Abstract Female mammalian cells have two X chromosomes, one of maternal origin and one of paternal origin. During development, one X chromosome randomly becomes inactivated 1–4 . This renders either the maternal X (X m ) chromosome or the paternal X (X p ) chromosome inactive, causing X mosaicism that varies between female individuals, with some showing considerable or complete skew of the X chromosome that remains active 5–7 . Parent-of-X origin can modify epigenetics through DNA methylation 8,9 and possibly gene expression; thus, mosaicism could buffer dysregulated processes in ageing and disease. However, whether X skew or its mosaicism alters functions in female individuals is largely unknown. Here we tested whether skew towards an active X m chromosome influences the brain and body—and then delineated unique features of X m neurons and X p neurons. An active X m chromosome impaired cognition in female mice throughout the lifespan and led to worsened cognition with age. Cognitive deficits were accompanied by X m -mediated acceleration of biological or epigenetic ageing of the hippocampus, a key centre for learning and memory, in female mice. Several genes were imprinted on the X m chromosome of hippocampal neurons, suggesting silenced cognitive loci. CRISPR-mediated activation of X m -imprinted genes improved cognition in ageing female mice. Thus, the X m chromosome impaired cognition, accelerated brain ageing and silenced genes that contribute to cognition in ageing. Understanding how X m impairs brain function could lead to an improved understanding of heterogeneity in cognitive health in female individuals and to X-chromosome-derived pathways that protect against cognitive deficits and brain ageing.
Epigenetic regulation of chromatin is dependent on both the histone protein isoforms and state of their post-translational modifications. The assignment of all post-translational modification sites for each individual intact protein isoform remains an experimental challenge. We present an on-line reversed phase LC tandem mass spectrometry approach for the separation of intact, unfractionated histones and a high resolution mass analyzer, the Orbitrap, with electron transfer dissociation capabilities to detect and record accurate mass values for the molecular and fragment ions observed. From a single LC-electron transfer dissociation run, this strategy permits the identification of the most abundant intact proteins, determination of the isoforms present, and the localization of post-translational modifications.
Abstract CRISPR/Cas technologies have transformed our ability to add functionality to the genome by knock-in of payload via homology-directed repair (HDR). However, a systematic and quantitative profiling of the knock-in integration landscape is still lacking. Here, we present a framework based on long-read sequencing and an integrated computational pipeline (knock-knock) to analyze knock-in repair outcomes across a wide range of experimental parameters. Our data uncover complex repair profiles, with perfect HDR often accounting for a minority of payload integration events, and reveal markedly distinct mis-integration patterns between cell-types or forms of HDR templates used. Our analysis demonstrates that the two sides of a given double-strand break can be repaired by separate pathways and identifies a major role for sequence micro-homology in driving donor mis-integration. Altogether, our comprehensive framework paves the way for investigating repair mechanisms, monitoring accuracy, and optimizing the precision of genome engineering.
The monosaccharide addition of an N -acetylglucosamine to serine and threonine residues of nuclear and cytosolic proteins ( O -GlcNAc) is a posttranslational modification emerging as a general regulator of many cellular processes, including signal transduction, cell division, and transcription. The sole mouse O -GlcNAc transferase (OGT) is essential for embryonic development. To understand the role of OGT in mouse development better, we mapped sites of O -GlcNAcylation of nuclear proteins in mouse embryonic stem cells (ESCs). Here, we unambiguously identify over 60 nuclear proteins as O -GlcNAcylated, several of which are crucial for mouse ESC cell maintenance. Furthermore, we extend the connection between OGT and Polycomb group genes from flies to mammals, showing Polycomb repressive complex 2 is necessary to maintain normal levels of OGT and for the correct cellular distribution of O -GlcNAc. Together, these results provide insight into how OGT may regulate transcription in early development, possibly by modifying proteins important to maintain the ESC transcriptional repertoire.
Post-translational histone modifications modulate chromatin-templated processes and therefore affect cellular proliferation, growth, and development. Although post-translational modifications on the core histones have been under intense investigation for several years, the modifications on variant histones are poorly understood. We used tandem mass spectrometry to identify covalent modifications on a histone H2A variant, macroH2A1.2. MacroH2A1.2 can be monoubiquitinated; however, the site of monoubiquitination has not been documented. In this study we used green fluorescent protein-tagged macroH2A1.2 to determine that Lys(115) is a site of ubiquitination. In addition, we found that this variant H2A is methylated on the epsilon amino group of lysine residues Lys(17), Lys(122), and Lys(238) and phosphorylated on Thr(128). Three of these modifications were also found to be present in the endogenous protein by mass spectrometric analysis. These results provide the first direct evidence that multiple post-translational modifications are imposed on macroH2A1.2, suggesting that, like canonical H2A, this variant H2A is subject to regulation by combinatorial use of covalent modifications.
Polycomb group (PcG) proteins belonging to the polycomb (Pc) repressive complexes 1 and 2 (PRC1 and PRC2) maintain homeotic gene silencing. In Drosophila, PRC2 methylates histone H3 on lysine 27, and this epigenetic mark facilitates recruitment of PRC1. Mouse PRC2 (mPRC2) has been implicated in X inactivation, as mPRC2 proteins transiently accumulate on the inactive X chromosome (Xi) at the onset of X inactivation to methylate histone H3 lysine 27 (H3-K27). In this study, we demonstrate that mPRC1 proteins localize to the Xi, and that different mPRC1 proteins accumulate on the Xi during initiation and maintenance of X inactivation in embryonic cells. The Xi accumulation of mPRC1 proteins requires Xist RNA and is not solely regulated by the presence of H3-K27 methylation, as not all cells that exhibit this epigenetic mark on the Xi show Xi enrichment of mPRC1 proteins. Our results implicate mPRC1 in X inactivation and suggest that the regulated assembly of PcG protein complexes on the Xi contributes to this multistep process.
