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Temporal recording of mammalian development and precancer

ABSTRACT: Insights into many biological phenomena requires knowing the temporal order of cellular events, which is traditionally achieved through continuous direct observations [1, 2]. An alternative solution leverages irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using Native sgRNA Capture and sequencing (NSC-seq), a newly devised and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo while incorporating cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development, unconventional developmental relationships between cell types, and new epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas coupled to multi-omic and single-cell profiling of human precancers, with clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Our study presents a multimodal framework that lays the foundation for in vivo recording, integrating synthetic or natural indelible genetic changes with single-cell analyses to explore the origins and timing of development and tumorigenesis in mammalian systems. This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Mus musculus

PROVIDER: GSE235119 | GEO | 2024/07/07

REPOSITORIES: GEO

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Project description:The organization of mammalian DNA replication is poorly understood. We have produced genome-wide high-resolution dynamic maps of the timing of replication in human erythroid, mesenchymal and embryonic stem cells using TimEX, a method that relies on gaussian convolution of massive, highly redundant determinations of DNA copy number variations during S phase obtained using either high-density oligonucleotide tiling arrays or massively-parallel sequencing to produce replication timing profiles. We show that in untransformed human cells, timing of replication is highly regulated and highly synchronous, and that many genomic segments are replicated in temporal transition regions devoid of initiation where replication forks progress unidirectionally from origins that can be hundreds of kilobases away. Absence of initiation in one transition region is shown at the molecular level by SMARD analysis. Comparison of ES and erythroid cells replication patterns revealed that these cells replicate about 20% of their genome in different quarter of S phase and that ES cells replicate a larger proportion of their genome in early S phase than erythroid cells. Importantly, we detected a strong inverse relationship between timing of replication and distance to the closest expressed gene. This relationship can be used to predict tissue specific timing of replication profiles from expression data and genomic annotations. We also provide evidence that early origins of replication are preferentially located near highly expressed genes, that mid firing origins are located near moderately expressed genes and that late firing origins are located far from genes. Comparison of S/G1 from ES cells, MSCs and erythroid cells The purpose of the study was to measure replication timing by taking advantage of the fact that copy number of DNA increases from 2 two 4 during replication. Specifically, the study was designed to compare the number of copies of DNA of cells in the G1 phase of the cell cycle with the number of copies of DNA of cells in the S phase of the cell cycle. The cells in G1 and S of the cell cycle were obtained by sorting cells based on DNA content after staining with propidium iodide, a fluorescent compound that binds to DNA. We first tested the strategy with Nimblegen tiling arrays representing about 3% of the genome and found that it was indeed possible to measure such a small difference of copy number if a high-level of redundancy was used. We then extended the study genome-wide using massively parallel sequencing on either the SolID or the Illumina platform. Both platforms were tested and we found that for that application, both platforms were equivalent and provided sufficient number of reads to measure timing of replication genome-wide. We then attempted to better understand the correlation between timing of replication and transcription. We therefore obtained RNA from hESC and from erythroblast and hybridized them to standard Affymetrix U133plus2 arrays. This analysis revealed that it was possible to predict the replication timing patterns with relatively good accuracy (r=0.8) using expression data and distance of every genomic windows to express genes.

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Temporal recording of mammalian development and precancer

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