Reactivation of a silenced H19 gene in human rhabdomyosarcoma by demethylation of DNA but not by histone hyperacetylation

BACKGROUND: The active copy of the imprinted gene H19 is turned off by inappropriate methylation in several pediatric tumors including Wilms' Tumour and embryonal rhabdomyosarcoma. H19 controls in cis the linked Insulin-like Growth Factor 2 (IGF2) gene, encoding an important growth factor. Recent work has suggested that methylation of a gene may lead to deacetylation of its associated histones and that silenced genes can be reactivated by increasing histone acetylation levels. RESULTS: Treatment of a rhabdomyosarcoma cell line which has a silent, methylated H19 gene with histone deacetylase (HDAC) inhibitors under conditions which gave maximal hyperacetylation of histone 4, both globally and at the H19 gene itself could not reactivate H19 or affect the active Insulin-like Growth Factor 2 (IGF2) gene, but caused clear up-regulation of the Tissue-type Plasminogen Activator (TPA) gene, a non-imprinted gene known to respond to changes in histone acetylation. In contrast, mild treatment of the cells with the methylation inhibitor 5-AzaC-2'-deoxycytidine (AzaC) on its own was able to reactivate H19. Combining AzaC treatment with HDAC inhibitors gave a reduced rather than enhanced reactivation. These findings were confirmed in mouse primary liver and kidney explants which maintain normal imprinting, where we also found that the silent Igf2 gene could not be reactivated by HDAC inhibitors. CONCLUSION: These results suggest that DNA methylation rather than histone acetylation is the primary determinant of silencing of H19 in rhabdomyosarcoma

College Student Mental Health and Wellbeing Prior to and during the COVID-19 Pandemic

Student mental health was a growing concern globally prior to the onset of the COVID-19 pandemic. The aim of this study was to assess the impact of the pandemic and associated restrictions on the psychological wellbeing of college students. Baseline data were collected pre-pandemic in September 2019 among students attending a university in Northern Ireland and an Institute of Technology in the Republic of Ireland. Surveys were also conducted with this cohort during the pandemic, at the start of the academic years 2020 and 2021 (499 students fully completed all three waves). A follow-up survey was conducted at the end of their third year, in summer 2022 (n = 229). High levels of mental health problems were already present among students commencing college. The subsequent pandemic had a very negative impact on student’s academic experience and other aspects of life. Rates of depression (PHQ-9) increased significantly from the onset of the pandemic and remained high. Anxiety (GAD-7) initially decreased but then escalated at the end of college. The study highlights the importance of early intervention and makes recommendations for addressing the needs of students during times of stress. Additional supports may be required to deal with the long-lasting impact of the pandemic

Teaching efficacy of undergraduate PE students; what are the key predictors and what can PE educators learn from this?

IntroductionTeaching efficacy describes the belief in a teacher's ability to promote learning and this belief is an invaluable asset for all teachers. This study examined the contextual influences that predict the teaching efficacy of first-year undergraduate PE students wishing to enter teacher training programs.MethodUsing a mixed methods study design, 168 PE students completed an online questionnaire and 16 of these participants took part in semi-structured focus groups. The data collection procedures investigated students' perceptions of PE teaching efficacy and examined students' awareness of how their involvement in PE or sports influenced their decision to study PE.ResultsTeaching experiences and role model influences were the key predictors of students' perceived PE teaching efficacy.DiscussionsWe recommend that higher education PE programs should facilitate theoretically informed reflective learning opportunities to enable students to understand and make sense of the impact of these key predictors. These opportunities will enable students to understand their starting point in PE teaching efficacy and identify the requirements to develop it. The study extends the existing literature by identifying the key predictors of PE teaching efficacy derived from the acculturation experiences of undergraduate PE students

Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression

Additional file 3: Figure S2. Changes in methylation levels by genomic element. (A) Protein levels in knockdown lines by western blotting. As a control HCT116 colon cancer cells which are WT or have a homozygous mutation in DNMT1 (KO) are shown: the DNMT1-specific top band is indicated by the arrowhead at right. (B) Median levels of methylation are shown for each genomic element (listed at top). The positions of medians are also indicated at right (arrowheads). The differences between WT and KD medians were used to plot Fig. 1d. (C) Density distribution of methylation at the three main elements involved in gene regulation, shown by cell line. Demethylation seems most marked at gene bodies (Genes), indicated by increased density of probes at low methylation (β) values

Intragenic sequences in the trophectoderm harbour the greatest proportion of methylation errors in day 17 bovine conceptuses generated using assisted reproductive technologies

Abstract Background Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences. Results Of the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth. Conclusion By examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos

Supporting data for "CandiMeth: Powerful yet simple visualization and quantification of DNA methylation at candidate genes"

DNA methylation microarrays are widely used in clinical epigenetics and are often processed using R packages like ChAMP or RnBeads by trained bioinformaticians. However, looking at specific genes requires bespoke coding which wet-lab biologists or clinicians are not trained for. This leads to high demands on bioinformaticians, who in turn may lack insight into the specific biological problem. We therefore wished to develop a tool for mapping and quantification of methylation differences at candidate genomic features of interest, without using coding, to bridge this gap. We generated the workflow CandiMeth (CANDIdate METHylation) in the web-based environment Galaxy. CandiMeth takes as input any table listing differences in methylation generated by either of the popular R-based packages above and maps these to the human genome. A simple interface then allows the user to query the data using lists of gene names. CandiMeth generates 1)Tracks in the popular UCSC genome browser with an intuitive visual indicator of where differences in methylation occur between samples, or groups of samples 2) Tables containing quantitative data on the candidate regions, allowing interpretation of significance. In addition to genes and promoters, CandiMeth can analyse methylation differences at LINEs and SINEs. Cross-comparison to other open-resource genomic data at UCSC facilitates interpretation of the biological significance of the data and the design of wet lab assays to further explore methylation changes and their consequences for the candidate genes. CandiMeth (RRID:SCR_017974; Biotools:CandiMeth) allows rapid, quantitative analysis of methylation at user-specified features without the need for coding and is freely available, with extensive guidance, at CandiMeth GitHub repo

Alterations in the steroid hormone receptor co-chaperone FKBPL are associated with male infertility: a case-control study

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background Male infertility is a common cause of reproductive failure in humans. In mice, targeted deletions of the genes coding for FKBP6 or FKBP52, members of the FK506 binding protein family, can result in male infertility. In the case of FKBP52, this reflects an important role in potentiating Androgen Receptor (AR) signalling in the prostate and accessory glands, but not the testis. In infertile men, no mutations of FKBP52 or FKBP6 have been found so far, but the gene for FKBP-like (FKBPL) maps to chromosome 6p21.3, an area linked to azoospermia in a group of Japanese patients. Methods To determine whether mutations in FKBPL could contribute to the azoospermic phenotype, we examined expression in mouse and human tissues by RNA array blot, RT-PCR and immunohistochemistry and sequenced the complete gene from two azoospermic patient cohorts and matching control groups. FKBPL-AR interaction was assayed using reporter constructs in vitro. Results FKBPL is strongly expressed in mouse testis, with expression upregulated at puberty. The protein is expressed in human testis in a pattern similar to FKBP52 and also enhanced AR transcriptional activity in reporter assays. We examined sixty patients from the Japanese patient group and found one inactivating mutation and one coding change, as well as a number of non-coding changes, all absent in fifty-six controls. A second, Irish patient cohort of thirty showed another two coding changes not present in thirty proven fertile controls. Conclusions Our results describe the first alterations in the gene for FKBPL in azoospermic patients and indicate a potential role in AR-mediated signalling in the testis.Published versio