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Abstract BackgroundX chromosome inactivation (XCI) is a female-specific process in which one X chromosome is silenced to balance X-linked gene expression between the sexes. XCI is initiated in early development by upregulation of the lncRNAXiston the future inactive X (Xi). A subset of X-linked genes escape silencing and thus have higher expression in females, suggesting female-specific functions. One of these genes is the highly conserved geneKdm6a, which encodes a histone demethylase that removes methyl groups at H3K27 to facilitate gene expression.KDM6Amutations have been implicated in congenital disorders such as Kabuki Syndrome, as well as in sex differences in development and cancer. MethodsKdm6awas knocked out (KO) using CRISPR/Cas9 gene editing in hybrid female mouse embryonic stem (ES) cells derived either from a 129 × Mus castaneus(cast) cross or a BL6 xcastcross. In one of the lines a transcriptional stop signal inserted inTsixresults in completely skewed X silencing upon differentiation. The effects of both homozygous and heterozygousKdm6aKO onXistexpression during the onset of XCI were measured by RT-PCR and RNA-FISH. Changes in gene expression and in H3K27me3 enrichment were investigated using allele-specific RNA-seq and Cut&Run, respectively. KDM6A binding to theXistgene was characterized by Cut&Run. ResultsWe observed impaired upregulation ofXistand reduced coating of the Xi during early stages of differentiation inKdm6aKO cells, both homozygous and heterozygous, suggesting a threshold effect of KDM6A. This was associated with aberrant overexpression of genes from the Xi after differentiation, indicating loss of X inactivation potency. Consistent with KDM6A having a direct role inXistregulation, we found that the histone demethylase binds to theXistpromoter and KO cells show an increase in H3K27me3 atXist, consistent with reduced expression. ConclusionsThese results reveal a novel female-specific role for the X-linked histone demethylase, KDM6A in the initiation of XCI through histone demethylase-dependent activation ofXistduring early differentiation. Plain language summaryX chromosome inactivation is a female-specific mechanism that evolved to balance sex-linked gene dosage between females (XX) and males (XY) by silencing one X chromosome in females. X inactivation begins with the upregulation of the long noncoding RNAXiston the future inactive X chromosome. While most genes become silenced on the inactive X chromosome some genes escape inactivation and thus have higher expression in females compared to males, suggesting that escape genes may have female-specific functions. One such gene encodes the histone demethylase KDM6A which function is to turn on gene expression by removing repressive histone modifications. In this study, we investigated the role of KDM6A in the regulation ofXistexpression during the onset of X inactivation. We found that KDM6A binds to theXistgene to remove repressive histone marks and facilitate its expression in early development. Indeed, depletion of KDM6A prevents upregulation ofXistdue to abnormal persistence of repressive histone modifications. In turn, this results in aberrant overexpression of genes from the inactive X chromosome. Our findings point to a novel mechanism ofXistregulation during the initiation of X inactivation, which may lead to new avenues of treatment to alleviate congenital disorders such as Kabuki syndrome and sex-biased immune disorders where X-linked gene dosage is perturbed. 

Driven by the need for integrated management of groundwater (GW) and surface water (SW), quantification of GW–SW interactions and associated contaminant transport has become increasingly important. This is due to their substantial impact on water quantity and quality. In this review, we provide an overview of the methods developed over the past several decades to investigate GW–SW interactions. These methods include geophysical, hydrometric, and tracer techniques, as well as various modeling approaches. Different methods reveal valuable information on GW–SW interactions at different scales with their respective advantages and limitations. Interpreting data from these techniques can be challenging due to factors like scale effects, heterogeneous hydrogeological conditions, sediment variability, and complex spatiotemporal connections between GW and SW. To facilitate the selection of appropriate methods for specific sites, we discuss the strengths, weaknesses, and challenges of each technique, and we offer perspectives on knowledge gaps in the current science. 

A robust system should perform well under random failures or targeted attacks, and networks have been widely used to model the underlying structure of complex systems such as communication, infrastructure, and transportation networks. Hence, network robustness becomes critical to understanding system robustness. In this paper, we propose a spectral measure for network robustness: the second spectral moment m2 of the network. Our results show that a smaller second spectral moment m2 indicates a more robust network. We demonstrate both theoretically and with extensive empirical studies that the second spectral moment can help (1) capture various traditional measures of network robustness; (2) assess the robustness of networks; (3) design networks with controlled robustness; and (4) study how complex networked systems (e.g., power systems) behave under cascading failures.