CpG Island Methylation in Human Lymphocytes Is Highly Correlated with DNA Sequence, Repeats, and Predicted DNA Structure

CpG island methylation plays an important role in epigenetic gene control during mammalian development and is frequently altered in disease situations such as cancer. The majority of CpG islands is normally unmethylated, but a sizeable fraction is prone to become methylated in various cell types and pathological situations. The goal of this study is to show that a computational epigenetics approach can discriminate between CpG islands that are prone to methylation from those that remain unmethylated. We develop a bioinformatics scoring and prediction method on the basis of a set of 1,184 DNA attributes, which refer to sequence, repeats, predicted structure, CpG islands, genes, predicted binding sites, conservation, and single nucleotide polymorphisms. These attributes are scored on 132 CpG islands across the entire human Chromosome 21, whose methylation status was previously established for normal human lymphocytes. Our results show that three groups of DNA attributes, namely certain sequence patterns, specific DNA repeats, and a particular DNA structure, are each highly correlated with CpG island methylation (correlation coefficients of 0.64, 0.66, and 0.49, respectively). We predicted, and subsequently experimentally examined 12 CpG islands from human Chromosome 21 with unknown methylation patterns and found more than 90% of our predictions to be correct. In addition, we applied our prediction method to analyzing Human Epigenome Project methylation data on human Chromosome 6 and again observed high prediction accuracy. In summary, our results suggest that DNA composition of CpG islands (sequence, repeats, and structure) plays a significant role in predisposing CpG islands for DNA methylation. This finding may have a strong impact on our understanding of changes in CpG island methylation in development and disease

The nudibranch Berghia stephanieae (Valdés, 2005) is not able to initiate a functional symbiosome-like environment to maintain Breviolum minutum (J.E.Parkinson & LaJeunesse 2018)

Photosymbiosis is found in different animal lineages and is best understood in cnidarians. A successful initiation and maintenance of the symbiosis between the animal hosts and the photosymbiotic partners is based on a recognition by specific host receptors. This triggers signaling cascades that promote the photobiont tolerance by the host, including an interpartner nutrient exchange and the ability of the host to cope with increased levels of reactive oxygen species (ROS) generated by the photobiont. Key to the successful symbiosis is the inhibition of the phagosomal maturation resulting in the formation of the symbiosome. In animals other than cnidarians, little is known about the photosymbiosis initiation and maintenance, for instance in sea slugs belonging to the Nudibranchia. Here, we investigated the gene expression profile of Berghia stephanieae, which is able to incorporate Breviolum minutum from its cnidarian prey Exaiptasia diaphana (Rapp, 1829) but is not able to maintain the algae for more than a couple of days during starvation. We show that the recognition of the algae is based on similar mechanisms present in cnidarians, and we identified some additional candidate genes that might be molluscan specific for photobiont recognition. Downstream, B. stephanieae responds to increased levels of ROS but is not able to stop the phagosomal maturation or decrease the immune response against B. minutum, which seem to be the key factors missing in B. stephanieae that accounts for the unstable symbiosis in this slug. Hence, B. stephanieae can be considered a transitional state toward a stable photosymbiosis and can help to elucidate general aspects of the evolutionary processes involved in establishing photosymbioses in animals

Promiscuous gene expression in thymic epithelial cells is regulated at multiple levels

The role of central tolerance induction has recently been revised after the discovery of promiscuous expression of tissue-restricted self-antigens in the thymus. The extent of tissue representation afforded by this mechanism and its cellular and molecular regulation are barely defined. Here we show that medullary thymic epithelial cells (mTECs) are specialized to express a highly diverse set of genes representing essentially all tissues of the body. Most, but not all, of these genes are induced in functionally mature CD80hi mTECs. Although the autoimmune regulator (Aire) is responsible for inducing a large portion of this gene pool, numerous tissue-restricted genes are also up-regulated in mature mTECs in the absence of Aire. Promiscuously expressed genes tend to colocalize in clusters in the genome. Analysis of a particular gene locus revealed expression of clustered genes to be contiguous within such a cluster and to encompass both Aire-dependent and –independent genes. A role for epigenetic regulation is furthermore implied by the selective loss of imprinting of the insulin-like growth factor 2 gene in mTECs. Our data document a remarkable cellular and molecular specialization of the thymic stroma in order to mimic the transcriptome of multiple peripheral tissues and, thus, maximize the scope of central self-tolerance

DNA methylation biomarkers in colorectal cancer: Clinical applications for precision medicine

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide that is attributed to gradual long-term accumulation of both genetic and epigenetic changes. To reduce the mortality rate of CRC and to improve treatment efficacy, it will be important to develop accurate noninvasive diagnostic tests for screening, acute and personalized diagnosis. Epigenetic changes such as DNA methylation play an important role in the development and progression of CRC. Over the last decade, a panel of DNA methylation markers has been reported showing a high accuracy and reproducibility in various semi-invasive or noninvasive biosamples. Research to obtain comprehensive panels of markers allowing a highly sensitive and differentiating diagnosis of CRC is ongoing. Moreover, the epigenetic alterations for cancer therapy, as a precision medicine strategy will increase their therapeutic potential over time. Here, we discuss the current state of DNA methylation-based biomarkers and their impact on CRC diagnosis. We emphasize the need to further identify and stratify methylation-biomarkers and to develop robust and effective detection methods that are applicable for a routine clinical setting of CRC diagnostics particularly at the early stage of the disease

The composition of the pulmonary microbiota in sarcoidosis - an observational study

Background: Sarcoidosis is a systemic disease of unknown etiology. The disease mechanisms are largely speculative and may include the role microbial patterns that initiate and drive an underlying immune process. The aim of this study was to characterize the microbiota of the lung of patients with sarcoidosis and compare its composition and diversity with the results from patients with other interstitial lung disease (ILD) and historic healthy controls. Methods: Patients (sarcoidosis, n = 31; interstitial lung disease, n = 19) were recruited within the PULMOHOM study, a prospective cohort study to characterize inflammatory processes in pulmonary diseases. Bronchoscopy of the middle lobe or the lingula was performed and the recovered fluid was immediately sent for analysis of the pulmonary microbiota by 16sRNA gene sequencing. Subsequent bioinformatic analysis was performed to compare the groups. Results: There were no significant differences between patients with sarcoidosis or other ILDs with regard to microbiome composition and diversity. In addition, the abundance of the genera Atopobium, Fusobacterium, Mycobacterium or Propionibacterium were not different between the two groups. There were no gross differences to historical healthy controls. Conclusion: The analysis of the pulmonary microbiota based on 16sRNA gene sequencing did not show a significant dysbiosis in patients with sarcoidosis as compared to other ILD patients. These data do not exclude a microbiological component in the pathogenesis of sarcoidosis

Comparison of initial oral microbiomes of young adults with and without cavitated dentin caries lesions using an in situ biofilm model

Dental caries is caused by acids released from bacterial biofilms. However, the in vivo formation of initial biofilms in relation to caries remains largely unexplored. The aim of this study was to compare the oral microbiome during the initial phase of bacterial colonization for individuals with (CC) and without (NC) cavitated dentin caries lesions. Bovine enamel slabs on acrylic splints were worn by the volunteers (CC: 14, NC: 13) for in situ biofilm formation (2 h, 4 h, 8 h, 1 ml saliva as reference). Sequencing of the V1/V2 regions of the 16S rRNA gene was performed (MiSeq). The relative abundances of individual operational taxonomic units (OTUs) were compared between samples from the CC group and the NC group. Random forests models were furthermore trained to separate the groups. While the overall heterogeneity did not differ substantially between CC and NC individuals, several individual OTUs were found to have significantly different relative abundances. For the 8 h samples, most of the significant OTUs showed higher relative abundances in the CC group, while the majority of significant OTUs in the saliva samples were more abundant in the NC group. Furthermore, using OTU signatures enabled a separation between both groups, with area-under-the-curve (AUC) values of ~0.8. In summary, the results suggest that initial oral biofilms provide the potential to differentiate between CC and NC individuals

Bisulfite profiling of the MGMT promoter and comparison with routine testing in glioblastoma diagnostics

Background: Promoter methylation of the DNA repair gene O6 -methylguanine-DNA methyltransferase (MGMT) is an acknowledged predictive epigenetic marker in glioblastoma multiforme and anaplastic astrocytoma. Patients with methylated CpGs in the MGMT promoter beneft from treatment with alkylating agents, such as temozolomide, and show an improved overall survival and progression-free interval. A precise determination of MGMT promoter methyla‑ tion is of importance for diagnostic decisions. We experienced that diferent methods show partially divergent results in a daily routine. For an integrated neuropathological diagnosis of malignant gliomas, we therefore currently apply a combination of methylation-specifc PCR assays and pyrosequencing. Results: To better rationalize the variation across assays, we compared these standard techniques and assays to deep bisulfte sequencing results in a cohort of 80 malignant astrocytomas. Our deep analysis covers 49 CpG sites of the expanded MGMT promoter, including exon 1, parts of intron 1 and a region upstream of the transcription start site (TSS). We observed that deep sequencing data are in general in agreement with CpG-specifc pyrosequencing, while the most widely used MSP assays published by Esteller et al. (N Engl J Med 343(19):1350–1354, 2000. https://doi.org/ 10.1056/NEJM200011093431901) and Felsberg et al. (Clin Cancer Res 15(21):6683–6693, 2009. https://doi.org/10.1158/ 1078-0432.CCR-08-2801) resulted in partially discordant results in 22 tumors (27.5%). Local deep bisulfte sequencing (LDBS) revealed that CpGs located in exon 1 are suited best to discriminate methylated from unmethylated samples. Based on LDBS data, we propose an optimized MSP primer pair with 83% and 85% concordance to pyrosequencing and LDBS data. A hitherto neglected region upstream of the TSS, with an overall higher methylation compared to exon 1 and intron 1 of MGMT, is also able to discriminate the methylation status. Conclusion: Our integrated analysis allows to evaluate and redefne co-methylation domains within the MGMT pro‑ moter and to rationalize the practical impact on assays used in daily routine diagnostics

IGF2 mRNA Binding Protein 2 Transgenic Mice Are More Prone to Develop a Ductular Reaction and to Progress Toward Cirrhosis

The insulin-like growth factor 2 (IGF2) mRNA binding proteins (IMPs/IGF2BPs) IMP1 and 3 are regarded as oncofetal proteins, whereas the hepatic IMP2 expression in adults is controversially discussed. The splice variant IMP2-2/p62 promotes steatohepatitis and hepatocellular carcinoma. Aim of this study was to clarify whether IMP2 is expressed in the adult liver and influences progression toward cirrhosis. IMP2 was expressed at higher levels in embryonic compared to adult tissues as quantified in embryonic, newborn, and adult C57BL/6J mouse livers and suggested by analysis of publicly available human data. In an IMP2-2 transgenic mouse model microarray and qPCR analyses revealed increased expression of liver progenitor cell (LPC) markers Bex1, Prom1, Spp1, and Cdh1 indicating a de-differentiated liver cell phenotype. Induction of these LPC markers was confirmed in human cirrhotic tissue datasets. The LPC marker SPP1 has been described to play a major role in fibrogenesis. Thus, DNA methylation was investigated in order to decipher the regulatory mechanism of Spp1 induction. In IMP2-2 transgenic mouse livers single CpG sites were differentially methylated, as quantified by amplicon sequencing, whereas human HCC samples of a human publicly available dataset showed promoter hypomethylation. In order to study the impact of IMP2 on fibrogenesis in the context of steatohepatitis wild-type or IMP2-2 transgenic mice were fed either a methionine-choline deficient (MCD) or a control diet for 2-12 weeks. MCD-fed IMP2-2 transgenic mice showed a higher incidence of ductular reaction (DR), accompanied by hepatic stellate cell activation, extracellular matrix (ECM) deposition, and induction of the LPC markers Spp1, Cdh1, and Afp suggesting the occurrence of de-differentiated cells in transgenic livers. In human cirrhotic samples IMP2 overexpression correlated with LPC marker and ECM component expression. Progression of liver disease was induced by combined MCD and diethylnitrosamine (DEN) treatment. Combined MCD-DEN treatment resulted in shorter survival of IMP2-2 transgenic compared to wild-type mice. Only IMP2-2 transgenic livers progressed to cirrhosis, which was accompanied by strong DR. In conclusion, IMP2 is an oncofetal protein in the liver that promotes DR characterized by de-differentiated cells toward steatohepatitis-associated cirrhosis development with poor survival

DNA methylation-based prediction of response to immune checkpoint inhibition in metastatic melanoma

Background Therapies based on targeting immune checkpoints have revolutionized the treatment of metastatic melanoma in recent years. Still, biomarkers predicting long-term therapy responses are lacking. Methods A novel approach of reference-free deconvolution of large-scale DNA methylation data enabled us to develop a machine learning classifier based on CpG sites, specific for latent methylation components (LMC), that allowed for patient allocation to prognostic clusters. DNA methylation data were processed using reference-free analyses (MeDeCom) and reference-based computational tumor deconvolution (MethylCIBERSORT, LUMP). Results We provide evidence that DNA methylation signatures of tumor tissue from cutaneous metastases are predictive for therapy response to immune checkpoint inhibition in patients with stage IV metastatic melanoma. Conclusions These results demonstrate that LMC-based segregation of large-scale DNA methylation data is a promising tool for classifier development and treatment response estimation in cancer patients under targeted immunotherapy