Orchestrating Asymmetric Expression: Mechanisms behind **st Regulation
Department of Developmental Biology, Erasmus MC, University Medical Center, 3015 GD Rotterdam, The Netherlands
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Author to whom correspondence should be addressed.
Epigenomes 2024, 8(1), 6; https://doi.org/10.3390/epigenomes8010006
Submission received: 20 December 2023
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Revised: 19 January 2024
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Accepted: 22 January 2024
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Published: 1 February 2024
(This article belongs to the Special Issue X-Chromosome Inactivation)
Abstract
:Compensation for the gene dosage disequilibrium between sex chromosomes in mammals is achieved in female cells by repressing one of its X chromosomes through a process called X chromosome inactivation (XCI), exemplifying the control of gene expression by epigenetic mechanisms. A critical player in this mechanism is **st, a long, non-coding RNA upregulated from a single X chromosome during early embryonic development in female cells. Over the past few decades, many factors involved at different levels in the regulation of **st have been discovered. In this review, we hierarchically describe and analyze the different layers of **st regulation operating concurrently and intricately interacting with each other to achieve asymmetric and monoallelic upregulation of **st in murine female cells. We categorize these into five different classes: DNA elements, transcription factors, other regulatory proteins, long non-coding RNAs, and the chromatin and topological landscape surrounding **st.
1. Introduction
Dosage compensation of gene expression between chromosomes is essential for survival. Female mammalian cells carry two X chromosomes, while male cells carry an X and a Y chromosome, generating an X-linked gene dosage imbalance between the sexes. To achieve dosage compensation, female diploid epiblast cells inactivate a single X chromosome very early during embryonic development via a complex process known as X chromosome inactivation (XCI). This process results in the epigenetic silencing of one randomly selected X chromosome (** genes. Even though XCI happens in all mammals, different species show different patterns of it. While mice display both types of XCI, rabbits, monkeys, and humans, for instance, only show rXCI, and marsupials display iXCI only. This illustrates the variety of XCI mechanisms present within the mammalian class. New data are emerging on different mechanisms in other mammals, such as rabbits, monkeys and especially humans (reviewed in [1]). However, since most XCI research has been performed in mice and very exciting new data are still being generated nowadays, we focus this review on mouse rXCI.
1.1. Kicking off X Chromosome Inactivation: The Stochastic Model
In 1971, Mary Lyon proposed a model where a diffusible X-linked factor would guarantee XCI of a single X [2]. This model has been further developed into a stochastic model, where female cells sense X chromosome dosage, leading to the inactivation of a single X chromosome while preventing inactivation of the single X chromosome in male cells. The stochastic model proposes that XCI is achieved by a tightly controlled balance between X-encoded activators and autosomally encoded repressors [3]. Many autosomally encoded repressors are pluripotency factors or are linked to the pluripotent state and therefore prevent the inactivation of an X chromosome in the inner cell mass of the embryo (ICM) or in ESCs. ESCs are derived from the ICM, which will give rise to the embryo proper. Upon differentiation, the downregulation of repressors of XCI and upregulation of activators of XCI would tilt the balance towards XCI. Thus, female exclusive activation of XCI is mediated by the double dose of X-encoded XCI activators that are required to overcome the threshold set by the XCI repressors. Initiation of XCI is stochastic, and a negative feedback loop involving rapid silencing of some XCI activators prevents inactivation of the second X chromosome. The application of novel machine learning and mathematical modeling techniques indicates that XCI dynamics can be recapitulated based on the principles of the stochastic model with a limited number of activators and repressors regulating XCI [4,5].
1.2. The X Inactivation Center and the Tsix/** histone demethylases, KDM5C/JARID1C and KDM6A/UTX, implicated in sexual differentiation (see review [52]) with opposing roles in enhancing ** gene, Kdm6a, might be involved in ** XCI [90,91]. RNA FISH experiments indicated that when expressed from the ** XCI, with its nascent RNA detected adjacent to ** mechanisms. Given how essential XCI is for cell survival during female development, the presence of several redundant, “fool proof” mechanisms seems evolutionarily sensible.
Supplementary Materials
The following supporting information can be downloaded at: https://mdpi.longhoe.net/article/10.3390/epigenomes8010006/s1. Table S1: Overview of the different factors involved in ** DNA. Dev. Biol. 2008, 319, 416–425. [Google Scholar] [CrossRef]Van Bemmel, J.G.; Galupa, R.; Gard, C.; Servant, N.; Picard, C.; Davies, J.; Szempruch, A.J.; Zhan, Y.; Żylicz, J.J.; Nora, E.P.; et al. The Bipartite TAD Organization of the X-Inactivation Center Ensures Opposing Developmental Regulation of Tsix and ** Vole. Science 2021, 372, 592–600. [Google Scholar] [CrossRef] Guerra-Almeida, D.; Tschoeke, D.A.; Nunes-da-Fonseca, R. Understanding Small ORF Diversity through a Comprehensive Transcription Feature Classification. DNA Res. 2021, 28, dsab007. [Google Scholar] [CrossRef] [PubMed]
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© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Figure 1.
Schematic overview of the X inactivation center and the Tsix/**st tandem. The **c is located at ~103 Mb on the mouse X chromosome (mm10). It contains several lncRNAs, such as Linx/Lppnx, Tsx, **te, Tsix, **st, Jpx, Ftx, and Xert, and several coding genes, such as Nap1L2, Cdx4, Chic1, Slc16a2, and Rnf12. **st and Tsix are two antisense genes at the center of the **c. In the pluripotent state, their expression is symmetric between both X chromosomes: Tsix is biallelically expressed and represses **st, leading to low levels of **st expression. Upon XCI, Tsix is downregulated from the future **, resulting in monoallelic **st upregulation, while the future Xa maintains low levels of Tsix, further suppressing **st expression. The symmetry is broken, and XCI happens on a single chromosome in diploid female cells.
Figure 1.
Schematic overview of the X inactivation center and the Tsix/**st tandem. The **c is located at ~103 Mb on the mouse X chromosome (mm10). It contains several lncRNAs, such as Linx/Lppnx, Tsx, **te, Tsix, **st, Jpx, Ftx, and Xert, and several coding genes, such as Nap1L2, Cdx4, Chic1, Slc16a2, and Rnf12. **st and Tsix are two antisense genes at the center of the **c. In the pluripotent state, their expression is symmetric between both X chromosomes: Tsix is biallelically expressed and represses **st, leading to low levels of **st expression. Upon XCI, Tsix is downregulated from the future **, resulting in monoallelic **st upregulation, while the future Xa maintains low levels of Tsix, further suppressing **st expression. The symmetry is broken, and XCI happens on a single chromosome in diploid female cells.
Figure 2.
Overview of the different proteins and REs involved in **st and Tsix regulation. Genes that have an activating direct or indirect role on **st are shown in green, while genes involved in **st repression are shown in magenta. Several TF, other proteins, XCI activators, and RE are shown in green or magenta based on their effect on **st expression. (A) In pluripotency, the pluripotency factor network (OCT4, NANOG, SOX2, REX1, PRDM14) keeps Tsix active and represses **st, either directly by binding to Intron 1 or its promoter, or indirectly through Tsix or by inhibiting the **st activator RNF12. CTCF and KAP1 also work towards inhibiting its expression, while the MSL complex supports Tsix expression from its promoter. Another set of activating proteins, such as CHD8 and RIF1, supports low levels of **st expression. (B) At the onset of XCI, reduced pluripotency factor concentrations lead to decreased Tsix expression. Reduction in REX1 is further aided by increased RNF12 expression due to the disappearance of its pluripotent repressors. SPEN is required to shut down Tsix expression to allow **st upregulation. YY1, the paralog of REX1, binds to the P2 promoter of **st, activating it. KDM5C and KDM6A also seem to bind there, leading to the demethylation of H3K4me2/3 to H3K4me1, a mark of enhancers, while seemingly removing H3K27me3. CHD8, however, seems to have an opposing role at the onset of XCI during differentiation because it decreases YY1s accessibility to **st’s promoter. CHD8 seems to fine-tune **st expression depending on the developmental context. Several of the GATA binding factors are required for **st expression during differentiation by binding several of its regulatory sequences. Shades of magenta show **st repressors, while shades of green indicate **st activators and XCI activators.
Figure 2.
Overview of the different proteins and REs involved in **st and Tsix regulation. Genes that have an activating direct or indirect role on **st are shown in green, while genes involved in **st repression are shown in magenta. Several TF, other proteins, XCI activators, and RE are shown in green or magenta based on their effect on **st expression. (A) In pluripotency, the pluripotency factor network (OCT4, NANOG, SOX2, REX1, PRDM14) keeps Tsix active and represses **st, either directly by binding to Intron 1 or its promoter, or indirectly through Tsix or by inhibiting the **st activator RNF12. CTCF and KAP1 also work towards inhibiting its expression, while the MSL complex supports Tsix expression from its promoter. Another set of activating proteins, such as CHD8 and RIF1, supports low levels of **st expression. (B) At the onset of XCI, reduced pluripotency factor concentrations lead to decreased Tsix expression. Reduction in REX1 is further aided by increased RNF12 expression due to the disappearance of its pluripotent repressors. SPEN is required to shut down Tsix expression to allow **st upregulation. YY1, the paralog of REX1, binds to the P2 promoter of **st, activating it. KDM5C and KDM6A also seem to bind there, leading to the demethylation of H3K4me2/3 to H3K4me1, a mark of enhancers, while seemingly removing H3K27me3. CHD8, however, seems to have an opposing role at the onset of XCI during differentiation because it decreases YY1s accessibility to **st’s promoter. CHD8 seems to fine-tune **st expression depending on the developmental context. Several of the GATA binding factors are required for **st expression during differentiation by binding several of its regulatory sequences. Shades of magenta show **st repressors, while shades of green indicate **st activators and XCI activators.
Figure 3.
Overview of the different proteins, REs, lncRNAs, some local chromatin modifications, and topological organization of the **c. Same legend as in Figure 2, plus the role of the different lncRNAs on **st expression, the presence of different activating or repressing histone marks, and the bipartite TAD structure of the **c. (A) In pluripotency, Tsix represses **st thanks to transcription running through its gene body. Tsx seems to prop up **te and Tsix expression, while Linx/Lppnx seem to have a role on **st expression directly or through its RNA molecule at the level of OCT4 and REX1 displacement from the **st locus. The **c presents a bipartite TAD structure separated by a region at the 3′ end of **st known as RS14. Expression of Tsix deposits the heterochromatic and euchromatic histone PTMs H3K36me3 and H3K4me2 across the Tsix/**st gene body as well as the P1 and P2 promoters of **st, maintaining the promoter regions in a poised state. Other histone heterochromatic PTMs found at the P1 and P2 promoters of **st in pluripotency are H3K9me2, H3K9me3, H3K27me1, and H4K20me2. Repression of known **st lncRNA activators is achieved by extended deposition of the heterochromatic mark H3K27me3 across the **st TAD, while expression of known Tsix lncRNA activators is maintained by enrichment of the histone euchromatic marks H3K4me3 and H3K27ac. The Tsix TAD is depicted by the pink triangle, while the **st TAD is depicted by the green triangle. When the lncRNA gene acts on Tsix or **st through its lncRNA molecule, the lncRNA symbol has been added. (B) At the onset of XCI, in the future **, Ftx and Xert will help in **st upregulation through either transcription running through their gene bodies (Ftx) or through RE at their promoters (Xert), while the mechanism of action of Jpx through its RNA molecule is debated. Loss of Tsix expression results in the enrichment of the histone euchromatic marks H3K4me2, H3K4me3, and H3K27ac at the **st P1 and P2 promoters. Tsix downregulation is then maintained by the removal of the euchromatic mark H3K27ac and vague deposition of H3K27me3 at its promoter. Upregulation of known **st lncRNA activators is achieved by depletion of the H3K27me3 hotspot within the **st TAD and subsequent increase in the histone euchromatic PTMs H3K27ac and H3K4me3 at the lncRNA promoters. Subsequently, the downregulation of Tsix lncRNA activators is maintained by the deposition of H3K27me3. Finally, the bipartite TAD structure of the **c is lost (grey). Shades of magenta show **st repressors, while shades of green indicate **st activators and XCI activators. When the lncRNA gene acts on Tsix or **st through its lncRNA molecule, the lncRNA symbol has been added.
Figure 3.
Overview of the different proteins, REs, lncRNAs, some local chromatin modifications, and topological organization of the **c. Same legend as in Figure 2, plus the role of the different lncRNAs on **st expression, the presence of different activating or repressing histone marks, and the bipartite TAD structure of the **c. (A) In pluripotency, Tsix represses **st thanks to transcription running through its gene body. Tsx seems to prop up **te and Tsix expression, while Linx/Lppnx seem to have a role on **st expression directly or through its RNA molecule at the level of OCT4 and REX1 displacement from the **st locus. The **c presents a bipartite TAD structure separated by a region at the 3′ end of **st known as RS14. Expression of Tsix deposits the heterochromatic and euchromatic histone PTMs H3K36me3 and H3K4me2 across the Tsix/**st gene body as well as the P1 and P2 promoters of **st, maintaining the promoter regions in a poised state. Other histone heterochromatic PTMs found at the P1 and P2 promoters of **st in pluripotency are H3K9me2, H3K9me3, H3K27me1, and H4K20me2. Repression of known **st lncRNA activators is achieved by extended deposition of the heterochromatic mark H3K27me3 across the **st TAD, while expression of known Tsix lncRNA activators is maintained by enrichment of the histone euchromatic marks H3K4me3 and H3K27ac. The Tsix TAD is depicted by the pink triangle, while the **st TAD is depicted by the green triangle. When the lncRNA gene acts on Tsix or **st through its lncRNA molecule, the lncRNA symbol has been added. (B) At the onset of XCI, in the future **, Ftx and Xert will help in **st upregulation through either transcription running through their gene bodies (Ftx) or through RE at their promoters (Xert), while the mechanism of action of Jpx through its RNA molecule is debated. Loss of Tsix expression results in the enrichment of the histone euchromatic marks H3K4me2, H3K4me3, and H3K27ac at the **st P1 and P2 promoters. Tsix downregulation is then maintained by the removal of the euchromatic mark H3K27ac and vague deposition of H3K27me3 at its promoter. Upregulation of known **st lncRNA activators is achieved by depletion of the H3K27me3 hotspot within the **st TAD and subsequent increase in the histone euchromatic PTMs H3K27ac and H3K4me3 at the lncRNA promoters. Subsequently, the downregulation of Tsix lncRNA activators is maintained by the deposition of H3K27me3. Finally, the bipartite TAD structure of the **c is lost (grey). Shades of magenta show **st repressors, while shades of green indicate **st activators and XCI activators. When the lncRNA gene acts on Tsix or **st through its lncRNA molecule, the lncRNA symbol has been added.
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Luchsinger-Morcelle, S.J.; Gribnau, J.; Mira-Bontenbal, H. Orchestrating Asymmetric Expression: Mechanisms behind **st Regulation. Epigenomes 2024, 8, 6. https://doi.org/10.3390/epigenomes8010006
AMA Style
Luchsinger-Morcelle SJ, Gribnau J, Mira-Bontenbal H. Orchestrating Asymmetric Expression: Mechanisms behind **st Regulation. Epigenomes. 2024; 8(1):6. https://doi.org/10.3390/epigenomes8010006
Chicago/Turabian StyleLuchsinger-Morcelle, Samuel Jesus, Joost Gribnau, and Hegias Mira-Bontenbal. 2024. "Orchestrating Asymmetric Expression: Mechanisms behind **st Regulation" Epigenomes 8, no. 1: 6. https://doi.org/10.3390/epigenomes8010006
