Organization of the Pluripotent Genome

Eukaryotic genome DNA is wrapped around core histones and forms a nucleosome structure. Together with associated proteins and RNAs, these nucleosomes are organized three‐dimensionally in the cell as chromatin. Emerging evidence demonstrates that chromatin consists of rather irregular and variable nucleosome arrangements without the regular fiber structure and that its dynamic behavior plays a critical role in regulating various genome functions. Single‐nucleosome imaging is a promising method to investigate chromatin behavior in living cells. It reveals local chromatin motion, which reflects chromatin organization not observed in chemically fixed cells. The motion data is like a gold mine. Data analyses from many aspects bring us more and more information that contributes to better understanding of genome functions. In this review article, we describe imaging of single‐nucleosomes and their tracked behavior through oblique illumination microscopy. We also discuss applications of this technique, especially in elucidating nucleolar organization in living cells.

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“…[ 72,113–114 ] These findings strengthen the concept of chromatin plasticity in pluripotent cells. [ 115 ]…”

Section: Liquid‐like Chromatin Is Constrained By Many Factors In the ...mentioning

confidence: 99%

Chromatin behavior in living cells: Lessons from single‐nucleosome imaging and tracking

2022

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“…We next sought to test absolute scaling in previously established contexts of hypertranscription. The early mouse embryo is especially well characterized in this regard and can be modeled in-vitro using mESCs 24 . Specifically, serum/LIF-grown mESCs are hypertranscriptional and represent the rapidly proliferating early postimplantation epiblast, while mESCs under dual GSK/MEK inhibition (2i media) are comparatively hypotranscriptional and represent a pre-implantation-like state 9,24 .…”

Section: Validation Of Absolute Scaling Using Embryonic Hypertranscription Datamentioning

confidence: 99%

“…The early mouse embryo is especially well characterized in this regard and can be modeled in-vitro using mESCs 24 . Specifically, serum/LIF-grown mESCs are hypertranscriptional and represent the rapidly proliferating early postimplantation epiblast, while mESCs under dual GSK/MEK inhibition (2i media) are comparatively hypotranscriptional and represent a pre-implantation-like state 9,24 . We performed absolute scaling on an ERCC-spiked mESC dataset generated using Fluidigm C1 containing both serum and 2i cells 25 .…”

Section: Validation Of Absolute Scaling Using Embryonic Hypertranscription Datamentioning

confidence: 99%

Absolute Scaling of Single-Cell Transcriptomes Reveals Pervasive Hypertranscription in Adult Stem and Progenitor Cells

Kim

Ramalho-Santos

2021

Preprint

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Hypertranscription facilitates biosynthetically demanding cellular state transitions through global upregulation of the nascent transcriptome. Despite its potential widespread relevance, documented examples of hypertranscription remain few and limited predominantly to early development. This limitation is in large part due to the fact that modern sequencing approaches, including single-cell RNA sequencing (scRNA-seq), generally assume similar levels of transcriptional output per cell. Here, we use molecule counting and spike-in normalization to develop absolute scaling of single-cell RNA sequencing data. Absolute scaling enables an estimation of total transcript abundances per cell, which we validate in embryonic stem cell (ESC) and germline data and apply to adult mouse organs at steady-state or during regeneration. The results reveal a remarkable dynamic range in transcriptional output among adult cell types. We find that many different multipotent stem and progenitor cell populations are in a state of hypertranscription, including in the hematopoietic system, intestine and skin. Hypertranscription marks cells with multilineage potential in adult organs, is redeployed in conditions of tissue injury, and can precede by 1-2 days bursts of proliferation during regeneration. In addition to the association between hypertranscription and the stem/progenitor cell state, we dissect the relationship between transcriptional output and cell cycle, ploidy and secretory behavior. Our analyses reveal a common set of molecular pathways associated with hypertranscription across adult organs, including chromatin remodeling, DNA repair, ribosome biogenesis and translation. Our findings introduce an approach towards maximizing single-cell RNA-seq profiling. By applying this methodology across a diverse collection of cell states and contexts, we put forth hypertranscription as a general and dynamic cellular program that is pervasively employed during development, organ maintenance and regeneration.

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“…Chromatin is composed of chains of nucleosomes and is packed at various densities related to the transcriptional activity in each region [2] . Active chromatin regions are loosely packed in the nucleus, whereas repressed chromatin regions are tightly packed [3][4][5]. The formation of chromatin is a benefit for functional storage of nuclear DNA, but it also interferes with the binding of transcription factors to DNA [1,2].…”

Section: Introductionmentioning

confidence: 99%

Disruption of Smarce1, a component of the SWI/SNF chromatin remodeling complex, decreases nucleosome stability in mouse embryonic stem cells and impairs differentiation

Kashiwagi

Yoshida

Kimurâ

et al. 2022

Preprint

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The SWI/SNF chromatin remodeling complex consists of more than 10 component proteins that form a large protein complex of > 1 MDa. The catalytic proteins Smarca4 or Smarca2 work in concert with the component proteins to form a chromatin platform suitable for transcriptional regulation. However, the mechanism by which each component protein works synergistically with the catalytic proteins remains largely unknown. Here, we report on the function of Smarce1, a component of the SWI/SNF complex, through the phenotypic analysis of homozygous mutant embryonic stem (ES) cells. Disruption of Smarce1 induced the dissociation of other complex components from the SWI/SNF complex. Histone binding to DNA was loosened in homozygous mutant ES cells, indicating that disruption of Smarce1 decreased nucleosome stability. Sucrose gradient sedimentation analysis suggested an ectopic genomic distribution of the SWI/SNF complex, accounting for the misregulation of chromatin conformations. Unstable nucleosomes remained during ES cell differentiation, impairing the heterochromatin formation that is characteristic of the differentiation process. These results suggest that Smarce1 guides the SWI/SNF complex to the appropriate genomic regions to generate chromatin structures adequate for transcriptional regulation.

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Organization of the Pluripotent Genome

Cited by 15 publications

References 192 publications

Chromatin behavior in living cells: Lessons from single‐nucleosome imaging and tracking

Chromatin behavior in living cells: Lessons from single‐nucleosome imaging and tracking

Absolute Scaling of Single-Cell Transcriptomes Reveals Pervasive Hypertranscription in Adult Stem and Progenitor Cells

Disruption of Smarce1, a component of the SWI/SNF chromatin remodeling complex, decreases nucleosome stability in mouse embryonic stem cells and impairs differentiation

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