Much attention has focussed on the conversion of human pluripotent stem cells (PSCs) to a more naïve developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24 h. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from that of primed PSCs and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to a level equivalent to that in the ICM and is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of biallelic X-linked gene transcription indicates reactivation of the silenced X chromosome. On reconversion to primed status, XIST-induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells.

No abstract

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 OGlcNAcylated, 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.EED | Host Cell Factor C1 | electron transfer dissociation

Although purification of biotinylated molecules is highly efficient, identifying specific sites of biotinylation remains challenging. We show that anti-biotin antibodies enable unprecedented enrichment of biotinylated peptides from complex peptide mixtures. Live-cell proximity labeling using APEX peroxidase followed by anti-biotin enrichment and mass spectrometry yielded over 1,600 biotinylation sites on hundreds of proteins, an increase of more than 30-fold in the number of biotinylation sites identified compared to streptavidin-based enrichment of proteins.

Regulation of gene expression is primarily controlled by changes in the proteins that occupy their regulatory elements. Chromatin immunoprecipitation can confirm a protein’s occupancy at a genomic locus but requires specific, high-quality, IP-competent antibodies against nominated proteins, limiting its utility. Here, we combine, genome targeting, proximity labeling, and quantitative proteomics to develop genomic locus proteomics, a method able to identify proteins associated a specific genomic locus in native cellular contexts.

Gene conversion plays an important part in shaping genetic diversity in populations, yet estimating the rate at which it occurs is difficult because of the short lengths of DNA involved. We have developed a new statistical approach to estimating gene conversion rates from genetic variation, by extending an existing model for haplotype data in the presence of crossover events. We show, by simulation, that when the rate of gene conversion events is at least comparable to the rate of crossover events, the method provides a powerful approach to the detection of gene conversion and estimation of its rate. Application of the method to data from the telomeric X chromosome of Drosophila melanogaster, in which crossover activity is suppressed, indicates that gene conversion occurs $400 times more often than crossover events. We also extend the method to estimating variable crossover and gene conversion rates and estimate the rate of gene conversion to be $1.5 times higher than the crossover rate in a region of human chromosome 1 with known recombination hotspots.

A streamlined and readily accessible sample preparation protocol has been developed to enable TMT-based proteomic profiling of relatively low numbers of cells directly from a flow cytometer. These methods were applied to 12 freshly isolated immune cell types from mice to a depth of over 7000 quantified proteins. These data recapitulate many aspects of known immunology, nominate new cell type specific protein markers, and provide evidence for post-transcriptional regulation of gene expression across the immune system. Graphical Abstract+ Int. Ref IM M U -N O L O -G Y Collection microreactor On-column TMT labeling bRP fractionation Long gradient, long column, high resolution LC-MS/MS % ACN −20 −10 0 10 20 −10 0 1 0 20 PC 1 PC 2 B1a GN DC CD8T CD4T B cell MC Treg Tgd NKT NK MF Streamlined protocol for reduced cell numbers GN GN T4 T8 NKT MF MC NK DC B B1a Treg Tgd mRNA co-regulation Protein co-regulation Proteomic profiling of murine immune cells Highlights• A low input proteomic profiling sample preparation workflow has been developed.• The protocol components are accessible and are widely applicable.• Over 7000 proteins across 12 immune cell types were quantified from 3e5 cells.• The data provide evidence for global post-transcriptional regulation.

RNA–protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA–protein interactions in living cells in an unbiased manner. We used two approaches to target the engineered peroxidase APEX2 to specific cellular RNAs for RNA-centered proximity biotinylation of protein interaction partners. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were used to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured candidate binding partners for hTR, including more than a dozen proteins not previously linked to hTR. We validated the interaction between hTR and the N6-methyladenosine (m6A) demethylase ALKBH5 and showed that ALKBH5 is able to erase the m6A modification on endogenous hTR. ALKBH5 also modulates telomerase complex assembly and activity. MS2- and Cas13-targeted APEX2 may facilitate the discovery of novel RNA–protein interactions in living cells.