Biology subjects

Kremer, L. P.

Publications and source records attributed to Kremer, L. P..

3 recordsLinked to original sources

bioRxiv2022

Single-cell triple-omics uncovers DNA methylation as key feature of stemness in the healthy and ischemic adult brain

Stem cells in the adult brain are specialized astrocytes capable of generating neurons and glial cells. While neural stem cells (NSCs) and common astrocytes have clearly distinct functions, they share highly similar transcriptome profiles. How stemness is molecularly encoded is therefore unclear. Here we use single-cell NMT-seq to simultaneously characterize the transcriptome, DNA methylome and chromatin accessibility of astrocytes and the NSC lineage in the healthy and ischemic brain. Our data reveal distinct methylation profiles associated with either astrocyte or stem cell function. Stemness is conferred by methylation of astrocyte genes and demethylation of neurogenic genes that are expressed only later. Surprisingly, ischemic injury unlocks the stemness-methylome in common astrocytes enabling generation of neuroblasts. Furthermore, we show that oligodendrocytes employ Tet-mediated demethylation to regulate expression of myelin-related genes, many of which are abnormally methylated in multiple sclerosis. Overall, we show that DNA methylation is a promising target for regenerative medicine. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/499860v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@149fe11org.highwire.dtl.DTLVardef@c71902org.highwire.dtl.DTLVardef@13ae945org.highwire.dtl.DTLVardef@a8d37e_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Analyzing single-cell bisulfite sequencing data with scbs

Single-cell bisulfite sequencing (scBS) is a technique that enables the assessment of DNA methylation at single-base pair and single-cell resolution. The analysis of large datasets obtained from scBS requires preprocessing to reduce data size, improve signal-to-noise ratio, and provide interpretability. Typically, this is achieved by dividing the genome into large tiles and averaging the methylation signals within each tile. Here, we demonstrate that this coarse-graining approach can lead to signal dilution. As an alternative, we propose improved strategies to identify more informative regions for methylation quantification, and a more accurate quantitation method than simple averaging. Our approach enables better discrimination of cell types and other features of interest and reduces the need for large numbers of cells. We also present an approach to detect differentially methylated regions (DMRs) between groups of cells, and demonstrate its ability to identify biologically meaningful regions that are associated with genes involved in the core functions of specific cell types. To facilitate the analysis of scBS data, we have developed a software tool called Meth-SCAn that implements these methods and provides additional functionality.

bioinformatics↗

bioRxiv2021

In vivo high-throughput screening of novel adeno-associated viral capsids targeting adult neural stem cells in the subventricular zone

The adult mammalian brain entails a reservoir of neural stem cells (NSCs) generating glial cells and neurons. However, NSCs become increasingly quiescent with age, which hampers their regenerative capacity. New means are therefore required to genetically modify adult NSCs for re-enabling endogenous brain repair. Recombinant adeno-associated viruses (AAVs) are ideal gene therapy vectors due to an excellent safety profile and high transduction efficiency. We thus conducted a high-throughput screening of 177 intraventricularly injected barcoded AAV variants profiled by RNA sequencing. Quantification of barcoded AAV mRNAs identified two synthetic capsids, AAV9_A2 and AAV1_P5, both of which transduce active and quiescent NSCs. Further optimization of AAV1_P5 by judicious selection of promoter and dose of injected viral genomes enabled labeling of 30-60% of the NSC compartment, which was validated by FACS analyses and single cell RNA sequencing. Importantly, transduced NSC readily produced neurons. The present study identifies AAV variants with a high regional tropism towards the v-SVZ with high efficiency in targeting adult NSCs, thereby paving the way for preclinical testing of regenerative gene therapy.

neuroscience↗