Single-cell profiling of transcriptome and histone modifications with EpiDamID.

Recent advances in single-cell sequencing technologies have enabled simultaneous measurement of multiple cellular modalities, but the combined detection of histone post-translational modifications and transcription at single-cell resolution has remained limited. Here, we introduce EpiDamID, an experimental approach to target a diverse set of chromatin types by leveraging the binding specificities of single-chain variable fragment antibodies, engineered chromatin reader domains, and endogenous chromatin-binding proteins. Using these, we render the DamID technology compatible with the genome-wide identification of histone post-translational modifications. Importantly, this includes the possibility to jointly measure chromatin marks and transcription at the single-cell level. We use EpiDamID to profile single-cell Polycomb occupancy in mouse embryoid bodies and provide evidence for hierarchical gene regulatory networks. In addition, we map H3K9me3 in early zebrafish embryogenesis, and detect striking heterochromatic regions specific to notochord. Overall, EpiDamID is a new addition to a vast toolbox to study chromatin states during dynamic cellular processes.

Antagonism between H3K27me3 and genome-lamina association drives atypical spatial genome organization in the totipotent embryo.

In mammals, early embryonic development exhibits highly unusual spatial positioning of genomic regions at the nuclear lamina, but the mechanisms underpinning this atypical genome organization remain elusive. Here, we generated single-cell profiles of lamina-associated domains (LADs) coupled with transcriptomics, which revealed a striking overlap between preimplantation-specific LAD dissociation and noncanonical broad domains of H3K27me3. Loss of H3K27me3 resulted in a restoration of canonical LAD profiles, suggesting an antagonistic relationship between lamina association and H3K27me3. Tethering of H3K27me3 to the nuclear periphery showed that the resultant relocalization is partially dependent on the underlying DNA sequence. Collectively, our results suggest that the atypical organization of LADs in early developmental stages is the result of a tug-of-war between intrinsic affinity for the nuclear lamina and H3K27me3, constrained by the available space at the nuclear periphery. This study provides detailed insight into the molecular mechanisms regulating nuclear organization during early mammalian development.

The role of heterochromatin in 3D genome organization during preimplantation development.

During the early stages of mammalian development, the epigenetic state of the parental genome is completely reprogrammed to give rise to the totipotent embryo. An important aspect of this remodeling concerns the heterochromatin and the spatial organization of the genome. While heterochromatin and genome organization are intricately linked in pluripotent and somatic systems, little is known about their relationship in the totipotent embryo. In this review, we summarize the current knowledge on the reprogramming of both regulatory layers. In addition, we discuss available evidence on their relationship and put this in the context of findings in other systems.

Simultaneous quantification of protein-DNA interactions and transcriptomes in single cells with scDam&T-seq.

Protein-DNA interactions are essential for establishing cell type-specific chromatin architecture and gene expression. We recently developed scDam&T-seq, a multi-omics method that can simultaneously quantify protein-DNA interactions and the transcriptome in single cells. The method effectively combines two existing methods: DNA adenine methyltransferase identification (DamID) and CEL-Seq2. DamID works through the tethering of a protein of interest (POI) to the Escherichia coli DNA adenine methyltransferase (Dam). Upon expression of this fusion protein, DNA in proximity to the POI is methylated by Dam and can be selectively digested and amplified. CEL-Seq2, in contrast, makes use of poly-dT primers to reverse transcribe mRNA, followed by linear amplification through in vitro transcription. scDam&T-seq is the first technique capable of providing a combined readout of protein-DNA contact and transcription from single-cell samples. Once suitable cell lines have been established, the protocol can be completed in 5 d, with a throughput of hundreds to thousands of cells. The processing of raw sequencing data takes an additional 1-2 d. Our method can be used to understand the transcriptional changes a cell undergoes upon the DNA binding of a POI. It can be performed in any laboratory with access to FACS, robotic and high-throughput-sequencing facilities.

Simultaneous quantification of protein-DNA contacts and transcriptomes in single cells.

Protein-DNA interactions are critical to the regulation of gene expression, but it remains challenging to define how cell-to-cell heterogeneity in protein-DNA binding influences gene expression variability. Here we report a method for the simultaneous quantification of protein-DNA contacts by combining single-cell DNA adenine methyltransferase identification (DamID) with messenger RNA sequencing of the same cell (scDam&T-seq). We apply scDam&T-seq to reveal how genome-lamina contacts or chromatin accessibility correlate with gene expression in individual cells. Furthermore, we provide single-cell genome-wide interaction data on a polycomb-group protein, RING1B, and the associated transcriptome. Our results show that scDam&T-seq is sensitive enough to distinguish mouse embryonic stem cells cultured under different conditions and their different chromatin landscapes. Our method will enable the analysis of protein-mediated mechanisms that regulate cell-type-specific transcriptional programs in heterogeneous tissues.

From squiggle to basepair: computational approaches for improving nanopore sequencing read accuracy.

Nanopore sequencing is a rapidly maturing technology delivering long reads in real time on a portable instrument at low cost. Not surprisingly, the community has rapidly taken up this new way of sequencing and has used it successfully for a variety of research applications. A major limitation of nanopore sequencing is its high error rate, which despite recent improvements to the nanopore chemistry and computational tools still ranges between 5% and 15%. Here, we review computational approaches determining the nanopore sequencing error rate. Furthermore, we outline strategies for translation of raw sequencing data into base calls for detection of base modifications and for obtaining consensus sequences.

Causes and Consequences of Age-Related Changes in DNA Methylation: A Role for ROS?

Recent genome-wide analysis of C-phosphate-G (CpG) sites has shown that the DNA methylome changes with increasing age, giving rise to genome-wide hypomethylation with site­specific incidences of hypermethylation. This notion has received a lot of attention, as it potentially explains why aged organisms generally have a higher risk of age-related diseases. However, very little is known about the mechanisms that could cause the occurrence of these changes. Moreover, there does not appear to be a clear link between popular theories of aging and alterations in the methylome. Some of the most fruitful of these theories attribute an important role to reactive oxygen species, which seem to be responsible for an increase in oxidative damage to macromolecules, such as DNA, during the lifetime of an organism. In this review, the connection between changes in DNA methylation and these reactive oxygen species is discussed, as well as the effect of these changes on health. Deeper insights into the nature, causes and consequences of the aging methylome might provide a deeper understanding of the molecular mechanisms of aging and eventually contribute to the development of new diagnostic and therapeutic tools.