What Are the Three Phases of X-Chromosome Inactivation?

Cells in female mammals must balance gene expression between their two X chromosomes to prevent a double dose of X-linked genes. This is achieved through X-chromosome inactivation (XCI), which silences one X chromosome in each cell. Without this mechanism, an imbalance could lead to severe developmental issues.

This inactivation occurs in three distinct phases, each playing a crucial role in ensuring stable and heritable gene silencing.

Initiation

The initiation phase of XCI is driven by the long non-coding RNA Xist (X-inactive specific transcript), which marks the future inactive X chromosome (Xi). Xist is transcribed from the X-inactivation center (Xic), a regulatory locus that governs the silencing process. Once expressed, Xist RNA spreads across the chromosome, coating it and recruiting chromatin-modifying complexes to initiate transcriptional repression. This step is tightly regulated to ensure that only one X chromosome is silenced, preventing gene dosage imbalances.

The choice of which X chromosome is inactivated is largely random in embryonic cells but becomes fixed in subsequent divisions. In placental mammals, this random inactivation results in some cells expressing genes from the maternal X and others from the paternal X. In contrast, marsupials and certain extra-embryonic tissues exhibit imprinted XCI, where the paternal X is preferentially silenced. The molecular mechanisms behind this choice involve Xist and its antagonist, Tsix, a non-coding RNA transcribed in the opposite direction. Tsix suppresses Xist on the active X chromosome (Xa), preventing its inactivation. The balance between these RNAs, along with chromatin accessibility and transcription factor binding, determines which chromosome is silenced.

Once Xist is upregulated on the future Xi, it recruits chromatin remodelers and histone-modifying enzymes to initiate repression. The Polycomb repressive complexes PRC1 and PRC2 deposit histone modifications such as H3K27me3, a hallmark of facultative heterochromatin. These modifications create a repressive chromatin environment that blocks RNA polymerase II from accessing gene promoters, effectively silencing transcription. DNA methylation at CpG islands further reinforces this repression, ensuring stable and heritable gene silencing.

Spreading

After Xist RNA coats the future Xi, the spreading phase extends silencing across the entire chromosome. This process involves coordinated recruitment of chromatin-modifying complexes that reinforce repression. Xist interacts with protein partners like SPEN (SHARP), which recruits histone deacetylases such as HDAC3. These enzymes remove acetyl groups from histones, leading to chromatin compaction and transcriptional repression.

Xist RNA also serves as a scaffold for Polycomb group proteins, which deposit repressive histone marks like H3K27me3. PRC2, a key Polycomb repressive complex, is recruited through interactions with Xist and its associated proteins, ensuring gene silencing extends beyond the X-inactivation center (Xic) to distant regions. Some genes, particularly those in late-replicating regions, are more resistant to inactivation. RNA fluorescence in situ hybridization (RNA-FISH) studies show that genes near the Xic are silenced first, while those farther away undergo repression more gradually.

The three-dimensional organization of the Xi plays a major role in spreading. Chromosome conformation capture techniques such as Hi-C reveal that the inactive X adopts a bipartite structure, with one compartment enriched for genes that escape silencing and another consisting of tightly compacted heterochromatin. Cohesin and other architectural proteins help separate silenced regions from transcriptionally active domains. This spatial organization ensures Xist RNA remains localized to the Xi, preventing ectopic gene silencing.

Maintenance

Once XCI is established, silencing must be stably propagated through cell divisions to ensure consistent gene dosage compensation. The maintenance phase relies on epigenetic modifications and chromatin structural changes. Unlike earlier phases, which involve dynamic RNA-mediated processes, maintenance is primarily governed by heritable modifications that persist even without continuous Xist expression.

DNA methylation plays a central role in stability, particularly at CpG islands associated with X-linked genes. Methylation of promoter regions prevents transcription factor binding, maintaining gene silencing across generations.

Histone modifications further reinforce the inactive state. H3K27me3, deposited by PRC2 during spreading, remains a defining feature of the Xi. H2AK119ub, a modification by PRC1, enhances chromatin compaction. Additionally, macroH2A, a histone variant enriched on the Xi, promotes a repressive environment resistant to transcriptional activation.

Nuclear positioning also contributes to maintaining XCI. The Xi is typically localized to the nuclear periphery or within nucleolus-associated heterochromatin, regions associated with transcriptional repression. This spatial sequestration limits interactions between the Xi and transcriptional machinery, reinforcing silencing. 3D chromatin mapping techniques show the Xi adopts a highly compacted structure distinct from the active X, with silenced regions organized into dense chromatin domains that restrict transcriptional activators.

Epigenetic Modifications

Long-term silencing of one X chromosome in female mammals depends on epigenetic modifications that reinforce repression. DNA methylation is a key marker of XCI, particularly at CpG islands in promoter regions. Methylation at these sites blocks transcription factor and RNA polymerase II binding, locking genes in an inactive state. Unlike transient histone modifications, DNA methylation patterns are faithfully copied during replication by DNA methyltransferase 1 (DNMT1), preserving the inactivated state.

Histone modifications further regulate the Xi’s chromatin environment. H3K27me3, deposited by PRC2, maintains chromatin compaction. Ubiquitination of histone H2A at lysine 119 (H2AK119ub) by PRC1 prevents RNA polymerase II from initiating transcription. MacroH2A, a histone variant found predominantly on the Xi, stabilizes the repressed state by altering nucleosome dynamics and limiting chromatin accessibility.

Patterns in Different Cell Types

XCI patterns vary across cell types, with different tissues and developmental stages exhibiting distinct silencing mechanisms. In placental mammals, embryonic cells undergo random XCI, while extra-embryonic tissues such as the placenta display imprinted XCI, where the paternal X chromosome is preferentially silenced. This distinction arises from differences in Xist regulation, as imprinted XCI is controlled by maternal epigenetic marks that suppress Xist activation on the maternal X.

In some cell types, incomplete XCI allows certain genes to escape silencing. These escapee genes, which remain partially active on the Xi, are particularly prevalent in human cells, where up to 15% of X-linked genes exhibit variable expression. Escape from XCI is influenced by chromatin organization, with genes in euchromatic regions more likely to retain transcriptional activity. Many escapee genes play roles in metabolism, immune responses, and neurological function. The extent of escape varies between individuals, contributing to differences in gene expression patterns and potentially influencing susceptibility to X-linked disorders.