Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Molecular Biology Institute, Department of Biological Chemistry, David Geffen School of Medicine of the University of California, Los Angeles, CA, United States.
Cells face the challenge of storing two meters of DNA in the three-dimensional (3D) space of the nucleus that spans only a few microns. The nuclear organization that is required to overcome this challenge must allow for the accessibility of the gene regulatory machinery to the DNA and, in the case of embryonic stem cells (ESCs), for the transcriptional and epigenetic changes that accompany differentiation. Recent technological advances have allowed for the mapping of genome organization at an unprecedented resolution and scale. These breakthroughs have led to a deluge of new data, and a sophisticated understanding of the relationship between gene regulation and 3D genome organization is beginning to form. In this review we summarize some of the recent findings illuminating the 3D structure of the eukaryotic genome, as well as the relationship between genome topology and function from the level of whole chromosomes to enhancer-promoter loops with a focus on features affecting genome organization in ESCs and changes in nuclear organization during differentiation.
[Show abstract][Hide abstract] ABSTRACT: The pluripotent nature of embryonic stem cells (ESC) is associated with a dynamic open chromatin state and an irregular nuclear shape. It has been postulated that the absence of Lamin A/C contributes to these features. However, we show that mouse ESC express low, yet readily detectable, amounts of Lamin A/C at both the RNA and protein levels. Full-length transcripts of both isoforms were readily detected by Q-PCR and deep RNA sequencing. Additionally, protein expression was validated in multiple primary and established ESC lines by immunoblotting using several independent antibodies. Immunofluorescence labeling showed localization of Lamin A/C at the nuclear periphery of all Oct4/Nanog double-positive ESC lines examined, as well as in the inner cell mass of blastocysts. Our results demonstrate ESCs do express low levels of Lamin A/C, thus models linking pluripotency and chromatin dynamics with the absence of Lamin A/C need to be revisited.
[Show abstract][Hide abstract] ABSTRACT: The relationship between 3D organization of the genome and gene-regulatory networks is poorly understood. Here, we examined long-range chromatin interactions genome-wide in mouse embryonic stem cells (ESCs), iPSCs, and fibroblasts and uncovered a pluripotency-specific genome organization that is gradually reestablished during reprogramming. Our data confirm that long-range chromatin interactions are primarily associated with the spatial segregation of open and closed chromatin, defining overall chromosome conformation. Additionally, we identified two further levels of genome organization in ESCs characterized by colocalization of regions with high pluripotency factor occupancy and strong enrichment for Polycomb proteins/H3K27me3, respectively. Underlining the independence of these networks and their functional relevance for genome organization, loss of the Polycomb protein Eed diminishes interactions between Polycomb-regulated regions without altering overarching chromosome conformation. Together, our data highlight a pluripotency-specific genome organization in which pluripotency factors such as Nanog and H3K27me3 occupy distinct nuclear spaces and reveal a role for cell-type-specific gene-regulatory networks in genome organization.
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