The three-dimensional organization of chromosomes within the nucleus and its dynamics during differentiation are largely unknown. To visualize this process in molecular detail, we generated high-resolution maps of genome-nuclear lamina interactions during subsequent differentiation of mouse embryonic stem cells via lineage-committed neural precursor cells into terminally differentiated astrocytes. This reveals that a basal chromosome architecture present in embryonic stem cells is cumulatively altered at hundreds of sites during lineage commitment and subsequent terminal differentiation. This remodeling involves both individual transcription units and multigene regions and affects many genes that determine cellular identity. Often, genes that move away from the lamina are concomitantly activated; many others, however, remain inactive yet become unlocked for activation in a next differentiation step. These results suggest that lamina-genome interactions are widely involved in the control of gene expression programs during lineage commitment and terminal differentiation.
In metazoans, the nuclear lamina is thought to play an important role in the spatial organization of interphase chromosomes, by providing anchoring sites for large genomic segments named lamina-associated domains (LADs). Some of these LADs are cell-type specific, while many others appear constitutively associated with the lamina. Constitutive LADs (cLADs) may contribute to a basal chromosome architecture. By comparison of mouse and human lamina interaction maps, we find that the sizes and genomic positions of cLADs are strongly conserved. Moreover, cLADs are depleted of synteny breakpoints, pointing to evolutionary selective pressure to keep cLADs intact. Paradoxically, the overall sequence conservation is low for cLADs. Instead, cLADs are universally characterized by long stretches of DNA of high A/T content. Cell-type specific LADs also tend to adhere to this “A/T rule” in embryonic stem cells, but not in differentiated cells. This suggests that the A/T rule represents a default positioning mechanism that is locally overruled during lineage commitment. Analysis of paralogs suggests that during evolution changes in A/T content have driven the relocation of genes to and from the nuclear lamina, in tight association with changes in expression level. Taken together, these results reveal that the spatial organization of mammalian genomes is highly conserved and tightly linked to local nucleotide composition.
In mammalian interphase nuclei, more than one thousand large genomic regions are positioned at the nuclear lamina (NL). These lamina‐associated domains (LADs) are involved in gene regulation and may provide a backbone for the folding of interphase chromosomes. Little is known about the dynamics of LADs during interphase, in particular at the onset of G1 phase and during DNA replication. We developed an antibody‐based variant of the DamID technology (named pA‐DamID) that allows us to map and visualize genome–NL interactions with high temporal resolution. Application of pA‐DamID combined with synchronization and cell sorting experiments reveals that LAD–NL contacts are generally rapidly established early in G1 phase. However, LADs on the distal ~25 Mb of most chromosomes tend to contact the NL first and then gradually detach, while centromere‐proximal LADs accumulate gradually at the NL. Furthermore, our data indicate that S‐phase chromatin shows transiently increased lamin interactions. These findings highlight a dynamic choreography of LAD–NL contacts during interphase progression and illustrate the usefulness of pA‐DamID to study the dynamics of genome compartmentalization.
The nuclear lamina is a major structural component of metazoan nuclei that has long been thought to provide an anchoring site for interphase chromosomes and have a role in gene regulation. Recent genome-wide mapping studies and functional experimental data strongly support these roles of the nuclear lamina. Here, we discuss new insights into various aspects of genome-nuclear lamina interactions, with emphasis on the links with gene regulation and with dynamics during cellular differentiation.
Background Lamina-associated domains (LADs) are large genomic regions that are positioned at the nuclear lamina. It has remained largely unclear what drives the positioning and demarcation of LADs. Because the insulator protein CTCF is enriched at LAD borders, it was postulated that CTCF binding could position some LAD boundaries, possibly through its function in stalling cohesin and hence preventing cohesin invading into the LAD. To test this, we mapped genome–nuclear lamina interactions in mouse embryonic stem cells after rapid depletion of CTCF and other perturbations of cohesin dynamics. Results CTCF and cohesin contribute to a sharp transition in lamina interactions at LAD borders, while LADs are maintained after depletion of these proteins, also at borders marked by CTCF. CTCF and cohesin may thus reinforce LAD borders, but do not position these. CTCF binding sites within LADs are locally detached from the lamina and enriched for accessible DNA and active histone modifications. Remarkably, despite lamina positioning being strongly correlated with genome inactivity, this DNA remains accessible after the local detachment is lost following CTCF depletion. At a chromosomal scale, cohesin depletion and cohesin stabilization by depletion of the unloading factor WAPL quantitatively affect lamina interactions, indicative of perturbed chromosomal positioning in the nucleus. Finally, while H3K27me3 is locally enriched at CTCF-marked LAD borders, we find no evidence for an interplay between CTCF and H3K27me3 on lamina interactions. Conclusions These findings illustrate that CTCF and cohesin are not primary determinants of LAD patterns. Rather, these proteins locally modulate NL interactions.
Lamina associated domains (LADs) are large genomic regions that are positioned at the nuclear lamina (NL). It has remained largely unclear what drives the positioning and demarcation of LADs. Because the insulator protein CTCF is enriched at LAD borders, it was postulated that CTCF binding could position a subset of LAD boundaries, possibly through its function in stalling cohesin and hence preventing cohesin to invade into the LAD. To test this, we mapped genome - NL interactions in mouse embryonic stem cells after rapid depletion of CTCF and other perturbations of cohesin dynamics. CTCF and cohesin contribute to a sharp transition in NL interactions at LAD borders, whilst LADs are maintained after depletion of these proteins, also at borders marked by CTCF. CTCF and cohesin may thus reinforce LAD borders, but do not position these. CTCF binding sites within LADs are locally detached from the NL and enriched for accessible DNA and active histone modifications. Remarkably, even though NL positioning is strongly correlated with genome inactivity, this DNA remains accessible after the local detachment is lost following CTCF depletion. At a chromosomal scale, cohesin depletion and cohesin stabilization (depletion of the unloading factor WAPL) quantitatively affect NL interactions, indicative of perturbed chromosomal positioning in the nucleus. Finally, while H3K27me3 is locally enriched at CTCF-marked LAD borders, we find no evidence for an interplay between CTCF and H3K27me3 on NL interactions. Combined, these findings illustrate that CTCF and cohesin do not shape LAD patterns. Rather, these proteins mediate fine-tuning of NL interactions.
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