and enhancement of archaeological heritage in African countries, particularly in Benin, present significant challenges for researchers. The general lack of awareness among Beninese populations regarding this heritage, coupled with its minimal integration into development and cultural policies, has led to increased degradation and, in some cases, the irreversible destruction of millennia-old relics. These artifacts bear witness to civilizations once thought extinct but remain vibrant within contemporary societies. The ETOO fortified site in the Shabè region exemplifies an archaeological site whose patrimonial value warrants preservation and promotion. This study addresses the following questions: What are the stakes involved in enhancing archaeological heritage in Benin? What heritage enhancement plan is suitable for the ETOO site in the Shabè region? How can enhancement strategies be explored through cinema and audiovisual media? The methodology combines empirical research, archaeological survey reports, and a specialized literature review, as well as a study of cinematographic and audiovisual productions on similar heritage sites in Africa and elsewhere. The objective is to present the potential of this archaeological site for heritage enhancement, identify associated threats, and reflect on the importance of archaeological heritage in territorial development plans. The article begins with a brief overview of archaeological realities in Africa, followed by an examination of the status of archaeological heritage within Beninese legislation. The third section introduces the ETOO fortified site. The final sections discuss the site's patrimonialization and analyze its potential for enhancement.

The Role of Fiscal Decentralization in Enhancing Regional Financial Independence in Indonesia

This study aims to analyze the role of fiscal decentralization in enhancing regional financial independence in Indonesia. Using a qualitative approach and literature review methodology, the research examines the role of fiscal decentralization in promoting regional financial autonomy in Indonesia. The analysis compares the effectiveness of fiscal decentralization policies in fostering financial independence across various regions in the country. Fiscal decentralization has demonstrated positive impacts by granting regions greater freedom to determine development priorities and explore funding sources. Regions with strong economic potential and natural resources can increase their locally generated revenue (Pendapatan Asli Daerah or PAD) to support development. However, this policy also creates disparities among regions, where wealthier regions manage their finances independently while poorer regions remain reliant on central government transfers. Moreover, limited managerial capacity in some regions hampers the policy's effectiveness. Fiscal decentralization can strengthen regional financial independence if accompanied by enhanced financial management capacity and fiscal policy adjustments.

ASEAN's Role in Enhancing Regional Security and Economic Stability through Defense Cooperation

The role of ASEAN in strengthening regional security and economic stability through defense cooperation is becoming increasingly pivotal in response to evolving global geopolitical and economic challenges. This study employs a qualitative methodology, specifically a literature study approach, to analyze ASEAN's mechanisms for fostering defense collaboration and their impact on the region's stability. Central to this analysis are key initiatives such as the ASEAN Defence Ministers’ Meeting (ADMM) and ADMM-Plus, which have enhanced regional security through joint military exercises, intelligence sharing, and maritime security efforts. These initiatives have been instrumental in mitigating tensions, particularly in contested areas like the South China Sea, safeguarding critical trade routes, and promoting economic resilience. The findings indicate that ASEAN's defense cooperation directly supports economic stability by fostering a secure environment that encourages foreign investment, intra-ASEAN trade, and infrastructure development. However, the study identifies significant challenges, including disparities in member states' defense capabilities, limited inter-state trust, and a tendency to prioritize arms modernization over institutional collaboration. Moreover, ASEAN faces the growing need to address non-traditional security threats, such as cybercrime and terrorism, which require adaptive strategies and enhanced cooperation. The research concludes that while ASEAN’s defense collaboration bolsters military preparedness, it also significantly contributes to political stability and economic growth in Southeast Asia. To sustain and amplify these benefits, ASEAN must address existing challenges through measures such as enhancing transparency, fostering trust among member states, and investing in capacity-building initiatives.

Rapid degradation of histone deacetylase 1 (HDAC1) reveals essential roles in both gene repression and active transcription.

Histone Deacetylase 1 (HDAC1) removes acetyl groups from lysine residues on core histones, a critical step in regulating chromatin accessibility. Despite histone deacetylation being an apparently repressive activity, suppression of HDACs causes both up- and downregulation of gene expression. Here we exploited the degradation tag (dTAG) system to rapidly degrade HDAC1 in mouse embryonic stem cells (ESCs) lacking its paralog, HDAC2. The dTAG system allowed specific degradation and removal of HDAC1 in<1 h (100x faster than genetic knockouts). This rapid degradation caused increased histone acetylation in as little as 2 h, with H2BK5 and H2BK11 being the most sensitive. The majority of differentially expressed genes following 2 h of HDAC1 degradation were upregulated (275 genes up versus 15 down) with increased proportions of downregulated genes observed at 6 h (1153 up versus 443 down) and 24 h (1146 up versus 967 down), respectively. Upregulated genes showed increased H2BK5ac and H3K27ac around their transcriptional start site (TSS). In contrast, decreased acetylation and chromatin accessibility of super-enhancers was linked to the most strongly downregulated genes. These findings suggest a paradoxical role for HDAC1 in the maintenance of histone acetylation levels at critical enhancer regions required for the pluripotency-associated gene network.

Cis-Regulation of an m6A Eraser by an Insertion Variant Associated with Survival of Patients With Non-Small Cell Lung Carcinoma.

N6-methyladenosine (m6A) serves as one of the crucial RNA modifications for genes involved in cancer progression. Here, 7273 expression quantitative trait loci potentially regulating 30 m6A pathway genes are identified from the GTEx database, with 69 single nucleotide polymorphisms significantly associated with survival of non-small cell lung carcinoma (NSCLC) patients (n = 1523) from the ongoing genome-wide association study after false positive probability tests. Notably, the rs151198415 locus, situated in a potential enhancer region, demonstrated a prolonged survival effect with the C>CCACG insertion, which is validated in an independent prospective cohort (n = 237), yielding a pooled hazard ratio of 0.72 (p = 0.007). Mechanistically, the rs151198415 C>CCACG insertion engaged in long-range interaction with the promoter of m6A eraser ALKBH5, promoting ALKBH5 transcription by the creation of an EGR1 binding site. Then, ALKBH5 upregulated FBXL5 expression by m6A demethylation, which is dependent on the ALKBH5 H204 amino acid site and specific m6A sites on FBXL5 mRNA. Finally, the ALKBH5-FBXL5 axis reduces intracellular reactive oxygen species levels, leading to PI3K/AKT and NF-kB pathway inhibition and consequently suppresses NSCLC proliferation and metastasis in vitro and in vivo. Triggered by an insertion variant, this remote cis-regulation of m6A eraser and the downstream molecular events modulate the survival of NSCLC patients.

Porphyromonas gingivalis-Lipopolysaccharide Induced Gingival Fibroblasts Trained Immunity Sustains Inflammation in Periodontitis.

To investigate whether trained immunity occurs in gingival fibroblasts (GFs) and its relationship to the persistence of inflammation in periodontitis. Periodontally healthy and inflammatory gingival fibroblasts (HGFs and IGFs) were cultured through continuous adherence subculture of tissue blocks. Trained immunity in HGFs was evaluated via a classic invitro model, with relevant markers assessed via enzyme-linked immunosorbent assay, lactate content assay, glycolytic rate assay, and chromatin immunoprecipitation. A histone methyltransferase blocker and a PI3K inhibitor were added to investigate the mechanisms underlying trained immunity. The relationship between trained immunity and periodontitis was further examined via immunofluorescence staining and chromatin immunoprecipitation on IGFs. Compared with untrained cells, GFs trained with Porphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) exhibited a significant increase in IL-6 and TNF-α secretion, enhanced glycolytic metabolism, and enriched mono-methylation of lysine 4 on histone H3 (H3K4me1) at the enhancer regions of TNF-α and IL-6. The addition of a histone methyltransferase blocker and a PI3K inhibitor greatly reduced trained immunity. Additionally, the response of IGFs to P. gingivalis-LPS stimulation and their epigenetic modifications were similar to those observed in trained HGFs. This study novelly discovered that both P. gingivalis-LPS-stimulated HGFs and IGFs in periodontitis acquired trained immunity. Following P. gingivalis-LPS stimulation, HGFs underwent metabolic and epigenetic changes via the PI3K/AKT pathway, with these epigenetic changes also observed in IGFs. This finding suggests that trained immunity in GFs may be a key mechanism underlying the recurrence and persistence of periodontitis.

Comprehensive Predictions of Mef2-Mediated Chromatin Loops, Which May Inhibit Ubx Binding by Blocking Low-Affinity Binding Sites.

Gene regulation depends on the interaction between chromatin-associated factors, such as transcription factors (TFs), which promote chromatin loops to ensure tight contact between enhancer and promoter regions. So far, positive interactions that lead to gene activation have been the main focus of research, but regulations related to blocking or inhibiting factor binding are also essential to maintaining a defined cellular status. To understand these interactions in greater detail, I investigated the possibility of the muscle differentiation factor Mef2 to prevent early Hox factor binding, leading to the proper timing of regulatory processes and the activation of differentiation events. My investigations relied on a collection of publicly available genome-wide binding data sets of Mef2 and Ubx (as the Hox factor), Capture-C interactions, and ATAC-seq analysis in Mef2 mutant cells. The analysis indicated that Mef2 can form possible chromatin loops to Ubx-bound regions. These regions contain low-affinity Ubx binding sites, and the chromatin architecture is independent of Mef2's function. High levels of Ubx may disrupt the loops and allow specific Ubx bindings to regulate defined targets. In summary, my investigations highlight that the use of many publicly available data sets enables computational approaches to make robust predictions and, for the first time, suggest a molecular function of Mef2 as a preventer of Hox binding, indicating that it may act as a timer for muscle differentiation.

Leveraging functional annotations to map rare variants associated with Alzheimer's disease with gruyere.

The increasing availability of whole-genome sequencing (WGS) has begun to elucidate the contribution of rare variants (RVs), both coding and non-coding, to complex disease. Multiple RV association tests are available to study the relationship between genotype and phenotype, but most are restricted to per-gene models and do not fully leverage the availability of variant-level functional annotations. We propose Genome-wide Rare Variant EnRichment Evaluation (gruyere), a Bayesian probabilistic model that complements existing methods by learning global, trait-specific weights for functional annotations to improve variant prioritization. We apply gruyere to WGS data from the Alzheimer's Disease (AD) Sequencing Project, consisting of 7,966 cases and 13,412 controls, to identify AD-associated genes and annotations. Growing evidence suggests that disruption of microglial regulation is a key contributor to AD risk, yet existing methods have not had sufficient power to examine rare non-coding effects that incorporate such cell-type specific information. To address this gap, we 1) use predicted enhancer and promoter regions in microglia and other potentially relevant cell types (oligodendrocytes, astrocytes, and neurons) to define per-gene non-coding RV test sets and 2) include cell-type specific variant effect predictions (VEPs) as functional annotations. gruyere identifies 15 significant genetic associations not detected by other RV methods and finds deep learning-based VEPs for splicing, transcription factor binding, and chromatin state are highly predictive of functional non-coding RVs. Our study establishes a novel and robust framework incorporating functional annotations, coding RVs, and cell-type associated non-coding RVs, to perform genome-wide association tests, uncovering AD-relevant genes and annotations.

Craniofrontonasal syndrome in a patient with an inv(X)(p22.2q13.1), separating EFNB1 from its enhancer.

Craniofrontonasal syndrome (CFNS) is an X-linked developmental disorder caused by loss of function variants (LOFVs) in the ephrin B1 (EFNB1) gene located on Xq13.1. In CFNS, unlike in other X-linked disorders, females with heterozygous EFNB1 pathogenic variants (PVs) have a severe phenotype, whereas males carrying hemizygous EFNB1 PVs have a mild phenotype. Here we report a female CFNS patient who was diagnosed with the typical features of CFNS as a new-born. Chromosomal analysis revealed a de novo pericentric inversion of one X chromosome; inv(X)(p22q13). Molecular testing for EFNB1 mutations and a SNP-array test for genomic imbalances returned negative results. We identified the inversion breakpoints using whole genome sequencing (WGS). One of the breakpoints was about 97 kbp downstream of the 3' end of the EFNB1 gene, separating a potential EFNB1 enhancer region from the EFNB1 gene. To our knowledge, this is the first case of CFNS caused by a large structural variant, altering the genomic and regulatory context of EFNB1.

BCL11A+58/+55 enhancer-editing facilitates HSPC engraftment and HbF induction in rhesus macaques conditioned with a CD45 antibody-drug conjugate.

Editing the+58 region of the BCL11A erythroid enhancer has shown promise in treating β-globin disorders. To address variations in fetal hemoglobin (HbF) response, we investigated editing both+58 and+55 enhancers. Rhesus macaques transplanted with edited hematopoietic stem/progenitor cells (HSPCs) following busulfan conditioning exhibited durable, high-level (∼90%) editing frequencies post transplantation with sustained HbF reactivation over 4 years, without hematological perturbations. HbF levels were further boosted by stress erythropoiesis or hydroxyurea. Bone marrow analysis revealed that gene edits were predominantly programmed deletions, programmed inversions, and short indels, each disrupting the enhancer core TGN7-9WGATAR half E-box/GATA binding motifs. Nonprogrammed long deletions were disfavored in engrafting cells. CD45 antibody-drug conjugate (ADC) conditioning achieved comparable engraftment and HbF reactivation, whereas lentiviral vector tracking showed polyclonal reconstitution with dynamics similar to animals conditioned with total body irradiation (TBI) or busulfan. Joining CD45-ADC conditioning with combined enhancer editing presents an effective strategy for β-hemoglobinopathies, enabling durable HbF reactivation without chemotherapy.

Maternal malnutrition in mice impairs nephrogenesis by disrupting DNA methylation of regulatory regions.

Maternal caloric restriction during pregnancy significantly impacts kidney development, influencing susceptibility to chronic kidney disease in adulthood. This study explores DNA methylation changes in nephron progenitor cells resulting from caloric restriction and their implications for kidney health. Global DNA hypomethylation is observed in nephron progenitors from caloric-restricted embryos, with specific genomic regions displaying distinct methylation patterns, including hypomethylation and hypermethylation. Differentially methylated regions exhibit enhanced chromatin accessibility, indicating biological relevance. Hypomethylated regions are enriched for genes associated with developmental processes, reflecting changes in gene expression and highlighting their functional relevance in kidney development. The study also reveals that supplementing methionine, an essential amino acid, restores disrupted DNA methylation patterns, particularly in enhancer regions, emphasizing methionine's critical role in regulating nephron progenitor cell epigenetics and ensuring proper kidney development. The intricate relationship between maternal nutrition, dynamic DNA methylation, and kidney development is highlighted, emphasizing the enduring impact of early-life nutritional challenges on kidney function. This research elucidates epigenetic mechanisms as mediators for the lasting effects of maternal caloric restriction on kidney health. The study contributes valuable insights into the origins of chronic kidney diseases during early developmental stages, offering potential interventions to mitigate adverse outcomes.NEW & NOTEWORTHY Our study establishes a direct link between maternal caloric restriction, DNA methylation patterns in nephron progenitor cells, and kidney development. We reveal consistent alterations in methylation patterns, coupled with corresponding shifts in the expression of genes related to kidney development and cell proliferation. Methionine supplementation emerges as a promising intervention, effectively restoring disrupted DNA methylation patterns. These findings pave the way for potential therapeutics, optimizing kidney development and mitigating the burden of chronic kidney disease in adulthood.