Dissecting the structural secrets of the inactive X chromosome

Inactivated (red) X chromosomes (Xi) in cultured mouse cells, which were labeled with a fluorescent probe for the Xi-specific RNA marker, Xist. Credit: 2023 RIKEN Biosystem Dynamics Research Center

RIKEN cell biologists have provided unprecedented insight into the distinctive characteristics of an unusual chromosome: the inactivated copy of the X chromosome carried by every female cell. The results are published in the journal Nature Structural and molecular biology.

Not all X chromosomes are created equal. In each cell of female mammals, one of the two copies of the X chromosome is compacted into an inactive form called the inactive X chromosome (Xi). It differs in both structure and function from its active counterpart and other chromosomes.

Unlike most chromosomes, which replicate gradually throughout the “S phase” phase of cell division, which lasts several hours, the Xi is copied in the second half of this phase. The researchers hypothesized that this replication behavior is closely related to the unusual structure of the Xi, which is more uniform and compact than other chromosomes.

“However, this was still uncertain when we started our research,” says Rawin Poonperm from the RIKEN Center for Biosystem Dynamics Research (BDR). “We reasoned that a detailed analysis of the Xi’s replication schedule could reveal its purpose and provide new insights into its 3D organization.”

Researchers used two cutting-edge analytical methods to answer this question by differentiating cultured mouse embryonic stem cells. The first, developed by a group led by Ichiro Hiratani (also of the BDR), precisely determined when specific chromosome sequences undergo replication. In parallel, they used a second technique, which revealed the 3D structure of chromatin – the combination of DNA and proteins that forms chromosomes – providing insight into gene expression activity.

This combination of the two techniques has proven to be very effective. “Our high-resolution replication analysis revealed that the Xi is copied rapidly in the second half of S phase as it forms during differentiation. Additionally, our 3D structure results showed that the changes “Dynamics in the timing of Xi replication closely corresponded to changes in chromatin organization during differentiation,” says Poonperm. She adds that their structural analysis of the Xi also revealed some surprises, including differences in the extent of compaction and inactivation at different sites on the chromosome.

When the researchers analyzed differentiated cells lacking a gene called SmcHD1, which helps keep the Xi inactive, they observed the reactivation of normally dormant genes at the edge of the chromosome. Most other Xi genes, however, remained inactive, hinting at hidden structural complexity in this seemingly uniform chromosome.

Hiratani’s group next plans to examine the process of inactivation of the X chromosome itself. This will involve considerably more difficult experiments on real mouse embryos rather than cultured embryonic stem cells. But Poonperm sees an exciting opportunity to solve the mysteries that have surrounded this process for decades. “We firmly believe that there are even more surprising discoveries to be made,” she says.

More information:
Rawin Poonperm et al, Replication Dynamics identifies principles of inactive X chromosome folding, Nature Structural and molecular biology (2023). DOI: 10.1038/s41594-023-01052-1

Quote: Dissection of the structural secrets of the inactive

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