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A new production facility for a beverage manufacturer has to provide a storage volume for around 5 million bottles as a refrigerated warehouse. The maximum temperatures were not allowed to exceed 14 °C due to the quality requirements in the production process. To achieve highest energy efficiency and to avoid year-round heating and cooling, the warehouse is passively conditioned: by explicitly coupling it with adjacent soil, its buffering effect was activated via uninsulated wall and floor components in contact with the ground. The warehouse stock was also integrated into the concept as thermal mass. Furthermore, the remaining building envelope was optimized to reduce heat gains and losses to external air to a minimum.

Naïve pluripotency is sustained by a self-reinforcing gene regulatory network (GRN) comprising core and naïve pluripotency-specific transcription factors (TFs). Upon exiting naïve pluripotency, embryonic stem cells (ESCs) transition through a formative post-implantation-like pluripotent state, where they acquire competence for lineage choice. However, the mechanisms underlying disengagement from the naïve GRN and initiation of the formative GRN are unclear. Here, we demonstrate that phosphorylated AKT acts as a gatekeeper that prevents nuclear localisation of FoxO TFs in naïve ESCs. PTEN-mediated reduction of AKT activity upon exit from naïve pluripotency allows nuclear entry of FoxO TFs, enforcing a cell fate transition by binding and activating formative pluripotency-specific enhancers. Indeed, FoxO TFs are necessary and sufficient for the activation of the formative pluripotency-specific GRN. Our work uncovers a pivotal role for FoxO TFs in establishing formative post-implantation pluripotency, a critical early embryonic cell fate transition., (© 2024. The Author(s).)

Enhancers bind transcription factors, chromatin regulators, and non-coding transcripts to modulate the expression of target genes. Here, we report 3D genome structures of single mouse ES cells as they are induced to exit pluripotency and transition through a formative stage prior to undergoing neuroectodermal differentiation. We find that there is a remarkable reorganization of 3D genome structure where inter-chromosomal intermingling increases dramatically in the formative state. This intermingling is associated with the formation of a large number of multiway hubs that bring together enhancers and promoters with similar chromatin states from typically 5-8 distant chromosomal sites that are often separated by many Mb from each other. In the formative state, genes important for pluripotency exit establish contacts with emerging enhancers within these multiway hubs, suggesting that the structural changes we have observed may play an important role in modulating transcription and establishing new cell identities., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

While extended loop extrusion across the entire Igh locus controls V H -DJ H recombination, local regulatory sequences, such as the PAIR elements, may also activate V H gene recombination in pro-B-cells. Here, we show that PAIR-associated V H 8 genes contain a conserved putative regulatory element (V8E) in their downstream sequences. To investigate the function of PAIR4 and its V8.7E, we deleted 890 kb containing all 14 PAIRs in the Igh 5' region, which reduced distal V H gene recombination over a 100-kb distance on either side of the deletion. Reconstitution by insertion of PAIR4-V8.7E strongly activated distal V H gene recombination. PAIR4 alone resulted in lower induction of recombination, indicating that PAIR4 and V8.7E function as one regulatory unit. The pro-B-cell-specific activity of PAIR4 depends on CTCF, as mutation of its CTCF-binding site led to sustained PAIR4 activity in pre-B and immature B-cells and to PAIR4 activation in T-cells. Notably, insertion of V8.8E was sufficient to activate V H gene recombination. Hence, enhancers of the PAIR4-V8.7E module and V8.8E element activate distal V H gene recombination and thus contribute to the diversification of the BCR repertoire in the context of loop extrusion., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Huntington's disease (HD) is a neurodegenerative disorder caused by CAG-repeat expansions in the huntingtin (HTT) gene. The resulting mutant HTT (mHTT) protein induces toxicity and cell death via multiple mechanisms and no effective therapy is available. Here, we employ a genome-wide screening in pluripotent mouse embryonic stem cells (ESCs) to identify suppressors of mHTT toxicity. Among the identified suppressors, linked to HD-associated processes, we focus on Metal response element binding transcription factor 1 (Mtf1). Forced expression of Mtf1 counteracts cell death and oxidative stress caused by mHTT in mouse ESCs and in human neuronal precursor cells. In zebrafish, Mtf1 reduces malformations and apoptosis induced by mHTT. In R6/2 mice, Mtf1 ablates motor defects and reduces mHTT aggregates and oxidative stress. Our screening strategy enables a quick in vitro identification of promising suppressor genes and their validation in vivo, and it can be applied to other monogenic diseases., (© 2023. The Author(s).)

Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expression levels of the translation initiation factor Eif4a2 and its premature termination codon-encoding isoform ( Eif4a2 PTC ). NMD deficiency leads to translation of the truncated eIF4A2 PTC protein. eIF4A2 PTC elicits increased mTORC1 activity and translation rates and causes differentiation delays. This establishes a previously unknown feedback loop between NMD and translation initiation. Furthermore, our results show a clear hierarchy in the severity of target deregulation and differentiation phenotypes between NMD effector KOs ( Smg5 KO > Smg6 KO > Smg7 KO), which highlights heterodimer-independent functions for SMG5 and SMG7. Together, our findings expose an intricate link between mRNA homeostasis and mTORC1 activity that must be maintained for normal dynamics of cell state transitions., (© 2022 Huth et al.; Published by Cold Spring Harbor Laboratory Press.)

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay., (© 2021. The Author(s).)

In mammalian genomes, differentially methylated regions (DMRs) and histone marks including trimethylation of histone 3 lysine 27 (H3K27me3) at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. However, neither parent-of-origin-specific transcription nor imprints have been comprehensively mapped at the blastocyst stage of preimplantation development. Here, we address this by integrating transcriptomic and epigenomic approaches in mouse preimplantation embryos. We find that seventy-one genes exhibit previously unreported parent-of-origin-specific expression in blastocysts (nBiX: novel blastocyst-imprinted expressed). Uniparental expression of nBiX genes disappears soon after implantation. Micro-whole-genome bisulfite sequencing (µWGBS) of individual uniparental blastocysts detects 859 DMRs. We further find that 16% of nBiX genes are associated with a DMR, whereas most are associated with parentally-biased H3K27me3, suggesting a role for Polycomb-mediated imprinting in blastocysts. nBiX genes are clustered: five clusters contained at least one published imprinted gene, and five clusters exclusively contained nBiX genes. These data suggest that early development undergoes a complex program of stage-specific imprinting involving different tiers of regulation.

In the mammalian embryo, epiblast cells must exit the naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene networks involved in the exit from naïve pluripotency remains fragmentary. Here, we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large-scale transcriptomics and computational systems biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 ESC lines deficient for regulators of the exit from naïve pluripotency predominantly manifest delays on the trajectory from naïve to formative epiblast. We find that gene networks operative in ESCs are also active during transition from pre- to post-implantation epiblast in utero. We identified 496 naïve state-associated genes tightly connected to the in vivo epiblast state transition and largely conserved in primate embryos. Integrated analysis of mutant transcriptomes revealed funnelling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signalling pathways direct this pivotal mammalian cell state transition., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Many genes are regulated by multiple enhancers that often simultaneously activate their target gene. However, how individual enhancers collaborate to activate transcription is not well understood. Here, we dissect the functions and interdependencies of five enhancer elements that together activate Fgf5 expression during exit from naive murine pluripotency. Four intergenic elements form a super-enhancer, and most of the elements contribute to Fgf5 induction at distinct time points. A fifth, poised enhancer located in the first intron contributes to Fgf5 expression at every time point by amplifying overall Fgf5 expression levels. Despite low individual enhancer activity, together these elements strongly induce Fgf5 expression in a super-additive fashion that involves strong accumulation of RNA polymerase II at the intronic enhancer. Finally, we observe a strong anti-correlation between RNA polymerase II levels at enhancers and their distance to the closest promoter, and we identify candidate elements with properties similar to the intronic enhancer., Competing Interests: Declaration of interests J.W. is a member of the CAMP4 scientific advisory board and the advisory board of Molecular Cell., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Fluorescence imaging and chromosome conformation capture assays such as Hi-C are key tools for studying genome organization. However, traditionally, they have been carried out independently, making integration of the two types of data difficult to perform. By trapping individual cell nuclei inside a well of a 384-well glass-bottom plate with an agarose pad, we have established a protocol that allows both fluorescence imaging and Hi-C processing to be carried out on the same single cell. The protocol identifies 30,000-100,000 chromosome contacts per single haploid genome in parallel with fluorescence images. Contacts can be used to calculate intact genome structures to better than 100-kb resolution, which can then be directly compared with the images. Preparation of 20 single-cell Hi-C libraries using this protocol takes 5 d of bench work by researchers experienced in molecular biology techniques. Image acquisition and analysis require basic understanding of fluorescence microscopy, and some bioinformatics knowledge is required to run the sequence-processing tools described here.

The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.

One of the two X chromosomes in female mammals is inactivated by the noncoding Xist RNA. In mice, X chromosome inactivation (XCI) is regulated by the antisense RNA Tsix, which represses Xist on the active X chromosome. In the absence of Tsix, PRC2-mediated histone H3 lysine 27 trimethylation (H3K27me3) is established over the Xist promoter. Simultaneous disruption of Tsix and PRC2 leads to derepression of Xist and in turn silencing of the single X chromosome in male embryonic stem cells. Here, we identified histone H3 lysine 36 trimethylation (H3K36me3) as a modification that is recruited by Tsix cotranscriptionally and extends over the Xist promoter. Reduction of H3K36me3 by expression of a mutated histone H3.3 with a substitution of methionine for lysine at position 36 causes a significant derepression of Xist. Moreover, depletion of the H3K36 methylase Setd2 leads to upregulation of Xist, suggesting H3K36me3 as a modification that contributes to the mechanism of Tsix function in regulating XCI. Furthermore, we found that reduction of H3K36me3 does not facilitate an increase in H3K27me3 over the Xist promoter, indicating that additional mechanisms exist by which Tsix blocks PRC2 recruitment to the Xist promoter., (Copyright © 2015 Ohhata et al.)

Haploid genetics has facilitated new insights into mammalian pathways and disease mechanisms. Most animal cells are diploid, and mammalian haploid cell cultures have remained elusive for a long time. Recent methodological progress has enabled the routine derivation of haploid stem cell lines from mammalian haploid embryos. Here we provide detailed protocols for the establishment, culture, and manipulation of parthenogenetic and androgenetic haploid embryonic stem cells from mouse embryos., (Copyright © 2015 John Wiley & Sons, Inc.)

Self-renewal circuitry in embryonic stem cells (ESCs) is increasingly defined. How the robust pluripotency program is dissolved to enable fate transition is less appreciated. Here we develop a forward genetic approach using haploid ESCs. We created libraries of transposon integrations and screened for persistent self-renewal in differentiation-permissive culture. This yielded multiple mutants in the Fgf/Erk and GSK3/Tcf3 modules known to drive differentiation and in epigenetic modifiers implicated in lineage commitment. We also identified and validated factors not previously considered. These include the conserved small zinc finger protein Zfp706 and the RNA binding protein Pum1. Pum1 targets several mRNAs for naive pluripotency transcription factors and accelerates their downregulation at the onset of differentiation. These findings indicate that the dismantling of pluripotent circuitry proceeds at multiple levels. More broadly they exemplify the power of haploid ESCs for genetic interrogation of developmental processes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Sequencing projects have revealed the information of many animal genomes and thereby enabled the exploration of genome evolution. Insights into how genomes have been repeatedly modified provide a basis for understanding evolutionary innovation and the ever increasing complexity of animal developmental programs. Animal genomes are diploid in most cases, suggesting that redundant information in two copies of the genome increases evolutionary fitness. Genomes are well adapted to a diploid state. Changes of ploidy can be accommodated early in development but they rarely permit successful development into adulthood. In mammals, epigenetic mechanisms including imprinting and X inactivation restrict haploid development. These restrictions are relaxed in an early phase of development suggesting that dosage regulation appears less critical. Here we review the recent literature on haploid genomes and dosage effects and try to embed recent findings in an evolutionary perspective.

Background: Histone deacetylase inhibitors (HDACi) cause histone hyperacetylation and H3K4 hypermethylation in various cell types. They find clinical application as anti-epileptics and chemotherapeutic agents, but the pathways through which they operate remain unclear. Surprisingly, changes in gene expression caused by HDACi are often limited in extent and can be positive or negative. Here we have explored the ability of the clinically important HDACi valproic acid (VPA) to alter histone modification and gene expression, both globally and at specific genes, in mouse embryonic stem (ES) cells., Results: Microarray expression analysis of ES cells exposed to VPA (1 mM, 8 h), showed that only 2.4% of genes showed a significant, >1.5-fold transcriptional change. Of these, 33% were down-regulated. There was no correlation between gene expression and VPA-induced changes in histone acetylation or H3K4 methylation at gene promoters, which were usually minimal. In contrast, all Hoxb genes showed increased levels of H3K9ac after exposure to VPA, but much less change in other modifications showing bulk increases. VPA-induced changes were lost within 24 h of inhibitor removal. VPA significantly increased the low transcription of Hoxb4 and Hoxb7, but not other Hoxb genes. Expression of Hoxb genes increased in ES cells lacking functional Polycomb silencing complexes PRC1 and PRC2. Surprisingly, VPA caused no further increase in Hoxb transcription in these cells, except for Hoxb1, whose expression increased several fold. Retinoic acid (RA) increased transcription of all Hoxb genes in differentiating ES cells within 24 h, but thereafter transcription remained the same, increased progressively or fell progressively in a locus-specific manner., Conclusions: Hoxb genes in ES cells are unusual in being sensitive to VPA, with effects on both cluster-wide and locus-specific processes. VPA increases H3K9ac at all Hoxb loci but significantly overrides PRC-mediated silencing only at Hoxb4 and Hoxb7. Hoxb1 is the only Hoxb gene that is further up-regulated by VPA in PRC-deficient cells. Our results demonstrate that VPA can exert both cluster-wide and locus-specific effects on Hoxb regulation.

Haploid embryonic stem cells (ESCs) have recently been derived from parthenogenetic mouse embryos and offer new possibilities for genetic screens. The ability of haploid ESCs to give rise to a wide range of differentiated cell types in the embryo and in vitro has been demonstrated. However, it has remained unclear whether haploid ESCs can contribute to the germline. Here, we show that parthenogenetic haploid ESCs at high passage have robust germline competence enabling the production of transgenic mouse strains from genetically modified haploid ESCs. We also show that differentiation of haploid ESCs in the embryo correlates with the gain of a diploid karyotype and that diploidisation is the result of endoreduplication and not cell fusion. By contrast, we find that a haploid karyotype is maintained when differentiation to an extra-embryonic fate is forced by induction of Gata6.

The distinct cell types of the body are established from the fertilized egg in development and assembled into functional tissues. Functional characteristics and gene expression patterns are then faithfully maintained in somatic cell lineages over a lifetime. On the molecular level, transcription factors initiate lineage-specific gene expression programmmes and epigenetic regulation contributes to stabilization of expression patterns. Epigenetic mechanisms are essential for maintaining stable cell identities and their disruption can lead to disease or cellular transformation. Here, we discuss the role of epigenetic regulation in the early mouse embryo, which presents a relatively well-understood system. A number of studies have contributed to the understanding of the function of Polycomb group complexes and the DNA methylation system. The role of many other chromatin regulators in development remains largely unexplored. Albeit the current picture remains incomplete, the view emerges that multiple epigenetic mechanisms cooperate for repressing critical developmental regulators. Some chromatin modifications appear to act in parallel and others might repress the same gene at a different stage of cell differentiation. Studies in pluripotent mouse embryonic stem cells show that epigenetic mechanisms function to repress lineage specific gene expression and prevent extraembryonic differentiation. Insights into this epigenetic "memory" of the first lineage decisions help to provide a better understanding of the function of epigenetic regulation in adult stem cell differentiation.

Most animals are diploid, but haploid-only and male-haploid (such as honeybee and ant) species have been described. The diploid genomes of complex organisms limit genetic approaches in biomedical model species such as mice. To overcome this problem, experimental induction of haploidy has been used in fish. Haploid development in zebrafish has been applied for genetic screening. Recently, haploid pluripotent cell lines from medaka fish (Oryzias latipes) have also been established. In contrast, haploidy seems less compatible with development in mammals. Although haploid cells have been observed in egg cylinder stage parthenogenetic mouse embryos, most cells in surviving embryos become diploid. Here we describe haploid mouse embryonic stem cells and show their application in forward genetic screening.

How polycomb group proteins repress gene expression in vivo is not known. While histone-modifying activities of the polycomb repressive complexes (PRCs) have been studied extensively, in vitro data have suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that PRCs are required to maintain a compact chromatin state at Hox loci in embryonic stem cells (ESCs). There is specific decompaction in the absence of PRC2 or PRC1. This is due to a PRC1-like complex, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state and to repress Hox gene expression is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Mammals compensate X chromosome gene dosage between the sexes by silencing of one of the two female X chromosomes. X inactivation is initiated in the early embryo and requires the non-coding Xist RNA, which encompasses the inactive X chromosome (Xi) and triggers its silencing. In differentiated cells, several factors including the histone variant macroH2A and the scaffold attachment factor SAF-A are recruited to the Xi and maintain its repression. Consequently, in female somatic cells the Xi remains stably silenced independently of Xist. Here, we identify the Trithorax group protein Ash2l as a novel component of the Xi. Ash2l is recruited by Xist concomitantly with Saf-A and macroH2A at the transition to Xi maintenance. Recruitment of these factors characterizes a developmental transition point for the chromatin composition of the Xi. Surprisingly, expression of a mutant Xist RNA that does not cause gene repression can trigger recruitment of Ash2l, Saf-A and macroH2A to the X chromosome, and can cause chromosome-wide histone H4 hypoacetylation. This suggests that a chromatin configuration is established on non-genic chromatin on the Xi by Xist to provide a repressive compartment that could be used for maintaining gene silencing. Gene silencing is mechanistically separable from the formation of this repressive compartment and, thus, requires additional pathways. This observation highlights a crucial role for spatial organization of chromatin changes in the maintenance of X inactivation.

Polycomb complexes establish chromatin modifications for maintaining gene repression and are essential for embryonic development in mice. Here we use pluripotent embryonic stem (ES) cells to demonstrate an unexpected redundancy between Polycomb-repressive complex 1 (PRC1) and PRC2 during the formation of differentiated cells. ES cells lacking the function of either PRC1 or PRC2 can differentiate into cells of the three germ layers, whereas simultaneous loss of PRC1 and PRC2 abrogates differentiation. On the molecular level, the differentiation defect is caused by the derepression of a set of genes that is redundantly repressed by PRC1 and PRC2 in ES cells. Furthermore, we find that genomic repeats are Polycomb targets and show that, in the absence of Polycomb complexes, endogenous murine leukemia virus elements can mobilize. This indicates a contribution of the Polycomb group system to the defense against parasitic DNA, and a potential role of genomic repeats in Polycomb-mediated gene regulation.

Non-coding RNAs regulate dosage compensation in mammals by controlling transcriptional silencing of one of the two X chromosomes in females. The two major transcripts involved in this process are Xist and its antisense counterpart Tsix. Expression of Xist and Tsix from the X inactivation center is mutually exclusive. Xist expression triggers chromosome wide silencing of the X chromosome from which it is transcribed. Tsix is a repressor of Xist and is specifically expressed from the other X chromosome, maintaining its activity. Here, we review non-coding RNAs that have been implicated in X chromosome inactivation. Focusing on the best studied transcripts Xist and Tsix we portray a current perspective on chromosome wide gene regulation by non-coding RNAs.

The noncoding Xist RNA triggers silencing of one of the two female X chromosomes during X inactivation in mammals. Gene silencing by Xist is restricted to a special developmental context in early embryos and specific hematopoietic precursors. Here, we show that Xist can initiate silencing in a lymphoma model. We identify the special AT-rich binding protein SATB1 as an essential silencing factor. Loss of SATB1 in tumor cells abrogates the silencing function of Xist. In lymphocytes Xist localizes along SATB1-organized chromatin and SATB1 and Xist influence each other's pattern of localization. SATB1 and its homolog SATB2 are expressed during the initiation window for X inactivation in ES cells. Importantly, viral expression of SATB1 or SATB2 enables gene silencing by Xist in embryonic fibroblasts, which normally do not provide an initiation context. Thus, our data establish SATB1 as a crucial silencing factor contributing to the initiation of X inactivation.

The Polycomb group (PcG) gene Ring1B has been implicated in the repression of developmental control genes and X inactivation and is essential for embryogenesis. Ring1B protein contains a RING finger domain and functions as an E3 ubiquitin ligase that is crucial for the monoubiquitination of histone H2A (H2AK119ub1). Here, we study the function of Ring1B in mouse embryonic stem (ES) cells. The deletion of Ring1B causes the loss of several PcG proteins, showing an unanticipated function in the regulation of PcG protein levels. Derepression of lineage genes and an aberrant differentiation potential is observed in Ring1B-deficient ES cells. Despite a crucial function of Ring1B in establishing the chromosome-wide ubiquitination of histone H2A lysine 119 (H2AK119ub1) upon Xist expression in ES cells, the initiation of silencing by Xist is independent of Ring1B. Other chromatin marks associated with the initiation of X inactivation are not affected in Ring1B-deficient cells, suggesting compensation for the loss of Ring1B in X inactivation in contrast to the repression of lineage genes.

X inactivation is the mechanism by which mammals adjust the genetic imbalance that arises from the different numbers of gene-rich X-chromosomes between the sexes. The dosage difference between XX females and XY males is functionally equalized by silencing one of the two X chromosomes in females. This dosage-compensation mechanism seems to have arisen concurrently with early mammalian evolution and is based on the long functional Xist RNA, which is unique to placental mammals. It is likely that previously existing mechanisms for other cellular functions have been recruited and adapted for the evolution of X inactivation. Here, we critically review our understanding of dosage compensation in placental mammals and place these findings in the context of other cellular processes that intersect with mammalian dosage compensation.