{BiQ} Analyzer {HiMod}: An Interactive Software Tool for High-throughput Locus-specific Analysis of 5-Methylcytosine and its Oxidized Derivatives

Recent data suggest important biological roles for oxidative modifications of methylated cytosines, specifically hydroxymethylation, formylation and carboxylation. Several assays are now available for profiling these DNA modifications genome-wide as well as in targeted, locus-specific settings. Here we present BiQ Analyzer HiMod, a user-friendly software tool for sequence alignment, quality control and initial analysis of locus-specific DNA modification data. The software supports four different assay types, and it leads the user from raw sequence reads to DNA modification statistics and publication-quality plots. BiQ Analyzer HiMod combines well-established graphical user interface of its predecessor tool, BiQ Analyzer HT, with new and extended analysis modes. BiQ Analyzer HiMod also includes updates of the analysis workspace, an intuitive interface, a custom vector graphics engine and support of additional input and output data formats. The tool is freely available as a stand-alone installation package from http://biq-analyzer-himod.bioinf.mpi-inf.mpg.de/

Bone marrow MRI after autologous transplantation and the effect of residual tumor on progression-free survival of multiple myeloma patients

Background . The study of influence of residual tumor mass, determined by magnetic resonance imaging (MRI), on the progression-free survival (PFS) remains an actual problem. Since the visual assessment of tumor bone marrow lesion can be one of the criteria for the subsequent personalized treatment choice in multiple myeloma patients.The objective of study was to determine the effect of bone marrow lesions detected by MRI after autologous hematopoietic stem cells transplantation (auto-HSCT) on PFS in multiple myeloma patients.Materials and methods . The prospective study included 60 patients who underwent spine and pelvic bones MRI on the 100 th day after autoHSCT.Results . Focal bone marrow changes were found in 47 of them – from 1 to 56 lesions (mean 6 ± 9). Significant (p = 0.01) differences of PFS in multiple myeloma patients depending on the presence or absence of tumor mass on 100 th day after auto-HSCT were revealed: with MRI negative status, 2-year PFS was 89 % versus 50 % in a group of patients with residual tumor mass.Conclusion . MRI-negative status after auto-HSCT is a favorable prognostic factor contributing to prolonged disease-free survival

Epigenetic modulation of radiation-induced diacylglycerol kinase alpha expression prevents pro-fibrotic fibroblast response

Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis

In Vivo Control of CpG and Non-CpG DNA Methylation by DNA Methyltransferases

The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position–, cell type–, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs

Versatile workflow for cell-type resolved transcriptional and epigenetic profiles from cryopreserved human lung

Complexity of lung microenvironment and changes in cellular composition during disease make it exceptionally hard to understand molecular mechanisms driving development of chronic lung diseases. Although recent advances in cell type–resolved approaches hold great promise for studying complex diseases, their implementation relies on local access to fresh tissue, as traditional tissue storage methods do not allow viable cell isolation. To overcome these hurdles, we developed a versatile workflow that allows storage of lung tissue with high viability, permits thorough sample quality check before cell isolation, and befits sequencing-based profiling. We demonstrate that cryopreservation enables isolation of multiple cell types from both healthy and diseased lungs. Basal cells from cryopreserved airways retain their differentiation ability, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing and EPIC Array, we show that gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for omics profiling of lung cells. Moreover, we obtained high-quality single-cell RNA-sequencing data of cells from cryopreserved human lungs, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our workflow is well suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to viable human tissue

Globally altered epigenetic landscape and delayed osteogenic differentiation in H3.3-G34W-mutant giant cell tumor of bone

The neoplastic stromal cells of giant cell tumor of bone (GCTB) carry a mutation in H3F3A, leading to a mutant histone variant, H3.3-G34W, as a sole recurrent genetic alteration. We show that in patient-derived stromal cells H3.3-G34W is incorporated into the chromatin and associates with massive epigenetic alterations on the DNA methylation, chromatin accessibility and histone modification level, that can be partially recapitulated in an orthogonal cell line system by the introduction of H3.3-G34W. These epigenetic alterations affect mainly heterochromatic and bivalent regions and provide possible explanations for the genomic instability, as well as the osteolytic phenotype of GCTB. The mutation occurs in differentiating mesenchymal stem cells and associates with an impaired osteogenic differentiation. We propose that the observed epigenetic alterations reflect distinct differentiation stages of H3.3 WT and H3.3 MUT stromal cells and add to H3.3-G34W-associated changes