Julian Gutekunst

Chronic Inflammation Induces a Novel Epigenetic Program That Is Conserved in Intestinal Adenomas and in Colorectal Cancer

Chronic inflammation represents a major risk factor for tumor formation, but the underlying mechanisms have remained largely unknown. Epigenetic mechanisms can record the effects of environmental challenges on the genome level and could therefore play an important role in the pathogenesis of inflammation-associated tumors. Using single-base methylation maps and transcriptome analyses of a colitis-induced mouse colon cancer model, we identified a novel epigenetic program that is characterized by hypermethylation of DNA methylation valleys that are characterized by low CpG density and active chromatin marks. This program is conserved and functional in mouse intestinal adenomas and results in silencing of active intestinal genes that are involved in gastrointestinal homeostasis and injury response. Further analyses reveal that the program represents a prominent feature of human colorectal cancer and can be used to correctly classify colorectal cancer samples with high accuracy. Together, our results show that inflammatory signals establish a novel epigenetic program that silences a specific set of genes that contribute to inflammation-induced cellular transformation.

De Novo assembly and annotation of the freshwater crayfish Astacus astacus transcriptome

We generated RNA-seq data to assemble the transcriptome of the noble crayfish (Astacus astacus) from four combined tissues (abdominal muscle, hepatopancreas, ovaries, green glands). A total of 194 million read pairs with a length of 100 bp were generated. The transcriptome was assembled de novo using Trinity software, producing 158,649 non-redundant transcripts. Lowly expressed transcripts were filtered out leaving 45,415 transcripts of which 14,559 were found to contain open reading frames with predicted gene function. The Transrate software revealed that 91% of the total reads were realigned to the assembly. Furthermore, BUSCO analysis indicated that our assembly is 64% complete. A total of 13,770 transcripts were assigned at least one GO term. This first de novo transcriptome assembly is an important foundation for future genomic research on the noble crayfish and adds to the general knowledge and further characterization of transcriptomes of non-model organisms.

Reduced DNA methylation patterning and transcriptional connectivity define human skin aging

Epigenetic changes represent an attractive mechanism for understanding the phenotypic changes associated with human aging. Age‐related changes in DNA methylation at the genome scale have been termed ‘epigenetic drift’, but the defining features of this phenomenon remain to be established. Human epidermis represents an excellent model for understanding age‐related epigenetic changes because of its substantial cell‐type homogeneity and its well‐known age‐related phenotype. We have now generated and analyzed the currently largest set of human epidermis methylomes (N = 108) using array‐based profiling of 450 000 methylation marks in various age groups. Data analysis confirmed that age‐related methylation differences are locally restricted and characterized by relatively small effect sizes. Nevertheless, methylation data could be used to predict the chronological age of sample donors with high accuracy. We also identified discontinuous methylation changes as a novel feature of the aging methylome. Finally, our analysis uncovered an age‐related erosion of DNA methylation patterns that is characterized by a reduced dynamic range and increased heterogeneity of global methylation patterns. These changes in methylation variability were accompanied by a reduced connectivity of transcriptional networks. Our findings thus define the loss of epigenetic regulatory fidelity as a key feature of the aging epigenome.

Clonal genome evolution and rapid invasive spread of the marbled crayfish

The marbled crayfish Procambarus virginalis is a unique freshwater crayfish characterized by very recent speciation and parthenogenetic reproduction. Marbled crayfish also represent an emerging invasive species and have formed wild populations in diverse freshwater habitats. However, our understanding of marbled crayfish biology, evolution and invasive spread has been hampered by the lack of freshwater crayfish genome sequences. We have now established a de novo draft assembly of the marbled crayfish genome. We determined the genome size at approximately 3.5 gigabase pairs and identified >21,000 genes. Further analysis confirmed the close relationship to the genome of the slough crayfish, Procambarusfallax, and also established a triploid AA’B genotype with a high level of heterozygosity. Systematic fieldwork and genotyping demonstrated the rapid expansion of marbled crayfish on Madagascar and established the marbled crayfish as a potent invader of freshwater ecosystems. Furthermore, comparative whole-genome sequencing demonstrated the clonality of the population and their genetic identity with the oldest known stock from the German aquarium trade. Our study closes an important gap in the phylogenetic analysis of animal genomes and uncovers the unique evolutionary history of an emerging invasive species.The marbled crayfish is an emerging invasive species in freshwater habitats. Here, the authors sequence the genome of the marbled crayfish and show that evolution of this decapod crustacean involved genome duplication, triploidy and clonal expansion.

Impact of DLK1-DIO3 imprinted cluster hypomethylation in smoker patients with lung cancer

DNA methylation is important for gene expression and genome stability, and its disruption is thought to play a key role in the initiation and progression of cancer and other diseases. The DLK1-DIO3 cluster has been shown to be imprinted in humans, and some of its components are relevant to diverse pathological processes. The purpose of this study was to assess the methylation patterns of the DLK1-DIO3 cluster in patients with lung cancer to study its relevance in the pathogenesis of this disease. We found a characteristic methylation pattern of this cluster in smoking associated lung cancer, as compared to normal lung tissue. This methylation profile is not patent however in lung cancer of never smokers nor in lung tissue of COPD patients. We found 3 deregulated protein-coding genes at this locus: one was hypermethylated (DIO3) and two were hypomethylated (DLK1 and RTL1). Statistically significant differences were also detected in two different families of SNORDs, two miRNA clusters and four lncRNAs (MEG3, MEG8, MEG9 and LINC00524). These findings were validated using data from the cancer genome atlas (TCGA) database. We have then showed an inverse correlation between DNA methylation and expression levels in 5 randomly selected genes. Several targets of miRNAs included in the DLK1-DIO3 cluster have been experimentally verified as tumor suppressors. All of these results suggest that the dysmethylation of the imprinted DLK1-DIO3 cluster could have a relevant role in the pathogenesis of lung cancer in current and former smokers and may be used for diagnostic and/or therapeutic purposes.

Epigenetically Regulated Chromosome 14q32 miRNA Cluster Induces Metastasis and Predicts Poor Prognosis in Lung Adenocarcinoma Patients

Most lung cancer deaths are related to metastases, which indicates the necessity of detecting and inhibiting tumor cell dissemination. Here, we aimed to identify miRNAs involved in metastasis of lung adenocarcinoma as prognostic biomarkers and therapeutic targets. To that end, lymph node metastasis–associated miRNAs were identified in The Cancer Genome Atlas lung adenocarcinoma patient cohort (sequencing data; n = 449) and subsequently validated by qRT-PCR in an independent clinical cohort (n = 108). Overexpression of miRNAs located on chromosome 14q32 was associated with metastasis in lung adenocarcinoma patients. Importantly, Kaplan–Meier analysis and log-rank test revealed that higher expression levels of individual 14q32 miRNAs (mir-539, mir-323b, and mir-487a) associated with worse disease-free survival of never-smoker patients. Epigenetic analysis including DNA methylation microarray data and bisulfite sequencing validation demonstrated that the induction of 14q32 cluster correlated with genomic hypomethylation of the 14q32 locus. CRISPR activation technology, applied for the first time to functionally study the increase of clustered miRNA levels in a coordinated manner, showed that simultaneous overexpression of 14q32 miRNAs promoted tumor cell migratory and invasive properties. Analysis of individual miRNAs by mimic transfection further illustrated that miR-323b-3p, miR-487a-3p, and miR-539-5p significantly contributed to the invasive phenotype through the indirect regulation of different target genes. In conclusion, overexpression of 14q32 miRNAs, associated with the respective genomic hypomethylation, promotes metastasis and correlates with poor patient prognosis in lung adenocarcinoma. Implications: This study points to chromosome 14q32 miRNAs as promising targets to inhibit tumor cell dissemination and to predict patient prognosis in lung adenocarcinoma. Mol Cancer Res; 16(3); 390–402. ©2018 AACR.

Methylation profiling identifies two subclasses of squamous cell carcinoma related to distinct cells of origin

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer and usually progresses from a UV-induced precancerous lesion termed actinic keratosis (AK). Despite various efforts to characterize these lesions molecularly, the etiology of AK and its progression to cSCC remain partially understood. Here, we use Infinium MethylationEPIC BeadChips to interrogate the DNA methylation status in healthy, AK and cSCC epidermis samples. Importantly, we show that AK methylation patterns already display classical features of cancer methylomes and are highly similar to cSCC profiles. Further analysis identifies typical features of stem cell methylomes, such as reduced DNA methylation age, non-CpG methylation, and stem cell-related keratin and enhancer methylation patterns. Interestingly, this signature is detected only in half of the samples, while the other half shows patterns more closely related to healthy epidermis. These findings suggest the existence of two subclasses of AK and cSCC emerging from distinct keratinocyte differentiation stages.Cutaneous squamous cell carcinoma (cSCC) is a skin cancer that normally progresses from UV-induced actinic keratosis (AK). Here, the authors investigate the epigenomics of cSCC and highlight two distinct subclasses of AK and cSCC originating from distinct keratinocyte differentiation stages.

mIDH -associated DNA hypermethylation in acute myeloid leukemia reflects differentiation blockage rather than inhibition of TET-mediated demethylation

Isocitrate dehydrogenases 1 and 2 (IDH1/2) are recurrently mutated in acute myeloid leukemia (AML), but their mechanistic role in leukemogenesis is poorly understood. The inhibition of TET enzymes by D-2-hydroxyglutarate (D-2-HG), which is produced by mutant IDH1/2 (mIDH1/2), has been suggested to promote epigenetic deregulation during tumorigenesis. In addition, mIDH also induces a differentiation block in various cell culture and mouse models. Here we analyze the genomic methylation patterns of AML patients with mIDH using Infinium 450K data from a large AML cohort and found that mIDH is associated with pronounced DNA hypermethylation at tens of thousands of CpGs. Interestingly, however, myeloid leukemia cells overexpressing mIDH, cells that were cultured in the presence of D-2-HG or TET2 mutant AML patients did not show similar methylation changes. In further analyses, we also characterized the methylation landscapes of myeloid progenitor cells and analyzed their relationship to mIDH-associated hypermethylation. Our findings identify the differentiation state of myeloid cells, rather than inhibition of TET-mediated DNA demethylation, as a major factor of mIDH-associated hypermethylation in AML. Furthermore, our results are also important for understanding the mode of action of currently developed mIDH inhibitors.

Transforming growth factor β1-mediated functional inhibition of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia

Mesenchymal stromal cells are involved in the pathogenesis of myelodysplastic syndromes and acute myeloid leukemia, but the underlying mechanisms are incompletely understood. To further characterize the pathological phenotype we performed RNA sequencing of mesenchymal stromal cells from patients with myelodysplastic syndromes and acute myeloid leukemia and found a specific molecular signature of genes commonly deregulated in these disorders. Pathway analysis showed a strong enrichment of genes related to osteogenesis, senescence, inflammation and inhibitory cytokines, thereby reflecting the structural and functional deficits of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia on a molecular level. Further analysis identified transforming growth factor β1 as the most probable extrinsic trigger factor for this altered gene expression. Following exposure to transforming growth factor β1, healthy mesenchymal stromal cells developed functional deficits and adopted a phenotype reminiscent of that observed in patient-derived stromal cells. These suppressive effects of transforming growth factor β1 on stromal cell functionality were abrogated by SD-208, an established inhibitor of transforming growth factor β receptor signaling. Blockade of transforming growth factor β signaling by SD-208 also restored the osteogenic differentiation capacity of patient-derived stromal cells, thus confirming the role of transforming growth factor β1 in the bone marrow microenvironment of patients with myelodysplastic syndromes and acute myeloid leukemia. Our findings establish transforming growth factor β1 as a relevant trigger causing functional inhibition of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia and identify SD-208 as a candidate to revert these effects.

The methylome of the marbled crayfish links gene body methylation to stable expression of poorly accessible genes

BackgroundThe parthenogenetic marbled crayfish (Procambarus virginalis) is a novel species that has rapidly invaded and colonized various different habitats. Adaptation to different environments appears to be independent of the selection of genetic variants, but epigenetic programming of the marbled crayfish genome remains to be understood.ResultsHere, we provide a comprehensive analysis of DNA methylation in marbled crayfish. Whole-genome bisulfite sequencing of multiple replicates and different tissues revealed a methylation pattern that is characterized by gene body methylation of housekeeping genes. Interestingly, this pattern was largely tissue invariant, suggesting a function that is unrelated to cell fate specification. Indeed, integrative analysis of DNA methylation, chromatin accessibility and mRNA expression patterns revealed that gene body methylation correlated with limited chromatin accessibility and stable gene expression, while low-methylated genes often resided in chromatin with higher accessibility and showed increased expression variation. Interestingly, marbled crayfish also showed reduced gene body methylation and higher gene expression variability when compared with their noninvasive mother species, Procambarus fallax.ConclusionsOur results provide novel insights into invertebrate gene body methylation and its potential role in adaptive gene regulation.