Biology subjects

Holler, K.

Publications and source records attributed to Holler, K..

3 recordsLinked to original sources

bioRxiv2023

Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency

SummaryStochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of specific genomic loci after targeted purification in their native chromatin context. Applying MATAC-Seq to selected early-efficient (EE) and late-inefficient (LI) budding yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an optimal 100-115bp nucleosome-free region in combination with surrounding well-positioned nucleosomes and open +2 linker region is a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during DNA replication of eukaryotic cells. Consequently, our single-molecule assay for chromatin accessibility will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo.

biochemistry↗

bioRxiv2021

Transcription factor induction of vascular blood stem cell niches in vivo

The hematopoietic niche is a supportive microenvironment comprised of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses, we define a conserved gene expression signature and cis-regulatory landscape unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code involving members of the Ets, Sox and Nuclear Hormone Receptor families that is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance and division of HSPCs in vivo. These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo, and for effective therapies to modulate the endogenous niche.

developmental biology↗

bioRxiv2020

Spatio-temporal mRNA dynamics in the early zebrafish embryo

Early stages of embryogenesis depend heavily on subcellular localization and transport of maternally deposited mRNA. However, systematic analysis of these processes is currently hindered by a lack of spatio-temporal information in single-cell RNA sequencing. Here, we combined spatially-resolved transcriptomics and single-cell RNA labeling to study the spatio-temporal dynamics of the transcriptome during the first few hours of zebrafish development. We measured spatial localization of mRNA molecules with sub-single-cell resolution at the one-cell stage, which allowed us to identify a class of mRNAs that are specifically localized at an extraembryonic position, the vegetal pole. Furthermore, we established a method for high-throughput single-cell RNA labeling in early zebrafish embryos, which enabled us to follow the fate of individual maternal transcripts until gastrulation. This approach revealed that many localized transcripts are specifically transported to the primordial germ cells. Finally, we acquired spatial transcriptomes of two xenopus species, and we compared evolutionary conservation of localized genes as well as enriched sequence motifs. In summary, we established sub-single-cell spatial transcriptomics and single-cell RNA labeling to reveal principles of mRNA localization in early vertebrate development.

systems biology↗