XIST dampens X chromosome activity in a SPEN-dependent manner during early human development.

XIST (X-inactive specific transcript) long noncoding RNA (lncRNA) is responsible for X chromosome inactivation (XCI) in placental mammals, yet it accumulates on both X chromosomes in human female preimplantation embryos without triggering X chromosome silencing. The XACT (X-active coating transcript) lncRNA coaccumulates with XIST on active X chromosomes and may antagonize XIST function. Here, we used human embryonic stem cells in a naive state of pluripotency to assess the function of XIST and XACT in shaping the X chromosome chromatin and transcriptional landscapes during preimplantation development. We show that XIST triggers the deposition of polycomb-mediated repressive histone modifications and dampens the transcription of most X-linked genes in a SPEN-dependent manner, while XACT deficiency does not significantly affect XIST activity or X-linked gene expression. Our study demonstrates that XIST is functional before XCI, confirms the existence of a transient process of X chromosome dosage compensation and reveals that XCI and dampening rely on the same set of factors.

Unraveling hallmark suitability for staging pre- and post-implantation stem cell models.

The advent of novel 2D and 3D models for human development, including trophoblast stem cells and blastoids, has expanded opportunities for investigating early developmental events, gradually illuminating the enigmatic realm of human development. While these innovations have ushered in new prospects, it has become essential to establish well-defined benchmarks for the cell sources of these models. We aimed to propose a comprehensive characterization of pluripotent and trophoblastic stem cell models by employing a combination of transcriptomic, proteomic, epigenetic, and metabolic approaches. Our findings reveal that extended pluripotent stem cells share many characteristics with primed pluripotent stem cells, with the exception of metabolic activity. Furthermore, our research demonstrates that DNA hypomethylation and high metabolic activity define trophoblast stem cells. These results underscore the necessity of considering multiple hallmarks of pluripotency rather than relying on a single criterion. Multiplying hallmarks alleviate stage-matching bias.

Species-specific regulation of XIST by the JPX/FTX orthologs.

X chromosome inactivation (XCI) is an essential process, yet it initiates with remarkable diversity in various mammalian species. XIST, the main trigger of XCI, is controlled in the mouse by an interplay of lncRNA genes (LRGs), some of which evolved concomitantly to XIST and have orthologues across all placental mammals. Here, we addressed the functional conservation of human orthologues of two such LRGs, FTX and JPX. By combining analysis of single-cell RNA-seq data from early human embryogenesis with various functional assays in matched human and mouse pluripotent stem- or differentiated post-XCI cells, we demonstrate major functional differences for these orthologues between species, independently of primary sequence conservation. While the function of FTX is not conserved in humans, JPX stands as a major regulator of XIST expression in both species. However, we show that different entities of JPX control the production of XIST at various steps depending on the species. Altogether, our study highlights the functional versatility of LRGs across evolution, and reveals that functional conservation of orthologous LRGs may involve diversified mechanisms of action. These findings represent a striking example of how the evolvability of LRGs can provide adaptative flexibility to constrained gene regulatory networks.

X chromosome inactivation in human development.

X chromosome inactivation (XCI) is a key developmental process taking place in female mammals to compensate for the imbalance in the dosage of X-chromosomal genes between sexes. It is a formidable example of concerted gene regulation and a paradigm for epigenetic processes. Although XCI has been substantially deciphered in the mouse model, how this process is initiated in humans has long remained unexplored. However, recent advances in the experimental capacity to access human embryonic-derived material and in the laws governing ethical considerations of human embryonic research have allowed us to enlighten this black box. Here, we will summarize the current knowledge of human XCI, mainly based on the analyses of embryos derived from in vitro fertilization and of pluripotent stem cells, and highlight any unanswered questions.

Establishment of X chromosome inactivation and epigenomic features of the inactive X depend on cellular contexts.

X chromosome inactivation (XCI) is an essential epigenetic process that ensures X-linked gene dosage equilibrium between sexes in mammals. XCI is dynamically regulated during development in a manner that is intimately linked to differentiation. Numerous studies, which we review here, have explored the dynamics of X inactivation and reactivation in the context of development, differentiation and diseases, and the phenotypic and molecular link between the inactive status, and the cellular context. Here, we also assess whether XCI is a uniform mechanism in mammals by analyzing epigenetic signatures of the inactive X (Xi) in different species and cellular contexts. It appears that the timing of XCI and the epigenetic signature of the inactive X greatly vary between species. Surprisingly, even within a given species, various Xi configurations are found across cellular states. We discuss possible mechanisms underlying these variations, and how they might influence the fate of the Xi.

How Many Non-coding RNAs Does It Take to Compensate Male/Female Genetic Imbalance?

Genetic sex determination in mammals relies on dimorphic sex chromosomes that confer phenotypic/physiologic differences between males and females. In this heterogametic system, X and Y chromosomes diverged from an ancestral pair of autosomes, creating a genetic disequilibrium between XX females and XY males. Dosage compensation mechanisms alleviate intrinsic gene dosage imbalance, leading to equal expression levels of most X-linked genes in the two sexes. In therian mammals, this is achieved through inactivation of one of the two X chromosomes in females. Failure to undergo X-chromosome inactivation (XCI) results in developmental arrest and death. Although fundamental for survival, a surprising loose conservation in the mechanisms to achieve XCI during development in therian lineage has been, and continues, to be uncovered. XCI involves the concerted action of non-coding RNAs (ncRNAs), including the well-known Xist RNA, and has thus become a classical paradigm to study the mode of action of this particular class of transcripts. In this chapter, we will describe the processes coping with sex chromosome genetic imbalance and how ncRNAs underlie dosage compensation mechanisms and influence male-female differences in mammals. Moreover, we will discuss how ncRNAs have been tinkered with during therian evolution to adapt XCI mechanistic to species-specific constraints.

Erosion of X Chromosome Inactivation in Human Pluripotent Cells Initiates with XACT Coating and Depends on a Specific Heterochromatin Landscape.

Human pluripotent stem cells (hPSCs) display extensive epigenetic instability, particularly on the X chromosome. In this study, we show that, in hPSCs, the inactive X chromosome has a specific heterochromatin landscape that predisposes it to erosion of X chromosome inactivation (XCI), a process that occurs spontaneously in hPSCs. Heterochromatin remodeling and gene reactivation occur in a non-random fashion and are confined to specific H3K27me3-enriched domains, leaving H3K9me3-marked regions unaffected. Using single-cell monitoring of XCI erosion, we show that this instability only occurs in pluripotent cells. We also provide evidence that loss of XIST expression is not the primary cause of XCI instability and that gene reactivation from the inactive X (Xi) precedes loss of XIST coating. Notably, expression and coating by the long non-coding RNA XACT are early events in XCI erosion and, therefore, may play a role in mediating this process.

A prominent and conserved role for YY1 in Xist transcriptional activation.

Accumulation of the noncoding RNA Xist on one X chromosome in female cells is a hallmark of X-chromosome inactivation (XCI) in eutherians. Here we uncover an essential function for the ubiquitous autosomal transcription factor Yin-Yang 1 (YY1) in the transcriptional activation of Xist in both human and mouse. We show that loss of YY1 prevents Xist upregulation during the initiation and maintenance of X-inactivation, and that YY1 binds directly the Xist 5' region to trigger the activity of the Xist promoter. Binding of YY1 to the Xist 5' region before XCI competes with the Xist repressor REX1, whereas DNA methylation controls mono-allelic fixation of YY1 to Xist at the onset of XCI. YY1 is thus the first autosomal activating factor involved in a fundamental and conserved pathway of Xist regulation that ensures the asymmetric transcriptional upregulation of the master regulator of XCI.

Redox regulation by Pitx2 and Pitx3 is critical for fetal myogenesis.

During development, major metabolic changes occur as cells become more specialized within a lineage. In the case of skeletal muscle, differentiation is accompanied by a switch from a glycolytic proliferative progenitor state to an oxidative postmitotic differentiated state. Such changes require extensive mitochondrial biogenesis leading to increased reactive oxygen species (ROS) production that needs to be balanced by an antioxidant system. Our analysis of double conditional Pitx2/3 mouse mutants, both in vivo during fetal myogenesis and ex vivo in primary muscle cell cultures, reveals excessive upregulation of ROS levels leading to DNA damage and apoptosis of differentiating cells. This is a consequence of downregulation of Nrf1 and genes for antioxidant enzymes, direct targets of Pitx2/3, leading to decreased expression of antioxidant enzymes, as well as impairment of mitochondrial function. Our analysis identifies Pitx2 and Pitx3 as key regulators of the intracellular redox state preventing DNA damage as cells undergo differentiation.

Divergent transcriptional activities determine limb identity.

Limbs develop using a common genetic programme despite widely differing morphologies. This programme is modulated by limb-restricted regulators such as hindlimb (HL) transcription factors Pitx1 and Tbx4 and the forelimb (FL) Tbx5. Both Tbx factors have been implicated in limb patterning and growth, but their relative activities and underlying mechanisms remain unclear. In this paper, we show that Tbx4 and Tbx5 harbour conserved and divergent transcriptional regulatory domains that account for their roles in limb development. In particular, both factors share an activator domain and the ability to stimulate limb growth. However, we find that Tbx4 is the primary effector of HL identity for both skeletal and muscle development; this activity relies on a repressor domain that is inactivated by a human TBX4 small-patella syndrome mutation. We propose that limb identity is largely achieved by default in FL, whereas a specific repressor activity unique to Tbx4 determines HL identity.

Pitx2 defines alternate pathways acting through MyoD during limb and somitic myogenesis.

The MyoD gene is part of the core regulatory network that governs skeletal myogenesis and acts as an essential determinant of the myogenic cell fate. Although generic regulatory networks converging on this gene have been described, the specific mechanisms leading to MyoD expression in muscles of different ontology remain misunderstood. We now show that the homeobox gene Pitx2 is required for initial activation of the MyoD gene in limb muscle precursors through direct binding of Pitx2 to the MyoD core enhancer. Whereas Myf5 and Mrf4 are dispensable for limb muscle progenitor fate, inactivation of Myf5 and Mrf4 in Pitx2 mutants results in a drastic decrease of limb MyoD expression. Thus, Pitx2 and Myf5 define parallel genetic pathways for limb myogenesis. We show a similar dependence on Pitx2 and Myf5(Mrf4) in myotome, where MyoD expression is initially activated by Myf5 and Mrf4. In their absence, MyoD expression is eventually rescued by a Pax3-dependent mechanism. We now provide evidence that Pitx2 contributes to the rescue of MyoD expression and that it acts downstream of Pax3. We thus propose that myogenic differentiation of somite-derived muscle cells relies on two parallel genetic pathways, with the Pitx2 pathway being of primary importance for limb myogenesis but the Myf5 and Mrf4 pathway predominating in myotome. Muscle-specific wiring of regulatory networks composed of similar transcription factors thus underlies development of distinct skeletal muscles.

A muscle-specific promoter directs Pitx3 gene expression in skeletal muscle cells.

The Pitx homeobox transcription factor genes have been implicated in different developmental processes, including determination of hind limb identity for Pitx1, left-right asymmetry for Pitx2, and eye development and survival of midbrain dopaminergic neurons for Pitx3. Pitx1 and Pitx2 have partly redundant activities in craniofacial development, including in pituitary organogenesis, as indicated by their names. These genes also exhibit redundant activities in the control of hind limb bud growth. Recent studies have shown expression of the three Pitx genes in muscle, with Pitx3 being the most widely expressed in all skeletal muscles. We now report the identification of a muscle-specific promoter within the Pitx3 gene that is situated between the first exon for eye and brain expression and exon 2 that contains the initiator ATG codon. Sequences proximal to this muscle-specific exon 1 are essential and sufficient to confer muscle-specific expression in transgenic mice, they are responsive to myogenic basic helix-loop-helix regulatory factors, and they recruit these factors in vivo. In agreement with exclusive use of the muscle-specific promoter in aphakia mice that are deleted of the brain promoter, the trimethyl-lysine 4 histone H3 promoter signature shifts to this promoter in embryonic day 13 ak limb bud muscle cells. Myogenic basic helix-loop-helix regulatory factor activation of Pitx3 transcription may be part of a positive feedback loop contributing to establishment of the myogenic program.

Bone morphogenic protein (Smad)-mediated repression of proopiomelanocortin transcription by interference with Pitx/Tpit activity.

The signaling molecules bone morphogenic protein (BMP) 4 and 2 have been implicated in early organogenesis and cell differentiation of the pituitary. However, the use of different experimental paradigms has led to conflicting interpretations with regard to the action of these factors on differentiation of corticotroph cells and on expression of the proopiomelanocortin (POMC) gene. We have now directly assessed the action of BMP signaling on POMC expression and found that BMP4 represses POMC mRNA levels and promoter activity. This repression appears to be dependent on the classical BMP signaling pathway that involves the activin-like kinase 3/6 receptors and the Smad1/4 transcription factors. The repression is reversed by overexpression of the inhibitory Smads, Smad6 or Smad7. Collectively, the evidence suggests that autocrine BMP signaling may be acting upon AtT-20 cells to set the level of POMC expression. Upon BMP4 stimulation, activated phospho-Smad1 is recruited to the POMC promoter, where it apparently acts through interactions with the Pitx and Tpit transcription factors. It is postulated that these interactions interfere with the transcriptional activity of Pitx and/or Tpit, thus resulting in transcriptional repression.