Biology subjects

Horsthemke, B.

Publications and source records attributed to Horsthemke, B..

4 recordsLinked to original sources

bioRxiv2022

Epigenetic plasticity via adaptive DNA hypermethylation and clonal expansion underlie resistance to oncogenic pathway inhibition in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. Drug resistance is the major cause for therapeutic failure in PDAC patients with progressive disease. The mechanisms underlying resistance formation are complex and remain poorly understood. To gain insights into molecular changes during the formation of resistance to oncogenic MAPK pathway inhibition we utilized short-term passaged primary tumor cells from ten PDACs of genetically engineered mice. We followed gain and loss of resistance upon MEKi exposure and withdrawal by longitudinal integrative analysis of whole genome sequencing, whole genome bisulfite sequencing, RNA-sequencing and mass spectrometry data. We found that resistant cell populations under increasing MEKi treatment evolved by the expansion of a single clone but were not a direct consequence of known resistance-conferring mutations. Rather, resistant cells showed adaptive DNA hypermethylation of 209 and hypomethylation of 8 genomic sites, most of which overlap with regulatory elements known to be active in murine PDAC cells. Both DNA methylation changes and MEKi resistance were transient and reversible upon drug withdrawal. The effector caspase CASP3 is one of the 114 genes for which transcriptional downregulation inversely correlated with the methylation status of the associated DNA region. CASP3 inactivation in resistant cells led to attenuation of drug-induced apoptosis which could be reversed by DNA methyltransferase inhibition with remarkable sensitivity exclusively in the resistant cells. Overall, our data provide a context for characterization and targeting of epigenetically mediated resistance mechanisms in PDAC.

cancer biology↗

bioRxiv2020

The sperm epigenome does not display recurrent epimutations in patients with severely impaired spermatogenesis

BackgroundIn the past 15 years, numerous studies have described aberrant DNA methylation of imprinted genes (e.g. MEST and H19) in sperm of infertile patients, but the prevalence and genomic extent of abnormal methylation patterns have remained unknown. ResultsUsing deep bisulfite sequencing (DBS), we screened swim-up sperm samples from 40 normozoospermic and 93 oligoasthenoteratozoospermic (OAT) patients for H19 and MEST methylation. Based on this screening, we defined three patient groups: normal controls (NC), abnormally methylated infertile (AMI; n=7) and normally methylated infertile (NMI; n=86). Whole genome bisulfite sequencing (WGBS) of five NC and five AMI samples revealed abnormal methylation levels of all 50 imprinting control regions in each AMI sample. To investigate whether this finding reflected epigenetic germ line mosaicism or the presence of residual somatic DNA, we made a genome-wide inventory of soma-germ cell specific DNA methylation. We found that >2,000 germ cell-specific genes are promoter-methylated in blood and that AMI samples had abnormal methylation levels at these genes, consistent with the presence of somatic cell DNA. The comparison between the five NC and six NMI samples revealed 19 differentially methylated regions (DMRs), none of which could be validated in an independent cohort of 40 men. Previous studies reported a higher incidence of epimutations at single CpG sites in the CTCF-binding region 6 of H19 in infertile patients. DBS analysis of this locus, however, revealed an association between DNA methylation levels and genotype (rs2071094), but not fertility phenotype. ConclusionsOur results suggest that somatic DNA contamination and genetic variation confound methylation studies in sperm of infertile men. While we cannot exclude the existence of rare patients with slightly abnormal sperm methylation at non-recurrent CpG sites, the prevalence of aberrant methylation in swim-up purified sperm of infertile men has likely been overestimated, which is reassuring for patients undergoing assisted reproduction.

genetics↗

bioRxiv2019

wg-blimp: an end-to-end analysis pipeline for whole genome bisulfite sequencing data

BackgroundAnalysing whole genome bisulfite sequencing datasets is a data-intensive task that requires comprehensive and reproducible workflows to generate valid results. While many algorithms have been developed for tasks such as alignment, comprehensive end-to-end pipelines are still sparse. Furthermore, previous pipelines lack features or show technical deficiencies, thus impeding analyses. ResultsWe developed wg-blimp (whole genome bisulfite sequencing methylation analysis pipeline) as an end-to-end pipeline to ease whole genome bisulfite sequencing data analysis. It integrates established algorithms for alignment, quality control, methylation calling, detection of differentially methylated regions, and methylome segmentation, requiring only a reference genome and raw sequencing data as input. Comparing wg-blimp to previous end-to-end pipelines reveals similar setups for common sequence processing tasks, but shows differences for post-alignment analyses. We improve on previous pipelines by providing a more comprehensive analysis workflow as well as an interactive user interface. To demonstrate wg-blimps ability to produce correct results we used it to call differentially methylated regions for two publicly available datasets. We were able to replicate 112 of 114 previously published regions, and found results to be consistent with previous findings. We further applied wg-blimp to a publicly available sample of embryonic stem cells to showcase methylome segmentation. As expected, unmethylated regions were in close proximity of transcription start sites. Segmentation results were consistent with previous analyses, despite different reference genomes and sequencing techniques. Conclusionswg-blimp provides a comprehensive analysis pipeline for whole genome bisulfite sequencing data as well as a user interface for simplified result inspection. We demonstrated its applicability by analysing multiple publicly available datasets. Thus, wg-blimp is a relevant alternative to previous analysis pipelines and may facilitate future epigenetic research.

bioinformatics↗

bioRxiv2017

Preovulatory oocyte aging in mice affects fertilization rate and embryonic genome activation

Delayed ovulation, or preovulatory aging, can seriously compromise the developmental competence of oocytes. In the present study, we have investigated the effect of preovulatory aging on preimplantation embryos. Delaying ovulation with the gonadotropin releasing hormone (GnRH) antagonist Cetrorelix led to a decline in 2-cell rate from 76 to 46%. From control mice, an average of 17 embryos per mouse was retrieved. This number decreased to a mean of 5 embryos per mouse after preovulatory aging, suggesting that fertilization is impaired by aging. For analysis of zygotic genome activation, 2-cell embryos were incubated with BrUTP, which was incorporated into nascent RNA and detected by immunohistochemistry. A 2.85-fold increase in fluorescence intensity was detected after aging, pointing to a precocious activation of the genome. A possible effect of preovulatory aging on genomic imprint maintenance was investigated at the 8-cell stage. Deep amplicon bisulfite sequencing of Igf2r, Snrpn, H19 and Pou5f1 showed no significant changes between embryos derived from preovulatory-aged oocytes and control embryos, indicating stable imprint maintenance throughout epigenetic reprogramming. We conclude that preovulatory aging of the oocyte affects fertilization and embryonic genomic activation.

developmental biology↗