LINE-1 RNA causes heterochromatin erosion and is a target for amelioration of senescent phenotypes in progeroid syndromes.

Constitutive heterochromatin is responsible for genome repression of DNA enriched in repetitive sequences, telomeres, and centromeres. During physiological and pathological premature aging, heterochromatin homeostasis is profoundly compromised. Here, we showed that LINE-1 (Long Interspersed Nuclear Element-1; L1) RNA accumulation was an early event in both typical and atypical human progeroid syndromes. L1 RNA negatively regulated the enzymatic activity of the histone-lysine N-methyltransferase SUV39H1 (suppression of variegation 3-9 homolog 1), resulting in heterochromatin loss and onset of senescent phenotypes in vitro. Depletion of L1 RNA in dermal fibroblast cells from patients with different progeroid syndromes using specific antisense oligonucleotides (ASOs) restored heterochromatin histone 3 lysine 9 and histone 3 lysine 27 trimethylation marks, reversed DNA methylation age, and counteracted the expression of senescence-associated secretory phenotype genes such as p16, p21, activating transcription factor 3 (ATF3), matrix metallopeptidase 13 (MMP13), interleukin 1a (IL1a), BTG anti-proliferation factor 2 (BTG2), and growth arrest and DNA damage inducible beta (GADD45b). Moreover, systemic delivery of ASOs rescued the histophysiology of tissues and increased the life span of a Hutchinson-Gilford progeria syndrome mouse model. Transcriptional profiling of human and mouse samples after L1 RNA depletion demonstrated that pathways associated with nuclear chromatin organization, cell proliferation, and transcription regulation were enriched. Similarly, pathways associated with aging, inflammatory response, innate immune response, and DNA damage were down-regulated. Our results highlight the role of L1 RNA in heterochromatin homeostasis in progeroid syndromes and identify a possible therapeutic approach to treat premature aging and related syndromes.

Molecular basis of hUHRF1 allosteric activation for synergistic histone modification binding by PI5P.

Chromatin marks are recognized by distinct binding modules, many of which are embedded in multidomain proteins. How the different functionalities of such complex chromatin modulators are regulated is often unclear. Here, we delineated the interplay of the H3 amino terminus- and K9me-binding activities of the multidomain hUHRF1 protein. We show that the phosphoinositide PI5P interacts simultaneously with two distant flexible linker regions connecting distinct domains of hUHRF1. The binding is dependent on both, the polar head group, and the acyl part of the phospholipid and induces a conformational rearrangement juxtaposing the H3 amino terminus and K9me3 recognition modules of the protein. In consequence, the two features of the H3 tail are bound in a multivalent, synergistic manner. Our work highlights a previously unidentified molecular function for PI5P outside of the context of lipid mono- or bilayers and establishes a molecular paradigm for the allosteric regulation of complex, multidomain chromatin modulators by small cellular molecules.

Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA.

Background: Methylation of carbon-5 of cytosines (m 5C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. It can be further modified to yield 5-hydroxymethylcytidine (hm 5C), 5-formylcytidine (f 5C), 2´-O-methyl-5-hydroxymethylcytidine (hm 5Cm) and 2´-O-methyl-5-formylcytidine (f 5Cm). How m 5C, and specially its derivates, contribute to biology mechanistically is poorly understood. We recently showed that m 5C is required for Caenorhabditis elegans development and fertility under heat stress. m 5C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. Hence, identifying readers for RNA modifications can enhance our understanding in the biological roles of these modifications. Methods: To contribute to the understanding of how m 5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in C. elegans. Potential readers were identified using mass spectrometry. The interaction of two of the putative readers with m 5C was validated using immunoblotting. Results: Our mass spectrometry analyses revealed unique binding proteins for each of the modifications. In silico analysis for phenotype enrichments suggested that hm 5Cm unique readers are enriched in proteins involved in RNA processing, while readers for m 5C, hm 5C and f 5C are involved in germline processes. We validated our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m 5C in vitro. Finally, sequence alignment analysis showed that several of the putative m 5C readers contain the conserved RNA recognition motif (RRM), including ALY-1 and ALY-2. Conclusions: The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m 5C and its derivatives. Furthermore, we demonstrate that ALY-1 and ALY-2 bind to m 5C in C. elegans.

Narrow Precursor Mass Range for DIA-MS Enhances Protein Identification and Quantification in Arabidopsis.

Data independent acquisition-mass spectrometry (DIA-MS) is becoming widely utilised for robust and accurate quantification of samples in quantitative proteomics. Here, we describe the systematic evaluation of the effects of DIA precursor mass range on total protein identification and quantification. We show that a narrow mass range of precursors (~250 m/z) for DIA-MS enables a higher number of protein identifications. Subsequent application of DIA with narrow precursor range (from 400 to 650 m/z) on an Arabidopsis sample with spike-in known proteins identified 34.7% more proteins than in conventional DIA (cDIA) with a wide precursor range of 400-1200 m/z. When combining several DIA-MS analyses with narrow precursor ranges (i.e., 400-650, 650-900 and 900-1200 m/z), we were able to quantify 10,099 protein groups with a median coefficient of variation of <6%. These findings represent a 54.7% increase in the number of proteins quantified than with cDIA analysis. This is particularly important for low abundance proteins, as exemplified by the six-protein mix spike-in. In cDIA only five out of the six-protein mix were quantified while our approach allowed accurate quantitation of all six proteins.

Erosion of human X chromosome inactivation causes major remodeling of the iPSC proteome.

X chromosome inactivation (XCI) is a dosage compensation mechanism in female mammals whereby transcription from one X chromosome is repressed. Analysis of human induced pluripotent stem cells (iPSCs) derived from female donors identified that low levels of XIST RNA correlated strongly with erosion of XCI. Proteomic analysis, RNA sequencing (RNA-seq), and polysome profiling showed that XCI erosion resulted in amplified RNA and protein expression from X-linked genes, providing a proteomic characterization of skewed dosage compensation. Increased protein expression was also detected from autosomal genes without an mRNA increase, thus altering the protein-RNA correlation between the X chromosome and autosomes. XCI-eroded lines display an ∼13% increase in total cell protein content, with increased ribosomal proteins, ribosome biogenesis and translation factors, and polysome levels. We conclude that XCI erosion in iPSCs causes a remodeling of the proteome, affecting the expression of a much wider range of proteins and disease-linked loci than previously realized.

DEPS-1 is required for piRNA-dependent silencing and PIWI condensate organisation in Caenorhabditis elegans.

Membraneless organelles are sites for RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. How small ncRNAs utilise phase separated environments for their function is unclear. We investigated how the PIWI-interacting RNA (piRNA) pathway engages with the membraneless organelle P granule in Caenorhabditis elegans. Proteomic analysis of the PIWI protein PRG-1 reveals an interaction with the constitutive P granule protein DEPS-1. DEPS-1 is not required for piRNA biogenesis but piRNA-dependent silencing: deps-1 mutants fail to produce the secondary endo-siRNAs required for the silencing of piRNA targets. We identify a motif on DEPS-1 which mediates a direct interaction with PRG-1. DEPS-1 and PRG-1 form intertwining clusters to build elongated condensates in vivo which are dependent on the Piwi-interacting motif of DEPS-1. Additionally, we identify EDG-1 as an interactor of DEPS-1 and PRG-1. Our study reveals how specific protein-protein interactions drive the spatial organisation and piRNA-dependent silencing within membraneless organelles.

Population-scale proteome variation in human induced pluripotent stem cells.

Human disease phenotypes are driven primarily by alterations in protein expression and/or function. To date, relatively little is known about the variability of the human proteome in populations and how this relates to variability in mRNA expression and to disease loci. Here, we present the first comprehensive proteomic analysis of human induced pluripotent stem cells (iPSC), a key cell type for disease modelling, analysing 202 iPSC lines derived from 151 donors, with integrated transcriptome and genomic sequence data from the same lines. We characterised the major genetic and non-genetic determinants of proteome variation across iPSC lines and assessed key regulatory mechanisms affecting variation in protein abundance. We identified 654 protein quantitative trait loci (pQTLs) in iPSCs, including disease-linked variants in protein-coding sequences and variants with trans regulatory effects. These include pQTL linked to GWAS variants that cannot be detected at the mRNA level, highlighting the utility of dissecting pQTL at peptide level resolution.

Arabidopsis proteome and the mass spectral assay library.

Arabidopsis is an important model organism and the first plant with its genome completely sequenced. Knowledge from studying this species has either direct or indirect applications for agriculture and human health. Quantitative proteomics by data-independent acquisition mass spectrometry (SWATH/DIA-MS) was recently developed and is considered as a high-throughput, massively parallel targeted approach for accurate proteome quantification. In this approach, a high-quality and comprehensive spectral library is a prerequisite. Here, we generated an expression atlas of 10 organs of Arabidopsis and created a library consisting of 15,514 protein groups, 187,265 unique peptide sequences, and 278,278 precursors. The identified protein groups correspond to ~56.5% of the predicted proteome. Further proteogenomics analysis identified 28 novel proteins. We applied DIA-MS using this library to quantify the effect of abscisic acid on Arabidopsis. We were able to recover 8,793 protein groups of which 1,787 were differentially expressed. MS data are available via ProteomeXchange with identifier PXD012708 and PXD012710 for data-dependent acquisition and PXD014032 for DIA analyses.

Multibatch TMT Reveals False Positives, Batch Effects and Missing Values.

Multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent, tandem mass tags (TMT) in particular. Here we used a large iPSC proteomic experiment with twenty-four 10-plex TMT batches to evaluate the effect of integrating multiple TMT batches within a single analysis. We identified a significant inflation rate of protein missing values as multiple batches are integrated and show that this pattern is aggravated at the peptide level. We also show that without normalization strategies to address the batch effects, the high precision of quantitation within a single multiplexed TMT batch is not reproduced when data from multiple TMT batches are integrated.Further, the incidence of false positives was studied by using Y chromosome peptides as an internal control. The iPSC lines quantified in this data set were derived from both male and female donors, hence the peptides mapped to the Y chromosome should be absent from female lines. Nonetheless, these Y chromosome-specific peptides were consistently detected in the female channels of all TMT batches. We then used the same Y chromosome specific peptides to quantify the level of ion coisolation as well as the effect of primary and secondary reporter ion interference. These results were used to propose solutions to mitigate the limitations of multi-batch TMT analyses. We confirm that including a common reference line in every batch increases precision by facilitating normalization across the batches and we propose experimental designs that minimize the effect of cross population reporter ion interference.

Signal enhanced proteomics: a biological perspective on dissecting the functional organisation of cell proteomes.

Proteomes are highly dynamic and can respond rapidly to environmental and cellular signals. Within cells, proteins often form distinct pools with different functions and properties. However, in quantitative proteomics studies it is common to measure averaged values for proteins that do not reflect variations that may occur between different protein isoforms, different subcellular compartments, or in cells at different cell cycle stages and so on. Here we review experimental approaches that can be used to enhance the signal from specific pools of protein that may otherwise be obscured through averaging across protein populations. This signal enhancement can help to reveal functions associated with specific protein pools, providing insight into the regulation of cellular processes. We review different strategies for proteomic signal enhancement, with a focus on the analysis of protein pools in different subcellular locations. We describe how MS-based proteome analyses can be combined with a general physico-chemical cell fractionation procedure that can be applied to many cultured cell lines.

Corrigendum: Common genetic variation drives molecular heterogeneity in human iPSCs.

This corrects the article DOI: 10.1038/nature22403.

Comparative genetic, proteomic and phosphoproteomic analysis of C. elegans embryos with a focus on ham-1/STOX and pig-1/MELK in dopaminergic neuron development.

Asymmetric cell divisions are required for cellular diversity and defects can lead to altered daughter cell fates and numbers. In a genetic screen for C. elegans mutants with defects in dopaminergic head neuron specification or differentiation, we isolated a new allele of the transcription factor HAM-1 [HSN (Hermaphrodite-Specific Neurons) Abnormal Migration]. Loss of both HAM-1 and its target, the kinase PIG-1 [PAR-1(I)-like Gene], leads to abnormal dopaminergic head neuron numbers. We identified discrete genetic relationships between ham-1, pig-1 and apoptosis pathway genes in dopaminergic head neurons. We used an unbiased, quantitative mass spectrometry-based proteomics approach to characterise direct and indirect protein targets and pathways that mediate the effects of PIG-1 kinase loss in C. elegans embryos. Proteins showing changes in either abundance, or phosphorylation levels, between wild-type and pig-1 mutant embryos are predominantly connected with processes including cell cycle, asymmetric cell division, apoptosis and actomyosin-regulation. Several of these proteins play important roles in C. elegans development. Our data provide an in-depth characterisation of the C. elegans wild-type embryo proteome and phosphoproteome and can be explored via the Encyclopedia of Proteome Dynamics (EPD) - an open access, searchable online database.

Common genetic variation drives molecular heterogeneity in human iPSCs.

Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative. Our study outlines the major sources of genetic and phenotypic variation in iPS cells and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPS cell phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of genomic copy-number alterations that are repeatedly observed in iPS cells. In addition, we present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells.

The Chromatin Assembly Factor Complex 1 (CAF1) and 5-Azacytidine (5-AzaC) Affect Cell Motility in Src-transformed Human Epithelial Cells.

Tumor invasion into surrounding stromal tissue is a hallmark of high grade, metastatic cancers. Oncogenic transformation of human epithelial cells in culture can be triggered by activation of v-Src kinase, resulting in increased cell motility, invasiveness, and tumorigenicity and provides a valuable model for studying how changes in gene expression cause cancer phenotypes. Here, we show that epithelial cells transformed by activated Src show increased levels of DNA methylation and that the methylation inhibitor 5-azacytidine (5-AzaC) potently blocks the increased cell motility and invasiveness induced by Src activation. A proteomic screen for chromatin regulators acting downstream of activated Src identified the replication-dependent histone chaperone CAF1 as an important factor for Src-mediated increased cell motility and invasion. We show that Src causes a 5-AzaC-sensitive decrease in both mRNA and protein levels of the p150 (CHAF1A) and p60 (CHAF1B), subunits of CAF1. Depletion of CAF1 in untransformed epithelial cells using siRNA was sufficient to recapitulate the increased motility and invasive phenotypes characteristic of transformed cells without activation of Src. Maintaining high levels of CAF1 by exogenous expression suppressed the increased cell motility and invasiveness phenotypes when Src was activated. These data identify a critical role of CAF1 in the dysregulation of cell invasion and motility phenotypes seen in transformed cells and also highlight an important role for epigenetic remodeling through DNA methylation for Src-mediated induction of cancer phenotypes.

Analysis of Mass Spectrometry Data for Nucleolar Proteomics Experiments.

With recent advances in experiment design, sample preparation, separation and instruments, mass spectrometry (MS)-based quantitative proteomics is becoming increasingly more popular. This has the potential to usher a new revolution in biology, in which the protein complement of cell populations can be described not only with increasing coverage, but also in all of its dimensions with unprecedented precision. Indeed, while earlier proteomics studies aimed solely at identifying as many as possible of the proteins present in the sample, newer, so-called Next Generation Proteomics studies add to this the aim of determining and quantifying the protein variants present in the sample, their mutual associations within complexes, their posttranslational modifications, their variation across the cell-cycle or in response to stimuli or perturbations, and their subcellular distribution. This has the potential to make MS proteomics much more useful for researchers, but will also mean that researchers with no background in MS will increasingly be confronted with the less-than trivial challenges of preparing samples for MS analysis, then processing and interpreting the results. In Chapter 20 , we described a workflow for isolating the protein contents of a specific SILAC-labeled organelle sample (the nucleolus) and processing it into peptides suitable for bottom-up MS analysis. Here, we complete this workflow by describing how to use the freely available MaxQuant software to convert the spectra stored in the Raw files into peptide- and protein-level information. We also briefly describe how to visualize the data using the free R scripting language.

Quantitative Proteomic Analysis of the Human Nucleolus.

Recent years have witnessed spectacular progress in the field of mass spectrometry (MS)-based quantitative proteomics, including advances in instrumentation, chromatography, sample preparation methods, and experimental design for multidimensional analyses. It is now possible not only to identify most of the protein components of a cell proteome in a single experiment, but also to describe additional proteome dimensions, such as protein turnover rates, posttranslational modifications, and subcellular localization. Furthermore, by comparing the proteome at different time points, it is possible to create a "time-lapse" view of proteome dynamics. By combining high-throughput quantitative proteomics with detailed subcellular fractionation protocols and data analysis techniques it is also now possible to characterize in detail the proteomes of specific subcellular organelles, providing important insights into cell regulatory mechanisms and physiological responses. In this chapter we present a reliable workflow and protocol for MS-based analysis and quantitation of the proteome of nucleoli isolated from human cells. The protocol presented is based on a SILAC analysis of human MCF10A-Src-ER cells with analysis performed on a Q-Exactive Plus Orbitrap MS instrument (Thermo Fisher Scientific). The subsequent chapter describes how to process the resulting raw MS files from this experiment using MaxQuant software and data analysis procedures to evaluate the nucleolar proteome using customized R scripts.

Enhanced snoMEN Vectors Facilitate Establishment of GFP-HIF-1α Protein Replacement Human Cell Lines.

The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins. Using multiplex 47snoMEM vectors that co-express multiple 47snoMEN in a single transcript, each of which can target different sites in the same mRNA, we document >3-fold increase in knock-down efficiency when compared with the original HBII-180C based snoMEN. The multiplex 47snoMEM vector allowed the construction of human protein replacement cell lines with improved efficiency, including the establishment of novel GFP-HIF-1α replacement cells. Quantitative mass spectrometry analysis confirmed the enhanced efficiency and specificity of protein replacement using the 47snoMEN-PR vectors. The 47snoMEN vectors expand the potential applications for snoMEN technology in gene expression studies, target validation and gene therapy.

CDK-dependent phosphorylation of PHD1 on serine 130 alters its substrate preference in cells.

PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression through the regulation of the crucial centrosomal protein Cep192, establishing a link between the oxygen-sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, CDK4 and CDK6 at S130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of hypoxia-inducible factor (HIF) protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, although phosphorylation of PHD1 at S130 lowers its activity towards HIF1α, this modification increases the activity of PHD1 towards Cep192. These results establish a mechanism by which cell cycle mediators, such as CDKs, temporally control the activity of PHD1, directly altering the regulation of HIF1α and Cep192.