Search

Background: Quadriceps tendon extensor mechanism disruption is an infrequent but devastating complication after total knee arthroplasty (TKA). Our knowledge of specific risk factors for this complication is limited by the current literature. Thus, this study aimed to identify potential risk factors for quadriceps tendon extensor mechanism disruption following TKA., Methods: A retrospective cohort analysis was performed using the PearlDiver Administrative Claims Database. Patients undergoing TKA without a prior history of quadriceps tendon extensor mechanism disruption were identified. Quadriceps tendon extensor mechanism disruption included rupture of the quadriceps tendon, patellar tendon, or fracture of the patella. Patients who had a minimum of 5 years of follow-up after TKA were included. A total of 126,819 patients were included. Among them, 517 cases of quadriceps tendon extensor mechanism disruption occurred (incidence 0.41%). Hypothesized risk factors were compared between those who had postoperative quadriceps tendon extensor mechanism disruption and those who did not., Results: On multivariate analysis, increased Charlson Comorbidity Index (odds ratio (OR): 1.10, 95% confidence interval (CI) [1.07 to 1.13]; P < .001), obesity (OR: 1.49, 95% CI [1.24 to 1.79]; P < .001), and fluoroquinolone use any time after TKA (OR: 1.24, 95% CI [1.01 to 1.52]; P = .036) were significantly associated with quadriceps tendon extensor mechanism disruption., Conclusions: Our study identified the incidence of quadriceps tendon extensor mechanism disruption following TKA as 0.41%. Identified risk factors for quadriceps tendon extensor mechanism disruption after TKA include an increased Charlson Comorbidity Index, obesity, and use of fluoroquinolones postoperatively., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The correct establishment of DNA methylation patterns is vital for mammalian development and is achieved by the de novo DNA methyltransferases DNMT3A and DNMT3B. DNMT3B localises to H3K36me3 at actively transcribing gene bodies via its PWWP domain. It also functions at heterochromatin through an unknown recruitment mechanism. Here, we find that knockout of DNMT3B causes loss of methylation predominantly at H3K9me3-marked heterochromatin and that DNMT3B PWWP domain mutations or deletion result in striking increases of methylation in H3K9me3-marked heterochromatin. Removal of the N-terminal region of DNMT3B affects its ability to methylate H3K9me3-marked regions. This region of DNMT3B directly interacts with HP1α and facilitates the bridging of DNMT3B with H3K9me3-marked nucleosomes in vitro. Our results suggest that DNMT3B is recruited to H3K9me3-marked heterochromatin in a PWWP-independent manner that is facilitated by the protein's N-terminal region through an interaction with a key heterochromatin protein. More generally, we suggest that DNMT3B plays a role in DNA methylation homeostasis at heterochromatin, a process which is disrupted in cancer, aging and Immunodeficiency, Centromeric Instability and Facial Anomalies (ICF) syndrome., (© 2024. The Author(s).)

Background: Postnatal steroids are used to prevent bronchopulmonary dysplasia in extremely preterm infants but may have adverse effects on brain development. We assessed connectivity metrics of major cerebral and cerebellar white matter pathways at near-term gestational age among infants who did or did not receive a standardized regimen of hydrocortisone during the first 10 days of life., Methods: Retrospective cohort study., Participants: Infants born <28 weeks: Protocol group (n = 33) received at least 50% and not more than 150% of an intended standard dose of 0.5 mg/kg hydrocortisone twice daily for 7 days, then 0.5 mg/kg per day for 3 days; Non-Protocol group (n = 22), did not receive protocol hydrocortisone or completed <50% of the protocol dose. We assessed group differences in near-term diffusion MRI mean fractional anisotropy (FA) and mean diffusivity (MD) across the corticospinal tract, inferior longitudinal fasciculus, corpus callosum and superior cerebellar peduncle., Results: Groups were comparable in gestational age, post-menstrual age at scan, medical complications, bronchopulmonary dysplasia, and necrotizing enterocolitis. No significant large effect group differences were identified in mean FA or MD in any cerebral or cerebellar tract., Conclusion(s): Low dose, early, postnatal hydrocortisone was not associated with significant differences in white matter tract microstructure at near-term gestational age., Impact: This study compared brain microstructural connectivity as a primary outcome among extremely preterm infants who did or did not receive early postnatal hydrocortisone. Low dose hydrocortisone in the first 10 days of life was not associated with significant differences in white matter microstructure in major cerebral and cerebellar pathways. Hydrocortisone did not have a significant effect on early brain white matter circuits., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Purpose: A debilitating complication following anterior cruciate ligament reconstruction is a secondary meniscus tear. Currently, the literature is mixed regarding the risk factors associated with the incidence of secondary meniscus tears. The aim of this study was to investigate risk factors associated with meniscus tears following an isolated primary anterior cruciate ligament reconstruction. ACL graft failure was hypothesized to be the strongest risk factor for secondary meniscal injury occurrence., Methods: A retrospective cohort analysis was performed using the PearlDiver Database. Patients with a primary anterior cruciate ligament reconstruction were identified in the database. Patients with concomitant knee ligament injury or meniscus injury present at the time the index procedure were excluded. Patients were grouped to those who had a secondary meniscus tear within 2 years following anterior cruciate ligament reconstruction and those who did not. Univariate analysis and multivariable regression analysis was conducted to identify significant risk factors for a secondary meniscus tear., Results: There were 25,622 patients meeting criteria for inclusion in this study. Within 2 years from the primary anterior cruciate ligament reconstruction, there were 1,781 patients (7.0%) that experienced a meniscus tear. Graft failure had the highest odds of having a postoperative meniscus tear within 2 years (OR: 4.1; CI 3.5-4.8; p < 0.002). Additional significant risk factors included tobacco use (OR: 2.0; CI 1.0-3.1; p < 0.001), increased Charlson Comorbidity Index (OR: 1.2; CI 1.1-1.4), male gender (OR: 1.1; CI 1.1-1.2; p < 0.001), obesity (OR: 1.1; CI 1.1-1.2; p < 0.001), delayed surgery (OR:1.1; CI 1.1-1.2; p < 0.002), and patients age 30 and older (OR: 1.0; CI 1.0-1.0; p < 0.001)., Conclusions: This study found that anterior cruciate ligament graft failure is the strongest predictor of post-operative meniscus tears. Other risk factors, including tobacco use, increased CCI, male gender, obesity, delayed surgery, and age 30 and older, were established, with several being modifiable. Therefore, targeted preoperative optimization of modifiable risk factors and postoperative protocols may reduce the risk of secondary meniscus tears., Level of Evidence: Level III, prognostic trial., (© 2023. The Author(s) under exclusive licence to European Society of Sports Traumatology, Knee Surgery, Arthroscopy (ESSKA).)

High-throughput sequencing technology is central to our current understanding of the human methylome. The vast majority of studies use chemical conversion to analyse bulk-level patterns of DNA methylation across the genome from a population of cells. While this technology has been used to probe single-molecule methylation patterns, such analyses are limited to short reads of a few hundred basepairs. DNA methylation can also be directly detected using Nanopore sequencing which can generate reads measuring megabases in length. However, thus far these analyses have largely focused on bulk-level assessment of DNA methylation. Here, we analyse DNA methylation in single Nanopore reads from human lymphoblastoid cells, to show that bulk-level metrics underestimate large-scale heterogeneity in the methylome. We use the correlation in methylation state between neighbouring sites to quantify single-molecule heterogeneity and find that heterogeneity varies significantly across the human genome, with some regions having heterogeneous methylation patterns at the single-molecule level and others possessing more homogeneous methylation patterns. By comparing the genomic distribution of the correlation to epigenomic annotations, we find that the greatest heterogeneity in single-molecule patterns is observed within heterochromatic partially methylated domains (PMDs). In contrast, reads originating from euchromatic regions and gene bodies have more ordered DNA methylation patterns. By analysing the patterns of single molecules in more detail, we show the existence of a nucleosome-scale periodicity in DNA methylation that accounts for some of the heterogeneity we uncover in long single-molecule DNA methylation patterns. We find that this periodic structure is partially masked in bulk data and correlates with DNA accessibility as measured by nanoNOMe-seq, suggesting that it could be generated by nucleosomes. Our findings demonstrate the power of single-molecule analysis of long-read data to understand the structure of the human methylome., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kerr et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

DNA repair defects underlie many cancer syndromes. We tested whether de novo germline mutations (DNMs) are increased in families with germline defects in polymerase proofreading or base excision repair. A parent with a single germline POLE or POLD1 mutation, or biallelic MUTYH mutations, had 3-4 fold increased DNMs over sex-matched controls. POLE had the largest effect. The DNMs carried mutational signatures of the appropriate DNA repair deficiency. No DNM increase occurred in offspring of MUTYH heterozygous parents. Parental DNA repair defects caused about 20-150 DNMs per child, additional to the ~60 found in controls, but almost all extra DNMs occurred in non-coding regions. No increase in post-zygotic mutations was detected, excepting a child with bi-allelic MUTYH mutations who was excluded from the main analysis; she had received chemotherapy and may have undergone oligoclonal haematopoiesis. Inherited DNA repair defects associated with base pair-level mutations increase DNMs, but phenotypic consequences appear unlikely., (© 2023. The Author(s).)

Background: DNA methylation is an epigenetic mark associated with the repression of gene promoters. Its pattern in the genome is disrupted with age and these changes can be used to statistically predict age with epigenetic clocks. Altered rates of aging inferred from these clocks are observed in human disease. However, the molecular mechanisms underpinning age-associated DNA methylation changes remain unknown. Local DNA sequence can program steady-state DNA methylation levels, but how it influences age-associated methylation changes is unknown., Results: We analyze longitudinal human DNA methylation trajectories at 345,895 CpGs from 600 individuals aged between 67 and 80 to understand the factors responsible for age-associated epigenetic changes at individual CpGs. We show that changes in methylation with age occur at 182,760 loci largely independently of variation in cell type proportions. These changes are especially apparent at 8322 low CpG density loci. Using SNP data from the same individuals, we demonstrate that methylation trajectories are affected by local sequence polymorphisms at 1487 low CpG density loci. More generally, we find that low CpG density regions are particularly prone to change and do so variably between individuals in people aged over 65. This differs from the behavior of these regions in younger individuals where they predominantly lose methylation., Conclusions: Our results, which we reproduce in two independent groups of individuals, demonstrate that local DNA sequence influences age-associated DNA methylation changes in humans in vivo. We suggest that this occurs because interactions between CpGs reinforce maintenance of methylation patterns in CpG dense regions., (© 2022. The Author(s).)

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy of the bile ducts within the liver characterized by high levels of genetic heterogeneity. In the context of such genetic variability, determining which oncogenic mutations drive ICC growth has been difficult, and developing modes of patient stratification and targeted therapies remains challenging. Here we model the interactions between rare mutations with more common driver genes and combine in silico analysis of patient data with highly multiplexed in vivo CRISPR-spCas9 screens to perform a functional in vivo study into the role genetic heterogeneity plays in driving ICC. Novel tumor suppressors were uncovered, which, when lost, cooperate with the RAS oncoprotein to drive ICC growth. Focusing on a set of driver mutations that interact with KRAS to initiate aggressive, sarcomatoid-type ICC revealed that tumor growth relies on Wnt and PI3K signaling. Pharmacologic coinhibition of Wnt and PI3K in vivo impeded ICC growth regardless of mutational profile. Therefore, Wnt and PI3K activity should be considered as a signature by which patients can be stratified for treatment independent of tumor genotype, and inhibitors of these pathways should be levied to treat ICC., Significance: This work shows that, despite significant genetic heterogeneity, intrahepatic cholangiocarcinoma relies on a limited number of signaling pathways to grow, suggesting common therapeutic vulnerabilities across patients., (©2022 The Authors; Published by the American Association for Cancer Research.)

The accurate establishment and maintenance of DNA methylation patterns is vital for mammalian development and disruption to these processes causes human disease. Our understanding of DNA methylation mechanisms has been facilitated by mathematical modelling, particularly stochastic simulations. Megabase-scale variation in DNA methylation patterns is observed in development, cancer and ageing and the mechanisms generating these patterns are little understood. However, the computational cost of stochastic simulations prevents them from modelling such large genomic regions. Here, we test the utility of three different mean-field models to predict summary statistics associated with large-scale DNA methylation patterns. By comparison to stochastic simulations, we show that a cluster mean-field model accurately predicts the statistical properties of steady-state DNA methylation patterns, including the mean and variance of methylation levels calculated across a system of CpG sites, as well as the covariance and correlation of methylation levels between neighbouring sites. We also demonstrate that a cluster mean-field model can be used within an approximate Bayesian computation framework to accurately infer model parameters from data. As mean-field models can be solved numerically in a few seconds, our work demonstrates their utility for understanding the processes underpinning large-scale DNA methylation patterns.

Individuals of the same chronological age exhibit disparate rates of biological ageing. Consequently, a number of methodologies have been proposed to determine biological age and primarily exploit variation at the level of DNA methylation (DNAm). A novel epigenetic clock, termed 'DNAm GrimAge' has outperformed its predecessors in predicting the risk of mortality as well as many age-related morbidities. However, the association between DNAm GrimAge and cognitive or neuroimaging phenotypes remains unknown. We explore these associations in the Lothian Birth Cohort 1936 (n = 709, mean age 73 years). Higher DNAm GrimAge was strongly associated with all-cause mortality over the eighth decade (Hazard Ratio per standard deviation increase in GrimAge: 1.81, P < 2.0 × 10 -16 ). Higher DNAm GrimAge was associated with lower age 11 IQ (β = -0.11), lower age 73 general cognitive ability (β = -0.18), decreased brain volume (β = -0.25) and increased brain white matter hyperintensities (β = 0.17). There was tentative evidence for a longitudinal association between DNAm GrimAge and cognitive decline from age 70 to 79. Sixty-nine of 137 health- and brain-related phenotypes tested were significantly associated with GrimAge. Adjusting all models for childhood intelligence attenuated to non-significance a small number of associations (12/69 associations; 6 of which were cognitive traits), but not the association with general cognitive ability (33.9% attenuation). Higher DNAm GrimAge associates with lower cognitive ability and brain vascular lesions in older age, independently of early-life cognitive ability. This epigenetic predictor of mortality associates with different measures of brain health and may aid in the prediction of age-related cognitive decline., (© 2019. The Author(s).)

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment., Competing Interests: Declaration of interests S.M.P is a co-founder and shareholder of Cellinta., a biotechnology start-up that is developing cancer therapeutics, including for glioblastoma, and acts as an advisor to the company. J.W.P is a co-founder and shareholder of Macomics. S.A.Q. is co-founder and shareholder and Chief Scientific Officer for Achilles Therapeutics. The other authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The aberrant gain of DNA methylation at CpG islands is frequently observed in colorectal tumours and may silence the expression of tumour suppressors such as MLH1. Current models propose that these CpG islands are targeted by de novo DNA methyltransferases in a sequence-specific manner, but this has not been tested. Using ectopically integrated CpG islands, here we find that aberrantly methylated CpG islands are subject to low levels of de novo DNA methylation activity in colorectal cancer cells. By delineating DNA methyltransferase targets, we find that instead de novo DNA methylation activity is targeted primarily to CpG islands marked by the histone modification H3K36me3, a mark associated with transcriptional elongation. These H3K36me3 marked CpG islands are heavily methylated in colorectal tumours and the normal colon suggesting that de novo DNA methyltransferase activity at CpG islands in colorectal cancer is focused on similar targets to normal tissues and not greatly remodelled by tumourigenesis.

Inappropriate stimulation or defective negative regulation of the type I interferon response can lead to autoinflammation. In genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome, we identified biallelic mutations in LSM11 and RNU7-1, which encode components of the replication-dependent histone pre-mRNA-processing complex. Mutations were associated with the misprocessing of canonical histone transcripts and a disturbance of linker histone stoichiometry. Additionally, we observed an altered distribution of nuclear cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and enhanced interferon signaling mediated by the cGAS-stimulator of interferon genes (STING) pathway in patient-derived fibroblasts. Finally, we established that chromatin without linker histone stimulates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) production in vitro more efficiently. We conclude that nuclear histones, as key constituents of chromatin, are essential in suppressing the immunogenicity of self-DNA.

Mouse embryonic stem cells (mESCs) cultured with MEK/ERK and GSK3β (2i) inhibitors transition to ground state pluripotency. Gene expression changes, redistribution of histone H3K27me3 profiles and global DNA hypomethylation are hallmarks of 2i exposure, but it is unclear whether epigenetic alterations are required to achieve and maintain ground state or occur as an outcome of 2i signal induced changes. Here we show that ESCs with three epitypes, WT, constitutively methylated, or hypomethylated, all undergo comparable morphological, protein expression and transcriptome changes independently of global alterations of DNA methylation levels or changes in H3K27me3 profiles. Dazl and Fkbp6 expression are induced by 2i in all three epitypes, despite exhibiting hypermethylated promoters in constitutively methylated ESCs. We identify a number of activated gene promoters that undergo 2i dependent loss of H3K27me3 in all three epitypes, however genetic and pharmaceutical inhibition experiments show that H3K27me3 is not required for their silencing in non-2i conditions. By separating and defining their contributions, our data suggest that repressive epigenetic systems play minor roles in mESC self-renewal and naïve ground state establishment by core sets of dominant pluripotency associated transcription factor networks, which operate independently from these epigenetic processes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Epigenetic reprogramming is a cancer hallmark, but how it unfolds during early neoplastic events and its role in carcinogenesis and cancer progression is not fully understood. Here we show that resetting from primed to naïve human pluripotency results in acquisition of a DNA methylation landscape mirroring the cancer DNA methylome, with gradual hypermethylation of bivalent developmental genes. We identify a dichotomy between bivalent genes that do and do not become hypermethylated, which is also mirrored in cancer. We find that loss of H3K4me3 at bivalent regions is associated with gain of methylation. Additionally, we observe that promoter CpG island hypermethylation is not restricted solely to emerging naïve cells, suggesting that it is a feature of a heterogeneous intermediate population during resetting. These results indicate that transition to naïve pluripotency and oncogenic transformation share common epigenetic trajectories, which implicates reprogramming and the pluripotency network as a central hub in cancer formation.

Parent-of-origin effects (POE) exist when there is differential expression of alleles inherited from the two parents. A genome-wide scan for POE on DNA methylation at 639,238 CpGs in 5,101 individuals identifies 733 independent methylation CpGs potentially influenced by POE at a false discovery rate ≤ 0.05 of which 331 had not previously been identified. Cis and trans methylation quantitative trait loci (mQTL) regulate methylation variation through POE at 54% (399/733) of the identified POE-influenced CpGs. The combined results provide strong evidence for previously unidentified POE-influenced CpGs at 171 independent loci. Methylation variation at 14 of the POE-influenced CpGs is associated with multiple metabolic traits. A phenome-wide association analysis using the POE mQTL SNPs identifies a previously unidentified imprinted locus associated with waist circumference. These results provide a high resolution population-level map for POE on DNA methylation sites, their local and distant regulators and potential consequences for complex traits.

DNA methylation and Polycomb are key factors in the establishment of vertebrate cellular identity and fate. Here we report de novo missense mutations in DNMT3A, which encodes the DNA methyltransferase DNMT3A. These mutations cause microcephalic dwarfism, a hypocellular disorder of extreme global growth failure. Substitutions in the PWWP domain abrogate binding to the histone modifications H3K36me2 and H3K36me3, and alter DNA methylation in patient cells. Polycomb-associated DNA methylation valleys, hypomethylated domains encompassing developmental genes, become methylated with concomitant depletion of H3K27me3 and H3K4me3 bivalent marks. Such de novo DNA methylation occurs during differentiation of Dnmt3a W326R pluripotent cells in vitro, and is also evident in Dnmt3a W326R/+ dwarf mice. We therefore propose that the interaction of the DNMT3A PWWP domain with H3K36me2 and H3K36me3 normally limits DNA methylation of Polycomb-marked regions. Our findings implicate the interplay between DNA methylation and Polycomb at key developmental regulators as a determinant of organism size in mammals.

Background: DNA methylation levels change along with age, but few studies have examined the variation in the rate of such changes between individuals., Methods: We performed a longitudinal analysis to quantify the variation in the rate of change of DNA methylation between individuals using whole blood DNA methylation array profiles collected at 2-4 time points (N = 2894) in 954 individuals (67-90 years)., Results: After stringent quality control, we identified 1507 DNA methylation CpG sites (rsCpGs) with statistically significant variation in the rate of change (random slope) of DNA methylation among individuals in a mixed linear model analysis. Genes in the vicinity of these rsCpGs were found to be enriched in Homeobox transcription factors and the Wnt signalling pathway, both of which are related to ageing processes. Furthermore, we investigated the SNP effect on the random slope. We found that 4 out of 1507 rsCpGs had one significant (P < 5 × 10 -8 /1507) SNP effect and 343 rsCpGs had at least one SNP effect (436 SNP-probe pairs) reaching genome-wide significance (P < 5 × 10 -8 ). Ninety-five percent of the significant (P < 5 × 10 -8 ) SNPs are on different chromosomes from their corresponding probes., Conclusions: We identified CpG sites that have variability in the rate of change of DNA methylation between individuals, and our results suggest a genetic basis of this variation. Genes around these CpG sites have been reported to be involved in the ageing process.

Although CEBPA double-mutated ( CEBPA DM ) acute myeloid leukemia is considered to be a favorable-risk disease, relapse remains a major cause of treatment failure. Most CEBPA DM patients have a classic biallelic mutant combination with an N-terminal mutation leading to production of p30 protein plus a C-terminal loss-of-function in-frame indel mutation ( CEBPA Classic-DM ), but approximately one-third of cases have one or more non-classic mutations, with diverse combinations reported, and there is little information on the consequences of such mutants. We evaluated outcome in a cohort of 104 CEBPA DM patients, 79 CEBPA Classic-DM and 25 with non-classic mutants, and found that the latter may have poorer survival (5-year overall survival 64% vs. 46%; P =0.05), particularly post relapse (41% vs. 0%; P =0.02). However, for this analysis, all non-classic cases were grouped together, irrespective of mutant combination. As CEBPA DM cases have been reported to be hypermethylated, we used methylation profiling to assess whether this could segregate the different mutants. We developed a CEBPA Classic-DM methylation signature from a preliminary cohort of 10 CEBPA DM (including 8 CEBPA Classic-DM ) and 30 CEBPA wild-type ( CEBPA WT ) samples, and independently validated the signature in 17 CEBPA Classic-DM cases. Assessment of the signature in 16 CEBPA DM cases with different non-classic mutant combinations showed that only 31% had a methylation profile equivalent to CEBPA Classic-DM whereas for 69% the profile was either intermediate between CEBPA Classic-DM and CEBPA WT or equivalent to CEBPA WT These results suggest that CEBPA DM cases with non-classic mutants may be functionally different from those with CEBPA Classic-DM mutants, and should not automatically be included in the same prognostic group. (AML12 is registered under ISRCTN17833622 and AML15 under ISRCTN17161961)., (Copyright© 2018 Ferrata Storti Foundation.)

Glioblastoma multiforme (GBM) is an aggressive brain tumor driven by cells with hallmarks of neural stem (NS) cells. GBM stem cells frequently express high levels of the transcription factors FOXG1 and SOX2. Here we show that increased expression of these factors restricts astrocyte differentiation and can trigger dedifferentiation to a proliferative NS cell state. Transcriptional targets include cell cycle and epigenetic regulators (e.g., Foxo3 , Plk1 , Mycn , Dnmt1 , Dnmt3b , and Tet3 ). Foxo3 is a critical repressed downstream effector that is controlled via a conserved FOXG1/SOX2-bound cis -regulatory element. Foxo3 loss, combined with exposure to the DNA methylation inhibitor 5-azacytidine, enforces astrocyte dedifferentiation. DNA methylation profiling in differentiating astrocytes identifies changes at multiple polycomb targets, including the promoter of Foxo3 In patient-derived GBM stem cells, CRISPR/Cas9 deletion of FOXG1 does not impact proliferation in vitro; however, upon transplantation in vivo, FOXG1 -null cells display increased astrocyte differentiation and up-regulate FOXO3. In contrast, SOX2 ablation attenuates proliferation, and mutant cells cannot be expanded in vitro. Thus, FOXG1 and SOX2 operate in complementary but distinct roles to fuel unconstrained self-renewal in GBM stem cells via transcriptional control of core cell cycle and epigenetic regulators., (© 2017 Bulstrode et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Age-associated epigenetic changes are implicated in aging. Notably, age-associated DNA methylation changes comprise a so-called aging "clock", a robust biomarker of aging. However, while genetic, dietary and drug interventions can extend lifespan, their impact on the epigenome is uncharacterised. To fill this knowledge gap, we defined age-associated DNA methylation changes at the whole-genome, single-nucleotide level in mouse liver and tested the impact of longevity-promoting interventions, specifically the Ames dwarf Prop1 df/df mutation, calorie restriction and rapamycin., Results: In wild-type mice fed an unsupplemented ad libitum diet, age-associated hypomethylation was enriched at super-enhancers in highly expressed genes critical for liver function. Genes harbouring hypomethylated enhancers were enriched for genes that change expression with age. Hypermethylation was enriched at CpG islands marked with bivalent activating and repressing histone modifications and resembled hypermethylation in liver cancer. Age-associated methylation changes are suppressed in Ames dwarf and calorie restricted mice and more selectively and less specifically in rapamycin treated mice., Conclusions: Age-associated hypo- and hypermethylation events occur at distinct regulatory features of the genome. Distinct longevity-promoting interventions, specifically genetic, dietary and drug interventions, suppress some age-associated methylation changes, consistent with the idea that these interventions exert their beneficial effects, in part, by modulation of the epigenome. This study is a foundation to understand the epigenetic contribution to healthy aging and longevity and the molecular basis of the DNA methylation clock.

Background: Epigenetic changes are being increasingly recognized as a prominent feature of cancer. This occurs not only at individual genes, but also over larger chromosomal domains. To investigate this, we set out to identify large chromosomal domains of epigenetic dysregulation in breast cancers., Results: We identify large regions of coordinate down-regulation of gene expression, and other regions of coordinate activation, in breast cancers and show that these regions are linked to tumor subtype. In particular we show that a group of coordinately regulated regions are expressed in luminal, estrogen-receptor positive breast tumors and cell lines. For one of these regions of coordinate gene activation, we show that regional epigenetic regulation is accompanied by visible unfolding of large-scale chromatin structure and a repositioning of the region within the nucleus. In MCF7 cells, we show that this depends on the presence of estrogen., Conclusions: Our data suggest that the liganded estrogen receptor is linked to long-range changes in higher-order chromatin organization and epigenetic dysregulation in cancer. This may suggest that as well as drugs targeting histone modifications, it will be valuable to investigate the inhibition of protein complexes involved in chromatin folding in cancer cells.

Gene expression in higher organisms is thought to be regulated by a complex network of transcription factor binding and chromatin modifications, yet the relative importance of these two factors remains a matter of debate. Here, we show that a computational approach allows surprisingly accurate prediction of histone modifications solely from knowledge of transcription factor binding both at promoters and at potential distal regulatory elements. This accuracy significantly and substantially exceeds what could be achieved by using DNA sequence as an input feature. Remarkably, we show that transcription factor binding enables strikingly accurate predictions across different cell lines. Analysis of the relative importance of specific transcription factors as predictors of specific histone marks recapitulated known interactions between transcription factors and histone modifiers. Our results demonstrate that reported associations between histone marks and gene expression may be indirect effects caused by interactions between transcription factors and histone-modifying complexes.

DNA methylation is a repressive epigenetic mark vital for normal development. Recent studies have uncovered an unexpected role for the DNA methylome in ensuring the correct targeting of the Polycomb repressive complexes throughout the genome. Here, we discuss the implications of these findings for cancer, where DNA methylation patterns are widely reprogrammed. We speculate that cancer-associated reprogramming of the DNA methylome leads to an altered Polycomb binding landscape, influencing gene expression by multiple modes. As the Polycomb system is responsible for the regulation of genes with key roles in cell fate decisions and cell cycle regulation, DNA methylation induced Polycomb mis-targeting could directly drive carcinogenesis and disease progression., (© 2014 The Authors. Bioessays published by WILEY Periodicals, Inc.)

LINE-1 retrotransposons are abundant repetitive elements of viral origin, which in normal cells are kept quiescent through epigenetic mechanisms. Activation of LINE-1 occurs frequently in cancer and can enable LINE-1 mobilization but also has retrotransposition-independent consequences. We previously reported that in cancer, aberrantly active LINE-1 promoters can drive transcription of flanking unique sequences giving rise to LINE-1 chimeric transcripts (LCTs). Here, we show that one such LCT, LCT13, is a large transcript (>300 kb) running antisense to the metastasis-suppressor gene TFPI-2. We have modelled antisense RNA expression at TFPI-2 in transgenic mouse embryonic stem (ES) cells and demonstrate that antisense RNA induces silencing and deposition of repressive histone modifications implying a causal link. Consistent with this, LCT13 expression in breast and colon cancer cell lines is associated with silencing and repressive chromatin at TFPI-2. Furthermore, we detected LCT13 transcripts in 56% of colorectal tumours exhibiting reduced TFPI-2 expression. Our findings implicate activation of LINE-1 elements in subsequent epigenetic remodelling of surrounding genes, thus hinting a novel retrotransposition-independent role for LINE-1 elements in malignancy.

Background: Aberrant CpG island promoter DNA hypermethylation is frequently observed in cancer and is believed to contribute to tumor progression by silencing the expression of tumor suppressor genes. Previously, we observed that promoter hypermethylation in breast cancer reflects cell lineage rather than tumor progression and occurs at genes that are already repressed in a lineage-specific manner. To investigate the generality of our observation we analyzed the methylation profiles of 1,154 cancers from 7 different tissue types., Results: We find that 1,009 genes are prone to hypermethylation in these 7 types of cancer. Nearly half of these genes varied in their susceptibility to hypermethylation between different cancer types. We show that the expression status of hypermethylation prone genes in the originator tissue determines their propensity to become hypermethylated in cancer; specifically, genes that are normally repressed in a tissue are prone to hypermethylation in cancers derived from that tissue. We also show that the promoter regions of hypermethylation-prone genes are depleted of repetitive elements and that DNA sequence around the same promoters is evolutionarily conserved. We propose that these two characteristics reflect tissue-specific gene promoter architecture regulating the expression of these hypermethylation prone genes in normal tissues., Conclusions: As aberrantly hypermethylated genes are already repressed in pre-cancerous tissue, we suggest that their hypermethylation does not directly contribute to cancer development via silencing. Instead aberrant hypermethylation reflects developmental history and the perturbation of epigenetic mechanisms maintaining these repressed promoters in a hypomethylated state in normal cells.

Chemical inhibitors of histone deacetylase (HDAC) activity are used as experimental tools to induce histone hyperacetylation and deregulate gene transcription, but it is not known whether the inhibition of HDACs plays any part in the normal physiological regulation of transcription. Using both in vitro and in vivo assays, we show that lactate, which accumulates when glycolysis exceeds the cell's aerobic metabolic capacity, is an endogenous HDAC inhibitor, deregulating transcription in an HDAC-dependent manner. Lactate is a relatively weak inhibitor (IC(50) 40 mM) compared to the established inhibitors trichostatin A and butyrate, but the genes deregulated overlap significantly with those affected by low concentrations of the more potent inhibitors. HDAC inhibition causes significant up and downregulation of genes, but genes that are associated with HDAC proteins are more likely to be upregulated and less likely to be downregulated than would be expected. Our results suggest that the primary effect of HDAC inhibition by endogenous short-chain fatty acids like lactate is to promote gene expression at genes associated with HDAC proteins. Therefore, we propose that lactate may be an important transcriptional regulator, linking the metabolic state of the cell to gene transcription.

High expression of Rac small GTPases in invasive breast ductal carcinoma is associated with poor prognosis, but its therapeutic value in human cancers is not clear. The aim of the current study was to determine the response of human primary breast cancers to Rac-based drug treatments ex vivo. Three-dimensional organotypic cultures were used to assess candidate therapeutic avenues in invasive breast cancers. Uniquely, in these primary cultures, the tumour is not disaggregated, with both epithelial and mesenchymal components maintained within a 3-dimensional matrix of type I collagen. EHT 1864, a small molecule inhibitor of Rac GTPases, prevents spread of breast cancers in this setting, and also reduces proliferation at the invading edge. Rac1+ epithelial cells in breast tumours also contain high levels of the phosphorylated form of the transcription factor STAT3. The small molecule Stattic inhibits activation of STAT3 and induces effects similar to those seen with EHT 1864. Pan-Rac inhibition of proliferation precedes down-regulation of STAT3 activity, defining it as the last step in Rac activation during human breast cancer invasion. Our data highlights the potential use of Rac and STAT3 inhibition in treatment of invasive human breast cancer and the benefit of studying novel cancer treatments using 3-dimensional primary tumour tissue explant cultures.

The discovery of substantial amounts of 5-hydroxymethylcytosine (5hmC), formed by the oxidation of 5-methylcytosine (5mC), in various mouse tissues and human embryonic stem (ES) cells has necessitated a reevaluation of our knowledge of 5mC/5hmC patterns and functions in mammalian cells. Here, we investigate the tissue specificity of both the global levels and locus-specific distribution of 5hmC in several human tissues and cell lines. We find that global 5hmC content of normal human tissues is highly variable, does not correlate with global 5mC content, and decreases rapidly as cells from normal tissue adapt to cell culture. Using tiling microarrays to map 5hmC levels in DNA from normal human tissues, we find that 5hmC patterns are tissue specific; unsupervised hierarchical clustering based solely on 5hmC patterns groups independent biological samples by tissue type. Moreover, in agreement with previous studies, we find 5hmC associated primarily, but not exclusively, with the body of transcribed genes, and that within these genes 5hmC levels are positively correlated with transcription levels. However, using quantitative 5hmC-qPCR, we find that the absolute levels of 5hmC for any given gene are primarily determined by tissue type, gene expression having a secondary influence on 5hmC levels. That is, a gene transcribed at a similar level in several different tissues may have vastly different levels of 5hmC (>20-fold) dependent on tissue type. Our findings highlight tissue type as a major modifier of 5hmC levels in expressed genes and emphasize the importance of using quantitative analyses in the study of 5hmC levels.

Overexpression of matriptase has been reported in a variety of human cancers and is sufficient to trigger tumor formation in mice, but the importance of matriptase in breast cancer remains unclear. We analysed matriptase expression in 16 human breast cancer cell lines and in 107 primary breast tumors. The data revealed considerable diversity in the expression level of this protein indicating that the significance of matriptase may vary from case to case. Matriptase protein expression was correlated with HER2 expression and highest expression was seen in HER2-positive cell lines, indicating a potential role in this subgroup. Stable overexpression of matriptase in two breast cancer cell lines had different consequences. In MDA-MB-231 human breast carcinoma cells the only noted consequence of matriptase overexpression was modestly impaired growth in vivo. In contrast, overexpression of matriptase in 4T1 mouse breast carcinoma cells resulted in visible changes in morphology, actin staining and cell to cell contacts. This correlated with downregulation of the cell-cell adhesion molecule E-cadherin. These results suggest that the functions of matriptase in breast cancer are likely to be variable and cell context dependent.

The biology underlying early-stage epithelial ovarian cancer (EOC) development is poorly understood. Identifying biomarkers associated with early-stage disease could have a significant impact on reducing mortality. Here, we describe establishment of a three-dimensional (3D) in vitro genetic model of EOC initiation and early-stage neoplastic progression. Normal primary ovarian epithelial (POE) cells, immortalized using hTERT (immortalised ovarian epithelial [IOE] cells), were partially transformed by overexpressing the CMYC oncogene (IOE(CMYC) cells). Subsequent expression of mutant alleles of KRAS (KRAS(G12V)) or BRAF (BRAF(V600E)) created double-mutant lines (IOE(CMYC.KRAS) and IOE(CMYC.BRAF)). The transformed phenotype of IOE(CMYC) cells was further enhanced in concert with KRAS(G12V)/BRAF(V600E) expression, as in vitro analyses indicated that IOE(CMYC) cells had undergone morphological and phenotypic changes characteristic of neoplastic progression. When cultured as 3D spheroids, IOE cells underwent growth arrest, reminiscent of nonproliferative, unstimulated POE in vivo. In contrast, IOSE(CMYC+BRAF/KRAS) cells formed highly proliferative, poly-aggregate spheroid structures, showing increased expression of the Wilms tumour 1 tumourigenic marker and MIB1 proliferation marker. Transcriptomic analyses identified different gene expression profiles between the different cell lines and novel candidate genes (e.g. RGS4, CTGF and THBS1) that are somatically altered in EOCs. Gene expression signatures were compared with signatures from primary EOCs; tumours with IOE(CMYC) 'like' signatures were more likely to be high grade (P = 0.018); tumours with BRAF signatures were associated with improved relapse-free survival (P = 0.003). In conclusion, we have established in vitro 3D models of early-stage EOCs, which reflect genetic and phenotypic heterogeneity of the disease. Molecular genetic characteristics of these models correlated with molecular and clinical features of primary EOCs.

Aberrant promoter hypermethylation is frequently observed in cancer. The potential for this mechanism to contribute to tumor development depends on whether the genes affected are repressed because of their methylation. Many aberrantly methylated genes play important roles in development and are bivalently marked in ES cells, suggesting that their aberrant methylation may reflect developmental processes. We investigated this possibility by analyzing promoter methylation in 19 breast cancer cell lines and 47 primary breast tumors. In cell lines, we defined 120 genes that were significantly repressed in association with methylation (SRAM). These genes allowed the unsupervised segregation of cell lines into epithelial (EPCAM+ve) and mesenchymal (EPCAM-ve) lineages. However, the methylated genes were already repressed in normal cells of the same lineage, and >90% could not be derepressed by treatment with 5-aza-2'-deoxycytidine. The tumor suppressor genes APC and CDH1 were among those methylated in a lineage-specific fashion. As predicted by the epithelial nature of most breast tumors, SRAM genes that were methylated in epithelial cell lines were frequently aberrantly methylated in primary tumors, as were genes specifically repressed in normal epithelial cells. An SRAM gene expression signature also correctly identified the rare claudin-low and metaplastic tumors as having mesenchymal characteristics. Our findings implicate aberrant DNA methylation as a marker of cell lineage rather than tumor progression and suggest that, in most cases, it does not cause the repression with which it is associated.

Using a genetic model, we present a high-resolution chromatin fiber analysis of transcriptionally active (Xa) and inactive (Xi) X chromosomes packaged into euchromatin and facultative heterochromatin. Our results show that gene promoters have an open chromatin structure that is enhanced upon transcriptional activation but the Xa and the Xi have similar overall 30 nm chromatin fiber structures. Therefore, the formation of facultative heterochromatin is dependent on factors that act at a level above the 30 nm fiber and transcription does not alter bulk chromatin fiber structures. However, large-scale chromatin structures on Xa are decondensed compared with the Xi and transcription inhibition is sufficient to promote large-scale chromatin compaction. We show a link between transcription and large-scale chromatin packaging independent of the bulk 30 nm chromatin fiber and propose that transcription, not the global compaction of 30 nm chromatin fibers, determines the cytological appearance of large-scale chromatin structures., (Copyright © 2010 Elsevier Inc. All rights reserved.)

How polycomb group proteins repress gene expression in vivo is not known. While histone-modifying activities of the polycomb repressive complexes (PRCs) have been studied extensively, in vitro data have suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that PRCs are required to maintain a compact chromatin state at Hox loci in embryonic stem cells (ESCs). There is specific decompaction in the absence of PRC2 or PRC1. This is due to a PRC1-like complex, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state and to repress Hox gene expression is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The spatial organisation of the genome in the nucleus has a role in the regulation of gene expression. In vertebrates, chromosomal regions with low gene-density are located close to the nuclear periphery. Correlations have also been made between the transcriptional state of some genes and their location near the nuclear periphery. However, a crucial issue is whether this level of nuclear organisation directly affects gene function, rather than merely reflecting it. To directly investigate whether proximity to the nuclear periphery can influence gene expression in mammalian cells, here we relocate specific human chromosomes to the nuclear periphery by tethering them to a protein of the inner nuclear membrane. We show that this can reversibly suppress the expression of some endogenous human genes located near the tethering sites, and even genes further away. However, the expression of many other genes is not detectably reduced and we show that location at the nuclear periphery is not incompatible with active transcription. The dampening of gene expression around the nuclear periphery is dependent on the activity of histone deacetylases. Our data show that the radial position within the nucleus can influence the expression of some, but not all, genes. This is compatible with the suggestion that re-localisation of genes relative to the peripheral zone of the nucleus could be used by metazoans to modulate the expression of selected genes during development and differentiation., Competing Interests: The authors have declared that no competing interests exist.

Much of what we know about the chromatin-based mechanisms that regulate gene expression in mammals has come from the study of what are, paradoxically, atypical genes. These are clusters of structurally and/or functionally related genes that are coordinately regulated during development, or between different cell types. Can unravelling the mechanisms of gene regulation at these gene clusters help us to understand how other genes are controlled? Moreover, can it explain why there is clustering of apparently unrelated genes in mammalian genomes?