Dirk Schübeler
Friedrich Miescher Institute, Epigenetics, Faculty Member
Research Interests:
Research Interests:
Trimethylation of histone H3 at lysine 27 (H3K27me3) is a chromatin mark associated with Polycomb-mediated gene repression. Despite its critical role in development, it remains largely unclear how this mark is targeted to defined loci in... more
Trimethylation of histone H3 at lysine 27 (H3K27me3) is a chromatin mark associated with Polycomb-mediated gene repression. Despite its critical role in development, it remains largely unclear how this mark is targeted to defined loci in mammalian cells. Here, we use iterative genome editing to identify small DNA sequences capable of autonomously recruiting Polycomb. We inserted 28 DNA elements at a defined chromosomal position in mouse embryonic stem cells and assessed their ability to promote H3K27me3 deposition. Combined with deletion analysis, we identified DNA elements as short as 220 nucleotides that correctly recapitulate endogenous H3K27me3 patterns. Functional Polycomb recruiter sequences are invariably CpG-rich but require protection against DNA methylation. Alternatively, their activity can be blocked by placement of an active promoter-enhancer pair in cis. Taken together, these data support the model whereby PRC2 recruitment at specific targets in mammals is positively r...
Research Interests:
In Drosophila melanogaster, dosage compensation relies on the targeting of the male-specific lethal (MSL) complex to hundreds of sites along the male X chromosome. Transcription-coupled methylation of histone H3 lysine 36 is enriched... more
In Drosophila melanogaster, dosage compensation relies on the targeting of the male-specific lethal (MSL) complex to hundreds of sites along the male X chromosome. Transcription-coupled methylation of histone H3 lysine 36 is enriched toward the 3' end of active genes, similar to the MSL proteins. Here, we have studied the link between histone H3 methylation and MSL complex targeting using RNA interference and chromatin immunoprecipitation. We show that trimethylation of histone H3 at lysine 36 (H3K36me3) relies on the histone methyltransferase Hypb and is localized promoter distal at dosage-compensated genes, similar to active genes on autosomes. However, H3K36me3 has an X-specific function, as reduction specifically decreases acetylation of histone H4 lysine 16 on the male X chromosome. This hypoacetylation is caused by compromised MSL binding and results in a failure to increase expression twofold. Thus, H3K36me3 marks the body of all active genes yet is utilized in a chromoso...
Research Interests: Cellular Biology, Drosophila melanogaster, Transcription Factors, Biological Sciences, RNA interference, and 14 moreCell line, Molecular and cellular biology, Methylation, Female, Animals, Male, Sex Determination, Histones, Lysine, Chromatin Immunoprecipitation, X chromosome, Sex Chromosome, Histone Acetylation, and Dosage compensation
The posttranslational modification of histones by acetylation or methylation regulates chromatin structure and can determine how a DNA sequence is interpreted by, for example, the transcriptional machinery. In this issue of Cell, Pokholok... more
The posttranslational modification of histones by acetylation or methylation regulates chromatin structure and can determine how a DNA sequence is interpreted by, for example, the transcriptional machinery. In this issue of Cell, Pokholok and colleagues (2005) describe a new and reliable genome-wide microarray study of histone modifications and gene expression in yeast. Their epigenetic map can be used to guide further research on the epigenome.
Research Interests:
The regulated expression of genes during development and differentiation is influenced by the availability of regulatory proteins and accessibility of the DNA to the transcriptional apparatus. There is growing evidence that the... more
The regulated expression of genes during development and differentiation is influenced by the availability of regulatory proteins and accessibility of the DNA to the transcriptional apparatus. There is growing evidence that the transcriptional activity of genes is influenced by nuclear organization, which itself changes during differentiation. How do these changes in nuclear organization help to establish specific patterns of gene expression?
Research Interests:
We have investigated the mechanism, structural correlates, and cis-acting elements involved in chromatin opening and gene activation, using the human beta-globin locus as a model. Full transcriptional activity of the human beta-globin... more
We have investigated the mechanism, structural correlates, and cis-acting elements involved in chromatin opening and gene activation, using the human beta-globin locus as a model. Full transcriptional activity of the human beta-globin locus requires the locus control region (LCR), composed of a series of nuclease hypersensitive sites located upstream of this globin gene cluster. Our previous analysis of naturally occurring and targeted LCR deletions revealed that chromatin opening and transcriptional activity in the endogenous beta-globin locus are dissociable and dependent on distinct cis-acting elements. We now report that general histone H3/H4 acetylation and relocation of the locus away from centromeric heterochromatin in the interphase nucleus are correlated and do not require the LCR. In contrast, LCR-dependent promoter activation is associated with localized histone H3 hyperacetylation at the LCR and the transcribed beta-globin-promoter and gene. On the basis of these results...
Research Interests:
Biological insights can be obtained through computational integration of genomics data sets consisting of diverse types of information. The integration is often hampered by a large variety of existing file formats, often containing... more
Biological insights can be obtained through computational integration of genomics data sets consisting of diverse types of information. The integration is often hampered by a large variety of existing file formats, often containing similar information, and the necessity to use complicated tools to achieve the desired results. We have built an R package, genomation, to expedite the extraction of biological information from high throughput data. The package works with a variety of genomic interval file types and enables easy summarization and annotation of high throughput data sets with given genomic annotations. Availability and implementation: The software is currently distributed under MIT artistic license and freely available at http://bioinformatics.mdc-berlin.de/genomation, and through the Bioconductor framework. dirk.schubeler@fmi.ch, chm2042@med.cornell.edu, altuna.akalin@fmi.ch, or aakalin@gmail.com.
Research Interests:
Research Interests:
Methylated DNA immunoprecipitation (MeDIP) is a versatile immunocapturing approach for unbiased detection of methylated DNA. In brief, genomic DNA is randomly sheared by sonication and immunoprecipitated with a monoclonal antibody that... more
Methylated DNA immunoprecipitation (MeDIP) is a versatile immunocapturing approach for unbiased detection of methylated DNA. In brief, genomic DNA is randomly sheared by sonication and immunoprecipitated with a monoclonal antibody that specifically recognizes 5-methylcytidine. The resulting enrichment of methylated DNA in the immunoprecipitated fraction can be determined by PCR to assess the methylation state of individual regions. Alternatively, MeDIP can be combined with large-scale analysis using microarrays as a genome-wide experimental readout. This protocol has been applied to generate comprehensive DNA methylation profiles on a genome-wide scale in mammals and plants, and further to identify abnormally methylated genes in cancer cells.
Research Interests:
Cytosine methylation is required for mammalian development and is often perturbed in human cancer. To determine how this epigenetic modification is distributed in the genomes of primary and transformed cells, we used an immunocapturing... more
Cytosine methylation is required for mammalian development and is often perturbed in human cancer. To determine how this epigenetic modification is distributed in the genomes of primary and transformed cells, we used an immunocapturing approach followed by DNA microarray analysis to generate methylation profiles of all human chromosomes at 80-kb resolution and for a large set of CpG islands. In primary cells we identified broad genomic regions of differential methylation with higher levels in gene-rich neighborhoods. Female and male cells had indistinguishable profiles for autosomes but differences on the X chromosome. The inactive X chromosome (Xi) was hypermethylated at only a subset of gene-rich regions and, unexpectedly, overall hypomethylated relative to its active counterpart. The chromosomal methylation profile of transformed cells was similar to that of primary cells. Nevertheless, we detected large genomic segments with hypomethylation in the transformed cell residing in ge...
Research Interests:
The majority of mammalian promoters are CpG islands; regions of high CG density that require protection from DNA methylation to be functional. Importantly, how sequence architecture mediates this unmethylated state remains unclear. To... more
The majority of mammalian promoters are CpG islands; regions of high CG density that require protection from DNA methylation to be functional. Importantly, how sequence architecture mediates this unmethylated state remains unclear. To address this question in a comprehensive manner, we developed a method to interrogate methylation states of hundreds of sequence variants inserted at the same genomic site in mouse embryonic stem cells. Using this assay, we were able to quantify the contribution of various sequence motifs towards the resulting DNA methylation state. Modeling of this comprehensive dataset revealed that CG density alone is a minor determinant of their unmethylated state. Instead, these data argue for a principal role for transcription factor binding sites, a prediction confirmed by testing synthetic mutant libraries. Taken together, these findings establish the hierarchy between the two cis-encoded mechanisms that define the DNA methylation state and thus the transcriptional competence of CpG islands.
Research Interests:
DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A... more
DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined genomic binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG-dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.
Research Interests:
Research Interests:
Research Interests: Genetics, Plasticity, Stem Cells, Molecular Evolution, Forecasting, and 15 moreRegulation, Epigenetics, Stability, Gene expression, Gene Silencing, Biological Sciences, DNA, Cell Differentiation, Methylation, Cellular differentiation, DNA methylation, Differentiation, Chromatin, Epigenetica, and Nucleic Acid
Research Interests:
The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and... more
The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, suggesting a role for β-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and β-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.
Research Interests:
Research Interests:
Research Interests:
Research Interests:
DNA methylation has emerged as an important component of transcriptional regulation. However, our understanding of how DNA methylation influences transcription, chromatin structure, replication timing, and imprinting has been limited by... more
DNA methylation has emerged as an important component of transcriptional regulation. However, our understanding of how DNA methylation influences transcription, chromatin structure, replication timing, and imprinting has been limited by the lack of experimental systems that permit control of the methylation state of genes in a chromosomal context. Here, we describe a novel technique that allows for efficient introduction of methylated and unmethylated DNA into defined sites in the mammalian genome. This protocol utilizes bacterial CpG methyltransferases to methylate the DNA of interest in vitro, followed by site-specific targeting using Cre recombinase. Long-term maintenance of the methylation state in vivo allows analysis of the biological consequences of methylation by direct comparison of the methylated and unmethylated state in the same genomic position.
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests: Multidisciplinary, Epigenomics, Liver, Mice, Animals, and 13 moreHistone Modification, Male, Polymerase Chain Reaction, DNA methylation, CpG islands, PLoS one, mRna expression levels, Chromatin Immunoprecipitation, Sensitivity and Specificity, CpG island, Phenobarbital, Constitutive Androstane Receptor, and Tissue Specificity
Research Interests:
Research Interests: Genetics, Human Embryonic Stem Cells, Transcription Factors, Gene Silencing, Biological Sciences, and 18 moreHigher Order Thinking, Cell Differentiation, Humans, DNA methylation, CpG islands, Long Range, Chromatin, Physical Interaction, Adult, Colon cancer, Tretinoin, CpG island, Protein Binding, DNA binding proteins, Nucleic Acid Conformation, Polycomb group proteins, ES cell, and Long-range Interaction
Research Interests:
Histone modifications are central to the regulation of all DNA-dependent processes. Lys64 of histone H3 (H3K64) lies within the globular domain at a structurally important position. We identify trimethylation of H3K64 (H3K64me3) as a... more
Histone modifications are central to the regulation of all DNA-dependent processes. Lys64 of histone H3 (H3K64) lies within the globular domain at a structurally important position. We identify trimethylation of H3K64 (H3K64me3) as a modification that is enriched at pericentric heterochromatin and associated with repeat sequences and transcriptionally inactive genomic regions. We show that this new mark is dynamic during the two main epigenetic reprogramming events in mammals. In primordial germ cells, H3K64me3 is present at the time of specification, but it disappears transiently during reprogramming. In early mouse embryos, it is inherited exclusively maternally; subsequently, the modification is rapidly removed, suggesting an important role for H3K64me3 turnover in development. Taken together, our findings establish H3K64me3 as a previously uncharacterized histone modification that is preferentially localized to repressive chromatin. We hypothesize that H3K64me3 helps to 'secure' nucleosomes, and perhaps the surrounding chromatin, in an appropriately repressed state during development.