X chromosome reactivation dynamics reveal stages of reprogramming to pluripotency.

Reprogramming to iPSCs resets the epigenome of somatic cells, including the reversal of X chromosome inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X inactivation, whereas others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming.

Does XIST safeguard against sex-biased human diseases?

Yu et al. (2021) demonstrate that a subset of X-linked immune genes is repressed on the inactive X chromosome (Xi) in a manner dependent on XIST RNA in B cells, and derepression of these genes upon XIST depletion could bias differentiation of naive B cells and be involved in etiology of female-biased autoimmune diseases.

SmcHD1 underlies the formation of H3K9me3 blocks on the inactive X chromosome in mice.

Stable silencing of the inactive X chromosome (Xi) in female mammals is crucial for the development of embryos and their postnatal health. SmcHD1 is essential for stable silencing of the Xi, and its functional deficiency results in derepression of many X-inactivated genes. Although SmcHD1 has been suggested to play an important role in the formation of higher-order chromatin structure of the Xi, the underlying mechanism is largely unknown. Here, we explore the epigenetic state of the Xi in SmcHD1-deficient epiblast stem cells and mouse embryonic fibroblasts in comparison with their wild-type counterparts. The results suggest that SmcHD1 underlies the formation of H3K9me3-enriched blocks on the Xi, which, although the importance of H3K9me3 has been largely overlooked in mice, play a crucial role in the establishment of the stably silenced state. We propose that the H3K9me3 blocks formed on the Xi facilitate robust heterochromatin formation in combination with H3K27me3, and that the substantial loss of H3K9me3 caused by SmcHD1 deficiency leads to aberrant distribution of H3K27me3 on the Xi and derepression of X-inactivated genes.

A passive exoskeleton can assist split-belt adaptation.

An exoskeletal device can assist walking in those with gait deficits. A passive exoskeleton can be a favorable choice for local or home rehabilitation settings because it is affordable, light weight, and less complex to utilize. While there is research that investigates the effects of exoskeleton on gait research examining the effects of such devices on gait adaptation, is rare. This is important because in diseases like stroke, the ability to flexibly adapt is affected, such that functional recovery becomes difficult. The purpose of this study was to characterize gait adaptation patterns that result from exoskeleton usage during a split-belt adaptation task. Healthy young participants were randomly assigned to a unilateral exoskeleton or a no-exoskeleton group. Each participant performed the specific split-belt adaptation tasks on the treadmill, where the speed of each belt could be controlled independently. Symmetry indices of spatiotemporal variables were calculated to quantify gait adaptation. To analyze the adaptation, trials were divided into early and late adaptation. We also analyzed degree of adaptation, and transfer effects. We also measured the symmetry of the positive power generated by the individual legs during the split-belt task to determine if using exoskeleton assistance reduced power in the exoskeleton group versus the no-exoskeleton group. Use of a passive exoskeleton device altered gait adaptation during a split-belt treadmill task in comparison to the control group. Such adaptation was found to be largely restricted to the temporal domain. Changes in the gait coordination patterns consisted of both early and late adaptive changes, especially in intra-limb patterns like stance time rather than inter-limb patterns like step time. Although the symmetry of the positive power generated during the split-belt task was found to be reduced for the exoskeleton-assistance group, it was shown that this was primarily the result of increased positive power generated by the side not receiving exoskeletal assistance. An unpowered assistive device can provide a unique solution for coordinating the lower limbs during different gait tasks. Such a solution could reduce the neural burden of adaptation consequently resulting in a reduction of the mechanical burden of walking during the bilateral gait coordination task. This may be useful for accelerating gait rehabilitation in different patient populations. However, balance control is important to consider during unilateral exoskeletal assistance.

Role of SmcHD1 in establishment of epigenetic states required for the maintenance of the X-inactivated state in mice.

X inactivation in mammals is regulated by epigenetic modifications. Functional deficiency of SmcHD1 has been shown to cause de-repression of X-inactivated genes in post-implantation female mouse embryos, suggesting a role of SmcHD1 in the maintenance of X inactivation. Here, we show that de-repression of X-inactivated genes accompanied a local reduction in the enrichment of H3K27me3 in mouse embryonic fibroblasts deficient for SmcHD1. Furthermore, many of these genes overlapped with those having a significantly lower enrichment of H3K27me3 at the blastocyst stage in wild type. Intriguingly, however, depletion of SmcHD1 did not compromise the X-inactivated state in immortalized female mouse embryonic fibroblasts, in which X inactivation had been established and maintained. Taking all these findings together, we suggest that SmcHD1 facilitates the incorporation of H3K27me3 and perhaps other epigenetic modifications at gene loci that are silenced even with the lower enrichment of H3K27me3 at the early stage of X inactivation. The epigenetic state at these loci would, however, remain as it is at the blastocyst stage in the absence of SmcHD1 after implantation, which would eventually compromise the maintenance of the X-inactivated state at later stages.

Characterization of genetic-origin-dependent monoallelic expression in mouse embryonic stem cells.

Monoallelic gene expression occurs in various mammalian cells and can be regulated genetically, epigenetically and/or stochastically. We identified 145 monoallelically expressed genes (MoEGs), including seven known imprinted genes, in mouse embryonic stem cells (ESCs) derived from reciprocal F1 hybrid blastocysts and cultured in 2i/LIF. As all MoEGs except for the imprinted genes were expressed in a genetic-origin-dependent manner, we focused on this class of MoEGs for mechanistic studies. We showed that a majority of the genetic-origin-dependent MoEGs identified in 2i/LIF ESCs remain monoallelically expressed in serum/LIF ESCs, but become more relaxed or even biallelically expressed upon differentiation. These MoEGs and their regulatory regions were highly enriched for single nucleotide polymorphisms. In addition, some MoEGs were associated with retrotransposon insertions/deletions, consistent with the fact that certain retrotransposons act as regulatory elements in pluripotent stem cells. Interestingly, most MoEGs showed allelic differences in enrichment of histone H3K27me and H3K4me marks, linking allelic epigenetic differences and monoallelic expression. In contrast, there was little or no allelic difference in CpG methylation or H3K9me. Taken together, our study highlights the impact of genetic variation including single nucleotide polymorphisms and retrotransposon insertions/deletions on monoallelic epigenetic marks and expression in ESCs.

The Kickstart Walk Assist System for improving balance and walking function in stroke survivors: a feasibility study.

BACKGROUND: Compared with traditional physical therapy for stroke patients, lower extremity exoskeletons can provide patients with greater endurance and more repeatable and controllable training, which can reduce the therapeutic burden of the therapist. However, most exoskeletons are expensive, heavy or require active power to be operated. Therefore, a lighter, easy to wear, easy to operate, low-cost technology for stroke rehabilitation would be a welcome opportunity for stroke survivors, caregivers and clinicians. One such device is the Kickstart Walk Assist system and the purpose of this study was to determine feasibility of using this unpowered exoskeleton device in a sample of stroke survivors. METHODS: Thirty stroke survivors were enrolled in the study and experienced walking with the Kickstart exoskeleton device that provided spring-loaded assistance during gait. After 5 days of wearing the exoskeleton, participants were evaluated in the two states of wearing and not wearing the exoskeleton. Outcome measures included: (a) spatio-temporal gait measures, (b) balance measures and (c) exoskeleton-use feedback questionnaire. RESULTS: In comparison to not wearing the device, when participants wore the Kickstart walking system, weight bearing asymmetry was reduced. The time spent on the 10-m walk test was also reduced, but there was no difference in the timed-up-and-go test (TUGT). Gait analysis data showed reduction in step time and double support time. Stroke survivors were positive about the Kickstart walking system's ability to improve their balance, speed and gait. In addition, their confidence level and willingness to use the device was also positive. CONCLUSIONS: These findings show the feasibility of using the Kickstart walking system for improving walking performance in stroke survivors. Our future goal is to perform a longer duration study with more comprehensive pre- and post-testing in a larger sample of stroke survivors. Trial registration Chinese Clinical Trial Registry, ChiCTR2000032665. Registered 5 May 2020-Retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=53288.

Defects in dosage compensation impact global gene regulation in the mouse trophoblast.

Xist RNA, which is responsible for X inactivation, is a key epigenetic player in the embryogenesis of female mammals. Of the several repeats conserved in Xist RNA, the A-repeat has been shown to be essential for its silencing function in differentiating embryonic stem cells. Here, we introduced a new Xist allele into mouse that produces mutated Xist RNA lacking the A-repeat (XistCAGΔ5' ). XistCAGΔ5' RNA expressed in the embryo coated the X chromosome but failed to silence it. Although imprinted X inactivation was substantially compromised upon paternal transmission, allele-specific RNA-seq in the trophoblast revealed that XistCAGΔ5' RNA still retained some silencing ability. Furthermore, the failure of imprinted X inactivation had more significant impacts than expected on genome-wide gene expression. It is likely that dosage compensation is required not only for equalizing X-linked gene expression between the sexes but also for proper global gene regulation in differentiated female somatic cells.

The 5' region of Xist RNA has the potential to associate with chromatin through the A-repeat.

X inactive-specific transcript (Xist) is a long noncoding RNA that plays an essential role in X chromosome inactivation. Although Xist RNA, like common protein-coding mRNAs, is transcribed by RNA polymerase II, spliced and polyadenylated, it is retained in the nucleus and associates with the X chromosome it originates from. It has been assumed that Xist RNA recruits proteins involved in epigenetic modifications and chromatin compaction to the X chromosome. One of the major proteins constituting the nuclear matrix, hnRNP U, has been shown to be required for the association of Xist RNA with the inactive X chromosome (Xi). In this study, we found that the first 950-nt sequence of Xist RNA had the potential to associate with chromatin in a manner independent of hnRNP U. Furthermore, its chromatin association is apparently dependent on the presence of an intact A-repeat sequence, which is one of the repeats in Xist/XIST RNA conserved among many mammalian species, and has been shown to be important for Xist RNA-mediated silencing. Taking this unexpected finding and a previous study demonstrating the effect of Xist RNA lacking the A-repeat on the formation of the silent heterochromatin domain together, we suggest that the A-repeat captures chromatin near the initial loading site of Xist RNA and relocates it into the core of the heterochromatin domain.

Maintenance of Xist Imprinting Depends on Chromatin Condensation State and Rnf12 Dosage in Mice.

In female mammals, activation of Xist (X-inactive specific transcript) is essential for establishment of X chromosome inactivation. During early embryonic development in mice, paternal Xist is preferentially expressed whereas maternal Xist (Xm-Xist) is silenced. Unlike autosomal imprinted genes, Xist imprinting for Xm-Xist silencing was erased in cloned or parthenogenetic but not fertilized embryos. However, the molecular mechanism underlying the variable nature of Xm-Xist imprinting is poorly understood. Here, we revealed that Xm-Xist silencing depends on chromatin condensation states at the Xist/Tsix genomic region and on Rnf12 expression levels. In early preimplantation, chromatin decondensation via H3K9me3 loss and histone acetylation gain caused Xm-Xist derepression irrespective of embryo type. Although the presence of the paternal genome during pronuclear formation impeded Xm-Xist derepression, Xm-Xist was robustly derepressed when the maternal genome was decondensed before fertilization. Once Xm-Xist was derepressed by chromatin alterations, the derepression was stably maintained and rescued XmXpΔ lethality, indicating that loss of Xm-Xist imprinting was irreversible. In late preimplantation, Oct4 served as a chromatin opener to create transcriptional permissive states at Xm-Xist/Tsix genomic loci. In parthenogenetic embryos, Rnf12 overdose caused Xm-Xist derepression via Xm-Tsix repression; physiological Rnf12 levels were essential for Xm-Xist silencing maintenance in fertilized embryos. Thus, chromatin condensation and fine-tuning of Rnf12 dosage were crucial for Xist imprint maintenance by silencing Xm-Xist.

A new Xist allele driven by a constitutively active promoter is dominated by Xist locus environment and exhibits the parent-of-origin effects.

The dosage difference of X-linked genes between the sexes in mammals is compensated for by genetic inactivation of one of the X chromosomes in XX females. A noncoding RNA transcribed from the Xist gene at the onset of X chromosome inactivation coats the X chromosome in cis and induces chromosome-wide heterochromatinization. Here, we report a new Xist allele (Xist(CAG)) driven by a CAG promoter, which is known to be constitutively active in many types of cells. The paternal transmission of Xist(CAG) resulted in the preferential inactivation of the targeted paternal X (Xp) not only in the extra-embryonic but also the embryonic lineage, whereas maternal transmission ended with embryonic lethality at the early postimplantation stage with a phenotype that resembled mutant embryos carrying a maternal deficiency in Tsix, an antisense negative regulator of Xist, in both sexes. Interestingly, we found that the upregulation of Xist(CAG) in preimplantation embryos temporally differed depending on its parental origin: its expression started at the 4- to 8-cell stages when paternally inherited, and Xist(CAG) was upregulated at the blastocyst stage when maternally inherited. This might indicate that the Xist locus on Xp is permissive to transcription, but the Xist locus on the maternal X (Xm) is not. We extrapolated from these findings that the maternal Xist allele might manifest a chromatin structure inaccessible by transcription factors relative to the paternal allele. This might underlie the mechanism for the maternal repression of Xist at the early cleavage stage when Tsix expression has not yet occurred on Xm.

Targeted gene silencing in mouse germ cells by insertion of a homologous DNA into a piRNA generating locus.

In germ cells, early embryos, and stem cells of animals, PIWI-interacting RNAs (piRNAs) have an important role in silencing retrotransposons, which are vicious genomic parasites, through transcriptional and post-transcriptional mechanisms. To examine whether the piRNA pathway can be used to silence genes of interest in germ cells, we have generated knock-in mice in which a foreign DNA fragment was inserted into a region generating pachytene piRNAs. The knock-in sequence was transcribed, and the resulting RNA was processed to yield piRNAs in postnatal testes. When reporter genes possessing a sequence complementary to portions of the knock-in sequence were introduced, they were greatly repressed after the time of pachytene piRNA generation. This repression mainly occurred at the post-transcriptional level, as degradation of the reporter RNAs was accelerated. Our results show that the piRNA pathway can be used as a tool for sequence-specific gene silencing in germ cells and support the idea that the piRNA generating regions serve as traps for retrotransposons, enabling the host cell to generate piRNAs against active retrotransposons.

The CENP-O complex requirement varies among different cell types.

CENP-U (CENP-50) is a component of the CENP-O complex, which includes CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-U and is constitutively localized at kinetochores throughout the cell cycle in vertebrates. Although CENP-U deficiency results in some mitotic defects in chicken DT40 cells, CENP-U-deficient chicken DT40 cells are viable. To examine the functional roles of CENP-U in an organism-dependent context, we generated CENP-U-deficient mice. The CENP-U-deficient mice died during early embryogenesis (approximately E7.5). Thus, conditional CENP-U-deficient mouse ES cells were generated to analyze CENP-U-deficient phenotypes at the cell level. When CENP-U was disrupted in the mouse ES cells, all CENP-O complex proteins disappeared from kinetochores. In contrast, other kinetochore proteins were recruited in CENP-U-deficient mouse ES cells as CENP-U-deficient DT40 cells. However, the CENP-U-deficient ES cells died after exhibiting abnormal mitotic behavior. Although CENP-U was essential for cell viability during mouse early embryogenesis, CENP-U-deficient mouse embryonic fibroblast cells were viable, similar to the DT40 cells. Thus, although both DT40 and ES cells with CENP-U deficiency have similar mitotic defects, cellular responses to mitotic defects vary among different cell types.

Incomplete X-inactivation initiated by a hypomorphic Xist allele in the mouse.

X chromosome inactivation (X-inactivation) in female mammals is triggered by differential upregulation of the Xist gene on one of the two X chromosomes and subsequent coating of the X in cis with its non-coding transcripts. Although targeted mutation has clearly shown that Xist is essential for X-inactivation in cis, the molecular mechanism by which Xist RNA induces chromosome silencing is largely unknown. Here, we demonstrate that an Xist mutant generated previously in mouse by gene targeting, Xist(IVS), is unique in that it partially retains the capacity to silence the X chromosome. Although Xist(IVS) is differentially upregulated and its mutated transcript coats the X chromosome in cis in embryonic and extra-embryonic tissues, X-inactivation thus initiated does not seem to be fully established. The state of such incomplete inactivation is probably unstable and the mutated X is apparently reactivated in a subset of extra-embryonic tissues and, perhaps, early epiblastic cells. Xist(IVS), which can be referred to as a partial loss-of-function mutation, would provide an opportunity to dissect the molecular mechanism of Xist RNA-mediated chromosome silencing.

Species-specific differences in X chromosome inactivation in mammals.

In female mammals, the dosage difference in X-linked genes between XX females and XY males is compensated for by inactivating one of the two X chromosomes during early development. Since the discovery of the X inactive-specific transcript (XIST) gene in humans and its subsequent isolation of the mouse homolog, Xist, in the early 1990s, the molecular basis of X chromosome inactivation (X-inactivation) has been more fully elucidated using genetically manipulated mouse embryos and embryonic stem cells. Studies on X-inactivation in other mammals, although limited when compared with those in the mice, have revealed that, while their inactive X chromosome shares many features with those in the mice, there are marked differences in not only some epigenetic modifications of the inactive X chromosome but also when and how X-inactivation is initiated during early embryonic development. Such differences raise the issue about what extent of the molecular basis of X-inactivation in the mice is commonly shared among others. Recognizing similarities and differences in X-inactivation among mammals may provide further insight into our understanding of not only the evolutionary but also the molecular aspects for the mechanism of X-inactivation. Here, we reviewed species-specific differences in X-inactivation and discussed what these differences may reveal.

Replication dynamics identifies the folding principles of the inactive X chromosome.

Chromosome-wide late replication is an enigmatic hallmark of the inactive X chromosome (Xi). How it is established and what it represents remains obscure. By single-cell DNA replication sequencing, here we show that the entire Xi is reorganized to replicate rapidly and uniformly in late S-phase during X-chromosome inactivation (XCI), reflecting its relatively uniform structure revealed by 4C-seq. Despite this uniformity, only a subset of the Xi became earlier replicating in SmcHD1-mutant cells. In the mutant, these domains protruded out of the Xi core, contacted each other and became transcriptionally reactivated. 4C-seq suggested that they constituted the outermost layer of the Xi even before XCI and were rich in escape genes. We propose that this default positioning forms the basis for their inherent heterochromatin instability in cells lacking the Xi-binding protein SmcHD1 or exhibiting XCI escape. These observations underscore the importance of 3D genome organization for heterochromatin stability and gene regulation.

Passive Exoskeleton-Assisted Gait Shows a Unique Interlimb Coordination Signature Without Restricting Regular Walking.

Exoskeleton assistive devices have been developed as a potential approach to solve gait deficits like paretic propulsion and reduced speed. However, it is unclear how these devices affect inter-limb coordination. The duration and the synchrony of gait coordination was assessed during passive exoskeleton-assisted walking in healthy young individuals. It was hypothesized that inter-limb coordination would be reduced in comparison to normal walking without assistance, thus demonstrating gait with exoskeleton to be more explorative and flexible. Eighteen participants were divided into two groups (EXO: n = 9; NO EXO: n = 9) and performed a 5-min walking trial at a preferred walking speed after a familiarization trial. The duration of inter-limb coordination was examined using cross-recurrence quantification analysis and the synchrony was measured using cross sample entropy. There were no significant differences in spatiotemporal measurements between the two groups. However, in comparison to the no exoskeleton group, there was a reduction in the duration of coordination (mean diagonal length: p < 0.01) and the synchrony of coordination (entropy value: p < 0.05) in the exoskeleton group. These results indicate that exoskeletal-assisted gait is characterized by reduced inter-limb coordination possibly for allowing gait patterns to be more explorative and flexible. This is important in rehabilitation of patients who suffer from coordination deficits.

Ectopic Splicing Disturbs the Function of Xist RNA to Establish the Stable Heterochromatin State.

Non-coding Xist RNA plays an essential role in X chromosome inactivation (XCI) in female mammals. It coats the X chromosome in cis and mediates the recruitment of many proteins involved in gene silencing and heterochromatinization. The molecular basis of how Xist RNA initiates chromosomal silencing and what proteins participate in this process has been extensively studied and elucidated. Its involvement in the establishment and maintenance of the X-inactivated state is, however, less understood. The Xist IVS allele we previously reported is peculiar in that it can initiate XCI but fails to establish the inactive state that is stably maintained and, therefore, may provide an opportunity to explore how Xist RNA contributes to establish a robust heterochromatin state. Here we demonstrate that ectopic splicing taking place to produce Xist IVS RNA disturbs its function to properly establish stable XCI state. This finding warrants the potential of Xist IVS RNA to provide further insight into our understanding of how Xist RNA contributes to establish sustainable heterochromatin.

Tsix silences Xist through modification of chromatin structure.

X inactivation is controlled by Xist and its antisense gene, Tsix, neither of which encodes a protein. Xist is essential for X inactivation to occur in cis, and its differential expression is a key event in the initiation of X inactivation. Xist and Tsix are imprinted in the extraembryonic tissues of mouse embryos so that they are expressed from the paternal and maternal X, respectively, resulting in the preferential inactivation of the paternal X. Targeted disruption of Tsix causes ectopic expression of Xist, suggesting that Tsix negatively regulates Xist in cis. However, the molecular mechanism of this antisense regulation remains unknown. Here, we demonstrate that Tsix transcriptionally silences Xist in both embryonic and extraembryonic tissues of mouse embryos. Moreover, we show that disruption of Tsix impairs establishment of repressive epigenetic modifications and chromatin structure at the Xist locus. We propose that Tsix silences Xist through modification of the chromatin structure.

Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting.

Imprinted genes are epigenetically marked during gametogenesis so that they are exclusively expressed from either the paternal or the maternal allele in offspring. Imprinting prevents parthenogenesis in mammals and is often disrupted in congenital malformation syndromes, tumours and cloned animals. Although de novo DNA methyltransferases of the Dnmt3 family are implicated in maternal imprinting, the lethality of Dnmt3a and Dnmt3b knockout mice has precluded further studies. We here report the disruption of Dnmt3a and Dnmt3b in germ cells, with their preservation in somatic cells, by conditional knockout technology. Offspring from Dnmt3a conditional mutant females die in utero and lack methylation and allele-specific expression at all maternally imprinted loci examined. Dnmt3a conditional mutant males show impaired spermatogenesis and lack methylation at two of three paternally imprinted loci examined in spermatogonia. By contrast, Dnmt3b conditional mutants and their offspring show no apparent phenotype. The phenotype of Dnmt3a conditional mutants is indistinguishable from that of Dnmt3L knockout mice, except for the discrepancy in methylation at one locus. These results indicate that both Dnmt3a and Dnmt3L are required for methylation of most imprinted loci in germ cells, but also suggest the involvement of other factors.