Simultaneous Quantification of Spatial Genome Positioning and Transcriptomics in Single Cells with scDam&amp;T-Seq

Lochs, Silke J A; Kind, Jop

doi:10.1007/978-1-0716-2497-5_11

Cited by 1 publication

(2 citation statements)

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“…Correct expression of the mAID-Dam-Lamin B1 fusion protein as well as the wildtype Lamin B1 protein was confirmed using Immunofluorescence staining using an anti-HA antibody at 1 in 500 dilution (Cell Signaling Technologies, C29F4) and an anti-LaminB1 antibody at 1 in 500 dilution (Abcam, ab16048), followed by confocal imaging. All successfully repaired clones were subsequently screened for their level of induction upon IAA removal by m6A-PCR, evaluated by gel electrophoresis 6, 35 , followed by DamID2 sequencing in bulk 31, 35 , to select a heterozygous clone with a correct karyotype with the best signal-to-noise ratio of enrichment over LAD regions. This clone is labelled as F1ES mAID-Dam-LaminB1 #2B4.…”

Section: Methodsmentioning

confidence: 99%

“…and an anti-LaminB1 antibody at 1 in 500 dilution (Abcam, ab16048), followed by confocal imaging. All successfully repaired clones were subsequently screened for their level of induction upon IAA removal by m6A-PCR, evaluated by gel electrophoresis 6,35 , followed by DamID2 sequencing in bulk 31,35 , to select a heterozygous clone with a correct karyotype with the best signal-to-noise ratio of enrichment over LAD regions. This clone is labelled as F1ES mAID-Dam-LaminB1 #2B4.…”

Section: Generation Of Mouse Embryonic Stem Cell Linesmentioning

confidence: 99%

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H3K27me3 dictates atypical genome-nuclear lamina interactions and allelic asymmetry during early embryogenesis

Guerreiro

Rang

Kawamura

et al. 2023

Preprint

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The very first days of mammalian embryonic development are accompanied by epigenetic reprogramming and extensive changes in nuclear organization. In particular, genomic regions located at the periphery of the nucleus, termed lamina-associated domains (LADs), undergo major rearrangements after fertilization. However, the role of LADs in regulating gene expression as well as the interplay with various chromatin marks during preimplantation development remains elusive. In this study, we obtained single-cell LAD profiles coupled with the corresponding gene expression readout throughout the first days of mouse development. We detect extensive cell-cell LAD variability at the 2-cell stage, which surprisingly does not seem to functionally affect gene expression. This suggests an unusual uncoupling between 3D-nuclear genome organization and gene expression during totipotent developmental stages. By analyzing LAD dynamics and chromatin states across early developmental stages in an allelic-specific manner, we identify genomic regions that transiently detach from the nuclear lamina and are enriched by non-canonical H3K27me3. Upon maternal knock-out of a component of the Polycomb repressive complex 2 and concomitant loss of H3K27me3 during early embryogenesis, these regions relocate to the lamina at the 2-cell stage. Our results suggest that H3K27me3 is the prime determinant in establishing the atypical distribution of the genome at the nuclear periphery during the first days of embryonic development. This study provides insight into the molecular mechanisms regulating nuclear organization of parental genomes during very early mammalian development.

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