Biology subjects

Nery, J. R.

Publications and source records attributed to Nery, J. R..

5 recordsLinked to original sources

bioRxiv2018

Multi-omic profiling of transcriptome and DNA methylome in single nuclei with molecular partitioning

Single-cell transcriptomic and epigenomic analyses provide powerful strategies for unbiased determination of cell types in mammalian tissues. Although previous studies have identified cell types using individual molecular signatures, the generation of consensus cell type classification requires the integration of multiple data types. Most existing single-cell techniques can only make one type of molecular measurement. Here we describe single-nucleus methylcytosine and transcriptome sequencing (snmCT-seq), a multi-omic method that requires no physical separation of DNA and RNA molecules. We demonstrated that snmCT-seq profiles generated from single cells or nuclei robustly distinguish human cell types and accurately measures cytosine DNA methylation and gene expression signatures of each cell type.

genomics↗

bioRxiv2018

Global DNA methylation remodeling during direct reprogramming of fibroblasts to neurons

Direct reprogramming of fibroblasts to neurons induces widespread cellular and transcriptional reconfiguration. In this study, we characterized global epigenomic changes during direct reprogramming using whole-genome base-resolution DNA methylome (mC) sequencing. We found that the pioneer transcription factor Ascl1 alone is sufficient for inducing the uniquely neuronal feature of non-CG methylation (mCH), but co-expression of Brn2 and Mytl1 was required to establish a global mCH pattern reminiscent of mature cortical neurons. Ascl1 alone induced strong promoter CG methylation (mCG) of fibroblast specific genes, while BAM overexpression additionally targets a competing myogenic program and directs a more faithful conversion to neuronal cells. Ascl1 induces local demethylation at its binding sites. Surprisingly, co-expression with Brn2 and Mytl1 inhibited the ability of Ascl1 to induce demethylation, suggesting a contextual regulation of transcription factor - epigenome interaction. Finally, we found that de novo methylation by DNMT3A is required for efficient neuronal reprogramming.

neuroscience↗

bioRxiv2018

Robust single-cell DNA methylome profiling with snmC-seq2

Single-cell DNA methylome profiling has enabled the study of epigenomic heterogeneity in complex tissues and during cellular reprogramming. However, broader applications of the method have been impeded by the modest quality of sequencing libraries. Here we report snmC-seq2, which provides improved read mapping, reduced artifactual reads, enhanced throughput, as well as increased library complexity and coverage uniformity compared to snmC-seq. snmC-seq2 is an efficient strategy suited for large scale single-cell epigenomic studies.

genomics↗

bioRxiv2018

The complex architecture of plant transgene insertions

Over the last 35 years the soil bacterium Agrobacterium tumefaciens has been the workhorse tool for plant genome engineering. Replacement of native tumor-inducing (Ti) plasmid elements with customizable cassettes enabled insertion of a sequence of interest called Transfer DNA (T-DNA) into any plant genome. Although these T-DNA transfer mechanisms are well understood, detailed understanding of structure and epigenomic status of insertion events was limited by current technologies. To fill this gap, we analyzed transgenic Arabidopsis thaliana lines from three widely used collections (SALK, SAIL and WISC) with two single molecule technologies, optical genome mapping and nanopore sequencing. Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements, and for the first time the actual length of individual transgene insertions from 27 to 236 kilobases. De novo nanopore sequencing-based genome assemblies for two segregating lines resolved T-DNA structures up to 36 kb into the insertions and revealed large-scale T-DNA associated translocations and exchange of chromosome arm ends. The multiple internally rearranged nature of T-DNA arrays made full assembly impossible, even with long nanopore reads. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. This unprecedented nucleotide-level definition of T-DNA insertions enabled the mapping of epigenome data. We identify variable small RNA transgene targeting and DNA methylation. SALK_059379 T-DNA insertions were enriched for 24nt siRNAs and contained dense cytosine DNA methylation. Transgene silencing via the RNA-directed DNA methylation pathway was confirmed by in planta assays. In contrast, SAIL_232 T-DNA insertions are predominantly targeted by 21/22nt siRNAs, with DNA methylation and silencing limited to a reporter, but not the resistance gene. With the emergence of genome editing technologies that rely on Agrobacterium for gene delivery, this study provides new insights into the structural impact of engineering plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes.

plant biology↗

bioRxiv2017

Spatiotemporal DNA Methylome Dynamics of the Developing Mammalian Fetus

Genetic studies have revealed an essential role for cytosine DNA methylation in mammalian development. However, its spatiotemporal distribution in the developing embryo remains obscure. Here, we profiled the methylome landscapes of 12 mouse tissues/organs at 8 developmental stages spanning from early embryogenesis to birth. Indepth analysis of these spatiotemporal epigenome maps systematically delineated ~2 million methylation variant regions and uncovered widespread methylation dynamics at nearly one-half million tissue-specific enhancers, whose human counterparts were enriched for variants involved in genetic diseases. Strikingly, these predicted regulatory elements predominantly lose CG methylation during fetal development, whereas the trend is reversed after birth. Accumulation of non-CG methylation within gene bodies of key developmental transcription factors coincided with their transcriptional repression during later stages of fetal development. These spatiotemporal epigenomic maps provide a valuable resource for studying gene regulation during mammalian tissue/organ progression and for pinpointing regulatory elements involved in human developmental diseases.

genomics↗