Dynamic Erasure of Random X-Chromosome Inactivation during iPSC Reprogramming

Background & Summary:

During embryo development and in the derivation of induced pluripotent stem cells (iPSCs), the transition of X-chromosome inactivation (XCI) and reactivation (XCR) in female cells is a key process that require accurate gene regulation to achieve the switch of chromatin states. In mouse embryos, XCI occurs twice. At the 4-cell stage, imprinted XCI inactivates the paternal X-chromosome while, after XCR in the inner cell mass (ICM) of the blastocyst, random XCI takes place in the epiblast (1). XCR has been associated with naïve pluripotency and, in fact, during iPSC reprogramming, when female cells acquire a robust pluripotency network, they repress the long noncoding RNA (lncRNA) Xist and reactivate the inactive X-chromosome (Xi) (2).

In this work, Janiszewski, Talon and colleagues focused on how random XCI shifts to an active state (that is XCR) during iPSC reprogramming using allele-specific transcriptomic approaches. They found that this is a dynamic process and silenced genes are gradually reactivated. The early activated genes did not need the lncRNA Xist to be completely repressed, but their reactivation was associated with pluripotency factor binding and specific chromatin topology.

 

Key findings of the preprint:

• Transcriptional activation of silenced genes in the inactive X chromosome follow gene-specific kinetics.

Female mouse embryonic fibroblasts (MEFs) with a silenced X-GFP allele were reprogrammed to iPSCs, and transcriptome profiling was performed at different time points. The authors found that X-linked genes are not reactivated simultaneously. According to the timing of reactivation, they can be classified in four different groups that comprise a time window of more than 5 days: early, intermediate, late and very late activated genes.

• Before the complete suppression of Xist, some genes can be reactivated.

The early subset of genes is activated when Xist expression has begun to decrease but it has not been lost. XCR had previously been reported to occur before the silencing of Xist in the ICM (3) but not when random XCI is reverted. Also, it is remarkable that XCR starts before the activation of the full pluripotency network is completed given that some genes, such as Prdm14, are activated later.

• Early reactivated genes show a tendency to be closer to escapee genes and to key pluripotency transcription factor binding sites.

There are some escapee genes in the Xi that maintain bi-allelic expression, and it had been previously shown that they are located outside of the silenced compartment. Interestingly, the average genomic distance to the nearest escapee gene was reduced in early reactivated genes compared to very late reactivated genes. Also, early genes showed a higher enrichment of KLF4, SOX2 and c-MYC binding sites compared to very late genes.

• Histone deacetylation restricts X-chromosome reactivation.

The use of a pharmacological compound that blocks histone deacetylases during iPSCs reprogramming increases the proportion of XCR. Furthermore, acquisition of H3K27ac on the X-chromosome is restricted until late stages of reprogramming.

 

Why I chose this work:

Transcriptional activation and repression are tightly regulated mechanisms that govern proper functioning of developmental processes and tissue homeostasis. XCI and XCR are a beautiful example of chromosome-wide silencing and reactivation, and the transition between both states make them an interesting model of study.

I have chosen this preprint because I like the way the authors have approached the question from three different layers: a transcriptomic, an epigenomic and a 3D chromatin architectural layer. It is possible to think that a combination of different inputs may be required to regulate a process, and gene regulation is a clear example of this. I have found it especially interesting that early reactivated genes are more prone to be near escapee genes, which suggests that 3D chromatin structure may play an important role. Given that most of the genes that are activated early or late in random XCR compared to imprinted XCR are different, it would indicate that although this is not dependent on the genomic position on the X-chromosome, it could be affected by different 3D interactions in each case.

Epigenetic drug screening is a very interesting tool to approach the effect of chromatin modifiers in a systematic and relatively easy way, which could give important clues to further explore specific regulators in future analyses.

 

Questions to the authors:

• It has been shown that the inactivation of the X-chromosome leads to a loss of topologically associated domains (4). After their hypothesis that 3D architecture may play an important role, do the authors think that, upon XCR, cells would maintain epigenetic memory to acquire a stable topology according to their cell type? Would it entail that the subset of early and late reactivated genes should change from one cell type to another?
• During iPSCs reprogramming, the authors show how the activation of pluripotency markers is sequential, with Prdm14 being one of the last to be expressed. Interestingly, this is a naïve pluripotency marker that is associated with XCR in vivo both in the ICM of the blastocyst and in the specification of the primordial germ cells (PGCs). Have the authors checked if other markers such as Dppa3 which is also important in PGCs follow the same pattern? Do the authors know if PRDM14 could regulate the expression of late reactivated genes similarly to how KLF4 or SOX2 could regulate early reactivated genes?

 

References:

1. Payer B. Developmental regulation of X-chromosome inactivation. Semin Cell Dev Biol. 56:88-99.
2. Pasque V. and Plath K. X chromosome reactivation in reprogramming and in development. Curr Opin Cell Biol. 37:75-83.
3. Borensztein M, Okamoto I, Syx L, Guilbaud G, Picard C, Ancelin K, et al. Contribution of epigenetic landscapes and transcription factors to X-chromosome reactivation in the inner cell mass. Nat Commun. 2017;8(1):1297.
4. Giorgetti L, Lajoie BR, Carter AC, Attia M, Zhan Y, Xu J, et al. Structural organization of the inactive X chromosome in the mouse. Nature. 2016;535(7613):575-9.

Tags: ipsc, reprogramming, x-chromosome

doi: https://doi.org/10.1242/prelights.9336

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