Unlocking the chromatin code by deciphering protein–DNA interactions

Characterizing the composition of protein complexes bound to different genomic loci is essential for advancing our mechanistic understanding of transcriptional regulation. In their recent study, Krijgsveld and colleagues (Rafiee et al, 2016) report ChIP‐SICAP, a powerful tool for deciphering the chromatin proteome by combining chromatin immunoprecipitation, selective isolation of chromatin‐associated proteins and mass spectrometry

Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA [version 1; peer review: 1 approved, 2 approved with reservations]

Background: Methylation of carbon-5 of cytosines (m5C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. It can be further modified to yield 5-hydroxymethylcytidine (hm5C), 5-formylcytidine (f5C), 2´-O-methyl-5-hydroxymethylcytidine (hm5Cm) and 2´-O-methyl-5-formylcytidine (f5Cm). How m5C, and specially its derivates, contribute to biology mechanistically is poorly understood. We recently showed that m5C is required for Caenorhabditis elegans development and fertility under heat stress. m5C 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 m5C 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 m5C 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 hm5Cm unique readers are enriched in proteins involved in RNA processing, while readers for m5C, hm5C and f5C are involved in germline processes. We validated our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m5C in vitro. Finally, sequence alignment analysis showed that several of the putative m5C 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 m5C and its derivatives. Furthermore, we demonstrate that ALY-1 and ALY-2 bind to m5C in C. elegans

LINE-1 RNA triggers matrix formation in bone cells via a PKR-mediated inflammatory response

Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells.</p

LINE-1 RNA triggers matrix formation in bone cells via a PKR-mediated inflammatory response

Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells

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

Abstract: 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

Targeted knock-down of miR21 primary transcripts using snoMEN vectors induces apoptosis in human cancer cell lines

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2

Evaluating the use of HILIC in large-scale, multi dimensional proteomics:horses for courses?

AbstractDespite many recent advances in instrumentation, the sheer complexity of biological samples remains a major challenge in large-scale proteomics experiments, reflecting both the large number of protein isoforms and the wide dynamic range of their expression levels. However, while the dynamic range of expression levels for different components of the proteome is estimated to be ∼107–8, the equivalent dynamic range of LC–MS is currently limited to ∼106. Sample pre-fractionation has therefore become routinely used in large-scale proteomics to reduce sample complexity during MS analysis and thus alleviate the problem of ion suppression and undersampling. There is currently a wide range of chromatographic techniques that can be applied as a first dimension separation. Here, we systematically evaluated the use of hydrophilic interaction liquid chromatography (HILIC), in comparison with hSAX, as a first dimension for peptide fractionation in a bottom-up proteomics workflow. The data indicate that in addition to its role as a useful pre-enrichment method for PTM analysis, HILIC can provide a robust, orthogonal and high-resolution method for increasing the depth of proteome coverage in large-scale proteomics experiments. The data also indicate that the choice of using either HILIC, hSAX, or other methods, is best made taking into account the specific types of biological analyses being performed

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