Biology subjects

Manke, T.

Publications and source records attributed to Manke, T..

16 recordsLinked to original sources

bioRxiv2026

ATACofthesnake: A scalable framework for analyzing multifactorial and time course chromatin accessibility data

Advances in sample extraction techniques, improvement of library preparation methods and the dramatic reduction in sequencing costs have made complex, multifactorial experimental design the de facto standard in epigenomic studies. In contrast to the plethora of tools, workflows and libraries available to analyze gene expression data, consolidated workflows for chromatin accessibility data are lacking and analysis of such data often occurs on an ad hoc basis. Here, a scalable end-to-end workflow, ATACofthesnake, is presented that simplifies analysis of multifactorial chromatin accessibility data considerably and provides a newly created framework to handle time series data reproducibly.

bioinformatics↗

bioRxiv2026

Epigenetic perturbation unravels diverse origins, state conversions and de-differentiation of cortical astrocytes

The role of epigenetic modifiers in astrogenesis is less understood compared to neurogenesis. For example, the Disruptor of telomeric silencing 1 like, DOT1L, conferring H3K79 methylation safeguards neural progenitors from premature differentiation in various brain regions, however, its role in astrogenesis has not yet been reported. Here, we decipher its role during astrogenesis using an Emx1-cre driven DOT1L conditional knock-out during the development of mouse cerebral cortex from E12.5 till P0. We describe two astrocyte types at P0, distinguishable by expression of phagocytosis markers genes. DOT1L loss-of-function (LOF) increased the number of those astrocytes that express fewer phagocytosis marker genes, while it affects a larger number of differentially expressed genes in those astrocytes that strongly express homeostasis marker genes. Our findings establish DOT1L as a barrier between these astrocyte states, limiting differentiation of the astrocytes expressing phagocytosis marker genes. We also identify multiple developmental origins of astrocytes. In addition to apical progenitors (AP), the prevailing stem cell population considered as origin of cortical astrocytes, we identify neural stem cells (NSC) in the dorsal telencephalon differentiating towards astrocytes, and Nkx2.1-lineage derived astrocytes, originating from the ventral telencephalon. This finding refines the prevailing view that dorsal telencephalon APs are the only source of embryonic cortical astrocytes, and enter astrogliogenesis after neurogenesis. We show that the expression of Emx1 discriminates different astrocyte origins during cortical development, and we describe a new set of transcription factors (TFs) that mark the state conversion between the homeostasis or phagocytosis expression programs in astrocytes. DOT1L orchestrates different TF networks, of which Lhx2, Nfia, Sox6, and Creb5 drive astrogliogenesis, and Nfia, Nfib, and Tcf4 affect astrocyte state conversion. Importantly, our analysis also shows that NSCs, APs and astrocytes de-differentiate within this lineage trajectory, and lowering the DOT1L-mediated epigenetic barrier strongly favours appearance of astrocytes with limited phagocytosis properties and reactivation of progenitor programs.

neuroscience↗

bioRxiv2024

Enhancing single-cell ATAC sequencing with formaldehyde fixation, cryopreservation, and multiplexing for flexible analysis

The assay for transposase-accessible chromatin using sequencing (ATAC-seq) revolutionized the field of epigenetics since its emergence by providing a means to uncover chromatin dynamics and other factors affecting gene expression. The development of single-cell (sc) applications in recent years led to an even deeper understanding of cell type specific gene regulatory mechanisms. One of the major challenges while running ATAC-seq experiments, bulk or sc, is the need for freshly collected cells for successful experiments. While various freezing methods have already been tested and established for bulk and sc ATAC-seq, quality metrics for preserved cells are rather poor or dependent on sampling time when compared to fresh samples. This makes it difficult to conduct all sorts of complex experiments i.e. with multiple conditions, patients, or time course studies. Especially, accounting for batch effects can be difficult if samples need to be processed at different time points of collection. We tackled this issue by adding a fixation step prior to the freezing method. The additional fixation step improved library quality and yield data comparable to fresh samples. The workflow was also tested on multiplexed sc ATAC experiments, set-up for cost-efficient low input sample handling. Sample cross-in, typically encountered in Tn5-based multiplex approaches, were tackled with a computational procedure specifically developed for this approach.

genomics↗

bioRxiv2023

Alternative mRNA splicing controls the functions of the histone H3K27 demethylase UTX/KDM6A

BackgroundThe UTX/KDM6A histone H3K27 demethylase plays an important role in development and is frequently mutated in cancers such as urothelial cancer. Despite many studies on UTX proteins, variations in mRNA splicing have been overlooked. MethodsUsing Nanopore sequencing, we present a comprehensive analysis of UTX/KDM6A splicing events in human cell lines and in tissue samples from bladder cancer and normal epithelium. ResultsThe central region of UTX mRNAs encoded by exons 12 to 17 undergoes extensive alternative splicing. Up to half of all stable mRNAs (8-48% in bladder tissues and 18-58% in cell lines) are represented by the UTX canonical isoform lacking exon 14 encoding a nuclear localization sequence, hence localize to the nucleus, unlike cytonuclear localization of the canonical isoform. Chromatin association was also higher for exon 14-containing isoform compared to the canonical UTX. Using quantitative mass spectrometry, we found that all UTX isoforms integrated into the MLL3 and MLL4, PR-DUB and MiDAC complexes. Interestingly, one of the novel UTX isoforms, which lacks exons 14 and 16, fails to interact with PR-DUB and MiDAC complex members. ConclusionUTX mRNAs undergo extensive alternative splicing that controls the subcellular localization of UTX and its interactions with other chromatin regulatory complexes. Simple SummaryUTX/KDM6A is a histone H3K27 demethylase and plays an important role in mammalian development and human diseases such as urothelial cancer. We identified a region encompassing exons 12-17 of UTX that undergoes extensive splicing events. As a result, a nuclear localization sequence located in exon14 is missing in a considerable part of UTX transcripts in different cell lines and tissues from normal bladder epithelium and bladder cancer. Mass spectrometry analysis showed a role for this region in binding to the epigenetic PR-DUB and MiDAC complexes. UTX was also more extensively bound to chromatin when the alternative splicing region presented. Our study showed that alternative splicing of UTX transcripts plays an important role in its functions.

biochemistry↗

bioRxiv2021

Integrative multi-omics analyses reveal multi-modal FOXG1 functions acting on epigenetic processes and in concert with NEUROD1 to regulate synaptogenesis in the mouse hippocampus

BackgroundFOXG1 has important functions for neuronal differentiation and balances excitatory/inhibitory network activity. Mutations in the human FOXG1 gene cause a rare neurodevelopmental disorder, FOXG1-syndrome, which manifests differing phenotypes, including severe cognitive dysfunction, microencephaly, social withdrawal, and communication and memory deficits. Changes at the molecular level underlying these functional abnormalities upon FOXG1 haploinsufficiency are largely unexplored, in human patients as well as in animals modelling the debilitating disease. MethodsWe present multi-omics data and explore comprehensively how FOXG1 impacts neuronal maturation at the chromatin level in the adult mouse hippocampus. We used RNA-, ATAC- and ChIP-sequencing of primary hippocampal neurons and co-immunoprecipitation to explore various levels of epigenetic changes and transcription factor networks acting to alter neuronal differentiation upon reduction of FOXG1. ResultsWe provide the first comprehensive multi-omics data set exploring FOXG1 presence at the chromatin and identifying the consequences of reduced FOXG1 expression in primary hippocampal neurons. Analyzing the multi-omics data, our study reveals that FOXG1 uses various different ways to regulate transcription at the chromatin level. On a genome-wide level, FOXG1 (i) both represses and activates transcription, (ii) binds mainly to enhancer regions, and (iii) bidirectionally alters the epigenetic landscape in regard to levels of H3K27ac, H3K4me3, and chromatin accessibility. Genes affected by the chromatin alterations upon FOXG1 reduction impact synaptogenesis and axonogenesis. This finding emphasizes the importance of FOXG1 to integrate and coordinate transcription of genes necessary for proper neuronal function by acting on a genome-wide level. Interestingly, FOXG1 acts through histone deacetylases (HDACs) and inhibition of HDACs partly rescued transcriptional alterations observed upon FOXG1 reduction. On a more detailed level of analysis, we show that FOXG1 (iv) operates synergistically with NEUROD1. Interestingly, we could not detect a clear hierarchy of these two key transcription factors, but instead provide first evidence that they act in highly concerted and orchestrated manner to control neuronal differentiation. ConclusionsThis integrative and multi-omics view of changes upon FOXG1 reduction reveals an unprecedented multimodality of FOXG1 functions converging on neuronal maturation, fueling novel therapeutic options based on epigenetic drugs to alleviate, at least in part, neuronal dysfunctions.

neuroscience↗

bioRxiv2020

AutoRELACS: Automated Generation And Analysis Of Ultra-parallel ChIP-seq

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a method used to profile protein-DNA interactions genome-wide. RELACS (Restriction Enzyme-based Labeling of Chromatin in Situ) is a recently developed ChIP-seq protocol that deploys a chromatin barcoding strategy to enable standardized and high-throughput generation of ChIP-seq data. The manual implementation of RELACS is constrained by human processivity in both data generation and data analysis. To overcome these limitations, we have developed AutoRELACS, an automated implementation of the RELACS protocol using the liquid handler Biomek i7 workstation. We match the unprecedented processivity in data generation allowed by AutoRELACS with the automated computation pipelines offered by snakePipes. In doing so, we build a continuous workflow that streamlines epigenetic profiling, from sample collection to biological interpretation. Here, we show that AutoRELACS successfully automates chromatin barcode integration, and is able to generate high-quality ChIP-seq data comparable with the standards of the manual protocol, also for limited amounts of biological samples.

genomics↗

bioRxiv2020

DOT1L Methyltransferase Activity Preserves SOX2-Enhancer Accessibility And Prevents Activation of Repressed Genes In Murine Stem Cells

BackgroundDuring cellular differentiation, the chromatin landscape changes dynamically and contributes to the activation of cell-type specific transcriptional programs. Disruptor of telomeric silencing 1-like (DOT1L) is a histone methyltransferase that mediates mono-, di- and trimethylation of lysine 79 of histone H3 (H3K79me1, 2, 3). Its enzymatic activity is critical for driving cellular differentiation into cardiomyocytes, chondrocytes and neurons, from embryonic or other type of stem cells in physiological settings. Ectopic localization of DOT1L in MLL-rearranged leukemias is causative for leukemogenesis and relapse. Little is known about the causal relevance of DOT1L methyltransferase activity in the global chromatin context and how its enzymatic function affects transcriptional and global chromatin states. Recent reports conducted in leukemia cell models have suggested that deposition of H3K79me2 may be critical to preserve histone H3K27 acetylation (ac) and enhancer activity, and to sustain expression of highly transcribed genes. If and to what extent DOT1L affects chromatin states and enhancer activity during physiological differentiation processes is currently unknown. ResultsWe measure global changes of seven histone modifications during the differentiation process via high-throughput and quantitative ChIP-seq in an in-vitro neuronal differentiation model of mouse embryonic stem cells (mESC). We observe that H3K27ac globally decreases, whereas H3K79me2 globally increases during differentiation, while other modifications remain globally unaltered. Pharmacological inhibition of DOT1L in mESC and mESC-derived neural progenitors results in decreased expression of highly transcribed genes and increased expression of normally repressed genes. Acute DOT1L inhibition primes neural progenitors towards a mature differentiation state. Transcriptional downregulation associates with decreased accessibility of enhancers specifically bound by the master regulator SOX2. ConclusionsIn-vitro neuronal differentiation couples with a genome-wide accumulation of H3K79me2, never described previously in mammalian cells. Acute inhibition of DOT1L is sufficient to initiate a defined transcriptional program, which biases the transcriptome of neural progenitor cells towards neuronal differentiation. H3K79me2 is not generally causative for maintaining transcriptional levels at a genome-wide scale. In contrast, DOT1L inactivation reduces the chromatin accessibility of enhancers bound by SOX2 in-vivo, thereby reducing the expression level of a restricted number of genes. Our work establishes that DOT1L activity gates differentiation of progenitors by allowing SOX2-dependent transcription of stemness programs.

genomics↗

bioRxiv2018

CRUP: A comprehensive framework to predict condition-specific regulatory units

We present the software CRUP (Condition-specific Regulatory Units Prediction) to infer from epigenetic marks a list of regulatory units consisting of dynamically changing enhancers with their target genes. The workflow consists of a novel pre-trained enhancer predictor that can be reliably applied across cell lines and species, solely based on histone modification ChIP-seq data. Enhancers are subsequently assigned to different conditions and correlated with gene expression to derive regulatory units. We thoroughly test and then apply CRUP to a rheumatoid arthritis model, identifying enhancer-gene pairs comprising known disease genes as well as new candidate genes.\n\nAvailabilityhttps://github.com/VerenaHeinrich/CRUP

bioinformatics↗

bioRxiv2018

snakePipes enable flexible, scalable and integrative epigenomic analysis

The scale and diversity of epigenomics data has been rapidly increasing and ever more studies now present analyses of data from multiple epigenomic techniques. Performing such integrative analysis is time-consuming, especially for exploratory research, since there are currently no pipelines available that allow fast processing of datasets from multiple epigenomic assays while also allow for flexibility in running or upgrading the workflows. Here we present a solution to this problem: snakePipes, which can process and perform downstream analysis of data from all common epigenomic techniques (ChIP-seq, RNA-seq, Bisulfite-seq, ATAC-seq, Hi-C and single-cell RNA-seq) in a single package. We demonstrate how snakePipes can simplify integrative analysis by reproducing and extending the results from a recently published large-scale epigenomics study with a few simple commands. snakePipes are available under an open-source license at https://github.com/maxplanck-ie/snakepipes.

genomics↗

bioRxiv2018

Parkour LIMS: facilitating high-quality sample preparation in next generation sequencing

SummaryThis paper presents Parkour, a software package for sample processing and quality management of next generation sequencing data and samples. Starting with user requests, Parkour allows tracking and assessing samples based on predefined quality criteria through different stages of the sample preparation workflow. Ideally suited for academic core laboratories, the software aims to maximize efficiency and reduce turnaround time by intelligent sample grouping and a clear assignment of staff to work units. Tools for automated invoicing, interactive statistics on facility usage and simple report generation minimize administrative tasks. Provided as a web application, Parkour is a convenient tool for both deep sequencing service users and laboratory personal. A set of web APIs allow coordinated information sharing with local and remote bioinformaticians. The flexible structure allows workflow customization and simple addition of new features as well as the expansion to other domains.\n\nAvailability and implementationThe code and documentation are available at https://github.com/maxplanck-ie/parkour\n\nContactboenisch@ie-freiburg.mpg.de

genomics↗

bioRxiv2018

Integrative analysis of single cell expression data reveals distinct regulatory states in bidirectional promoters

BackgroundBidirectional promoters (BPs) are prevalent in eukaryotic genomes. However, it is poorly understood how the cell integrates different epigenomic information, such as transcription factor (TF) binding and chromatin marks, to drive gene expression at BPs. Single cell sequencing technologies are revolutionizing the field of genome biology. Therefore, this study focuses on the integration of single cell RNA-seq data with bulk ChIP-seq and other epigenetics data, for which single cell technologies are not yet established, in the context of BPs.\n\nResultsWe performed integrative analyses of novel human single cell RNA-seq (scRNA-seq) data with bulk ChIP-seq and other epigenetics data. scRNA-seq data revealed distinct transcription states of BPs that were previously not recognized. We find associations between these transcription states to distinct patterns in structural gene features, DNA accessibility, histone modification, DNA methylation and TF binding profiles.\n\nConclusionsOur results suggest that a complex interplay of all of these elements is required to achieve BP-specific transcriptional output in this specialized promoter configuration. Further, our study implies that novel statistical methods can be developed to deconvolute masked subpopulations of cells measured with different bulk epigenomic assays using scRNA-seq data.

genomics↗

bioRxiv2018

Ultra-parallel ChIP-seq by barcoding of intact nuclei

Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is an invaluable tool for mapping chromatin-associated proteins. However, sample preparation is still a largely individual and labor-intensive process that hinders assay throughput and comparability. Here, we present a novel method for ultra-parallelized high-throughput ChIP-seq that addresses the aforementioned problems. The method, called RELACS (Restriction Enzyme-based Labeling of Chromatin in Situ), employs barcoding of chromatin within intact nuclei extracted from different sources (e.g. tissues, treatments, time points). Barcoded nuclei are pooled and processed within the same ChIP, for maximal comparability and significant workload reduction. The choice of user-friendly, straightforward, enzymatic steps for chromatin fragmentation and barcoding makes RELACS particularly suitable for implementation large-scale clinical studies and scarce samples. RELACS can generate ChIP-seq libraries from hundreds of samples within three days and with less than 1000 cells per sample.

genomics↗

bioRxiv2018

Partially methylated domains are hallmarks of a cell specific epigenome topology

BackgroundPartially methylated domains, PMDs, are extended regions in the genome exhibiting a reduced average DNA-methylation level. PMDs cover gene-poor and transcriptionally inactive regions and tend to be heterochromatic. Here, we present a first comprehensive comparative analysis of PMDs across more than 190 WGBS methylomes of human and mouse cells providing a deep insight into structural and functional features associated with PMDs.\n\nResultsPMDs are ubiquitous signatures covering up to 75% of the genome in human and mouse cells irrespective of their tissue or cell origin. Additionally, each cell type comes with a distinct set of specific PMDs, and genes expressed in such PMDs show a strong cell type effect. Demethylation strength varies in PMDs with a tendency towards a more pronounced effect in differentiating and replicating cells. The strongest demethylation is observed in highly proliferating and immortal cancer cell lines. A decrease of DNA-methylation within PMDs tends to be linked to an increase in heterochromatic histone marks and a decrease of gene expressions. Characteristic combinations of heterochromatic signatures in PMDs are linked to domains of early, middle and late DNA-replication.\n\nConclusionPMDs are prominent signatures of long-range epigenomic organization. Integrative analysis identifies PMDs as important general, lineage- and cell-type specific topological features. PMD changes are hallmarks of cell differentiation. Demethylation of PMDs combined with increased heterochromatic marks is a feature linked to enhanced cell proliferation. In combination with broad histone marks PMDs demarcate distinct domains of late DNA-replication.

bioinformatics↗

bioRxiv2017

Community-driven data analysis training for biology

The primary problem with the explosion of biomedical datasets is not the data itself, not computational resources, and not the required storage space, but the general lack of trained and skilled researchers to manipulate and analyze these data. Eliminating this problem requires development of comprehensive educational resources. Here we present a community-driven framework that enables modern, interactive teaching of data analytics in life sciences and facilitates the development of training materials. The key feature of our system is that it is not a static but a continuously improved collection of tutorials. By coupling tutorials with a web-based analysis framework, biomedical researchers can learn by performing computation themselves through a web-browser without the need to install software or search for example datasets. Our ultimate goal is to expand the breadth of training materials to include fundamental statistical and data science topics and to precipitate a complete re-engineering of undergraduate and graduate curricula in life sciences.

bioinformatics↗

bioRxiv2017

High-resolution TADs reveal DNA sequences underlying genome organization in flies

Eukaryotic chromatin is partitioned into domains called TADs that are broadly conserved between species and virtually identical among cell types within the same species. Previous studies in mammals have shown that the DNA binding protein CTCF and cohesin contribute to a fraction of TAD boundaries. Apart from this, the molecular mechanisms governing this partitioning remain poorly understood. Using our new software, HiCExplorer, we annotated high-resolution (570 bp) TAD boundaries in flies and identified eight DNA motifs enriched at boundaries. Known insulator proteins bind five of these motifs while the remaining three motifs are novel. We find that boundaries are either at core promoters of active genes or at non-promoter regions of inactive chromatin and that these two groups are characterized by different sets of DNA motifs. Most boundaries are present at divergent promoters of constitutively expressed genes and the gene expression tends to be coordinated within TADs. In contrast to mammals, the CTCF motif is only present on 2% of boundaries in flies. We demonstrate that boundaries can be accurately predicted using only the motif sequences, along with open chromatin, suggesting that DNA sequence encodes the 3D genome architecture in flies. Finally, we present an interactive online database to access and explore the spatial organization of fly, mouse and human genomes, available at http://chorogeome.ie-freiburg.mpg.de.

genomics↗

bioRxiv2016

normR: Regime enrichment calling for ChIP-seq data

ChIP-seq probes genome-wide localization of DNA-associated proteins. To mitigate technical biases ChIP-seq read densities are normalized to read densities obtained by a control. Our statistical framework \"normR\" achieves a sensitive normalization by accounting for the effect of putative protein-bound regions on the overall read statistics. Here, we demonstrate normRs suitability in three studies: (i) calling enrichment for high (H3K4me3) and low (H3K36me3) signal-to-ratio data; (ii) identifying two previously undescribed H3K27me3 and H3K9me3 heterochromatic regimes of broad and peak enrichment; and (iii) calling differential H3K4me3 or H3K27me3-enrichment between HepG2 hepatocarcinoma cells and primary human Hepatocytes. normR is readily available on http://bioconductor.org/packages/normr

bioinformatics↗