The FRIGG project: From intermediate galactic scales to self-gravitating cores

Abridged. Understanding the detailed structure of the interstellar gas is essential for our knowledge of the star formation process. The small-scale structure of the interstellar medium (ISM) is a direct consequence of the galactic scales and making the link between the two is essential. We perform adaptive mesh simulations that aim to bridge the gap between the intermediate galactic scales and the self-gravitating prestellar cores. For this purpose we use stratified supernova regulated ISM magneto-hydrodynamical (MHD) simulations at the kpc scale to set up the initial conditions. We then zoom, performing a series of concentric uniform refinement and then refining on the Jeans length for the last levels. This allows us to reach a spatial resolution of a few $10^{-3}$ pc. The cores are identified using a clump finder and various criteria based on virial analysis. Their most relevant properties are computed and, due to the large number of objects formed in the simulations, reliable statistics are obtained. The cores properties show encouraging agreements with observations. The mass spectrum presents a clear powerlaw at high masses with an exponent close to $\simeq -1.3$ and a peak at about 1-2 $M_\odot$. The velocity dispersion and the angular momentum distributions are respectively a few times the local sound speed and a few $10^{-2}$ pc km s$^{-1}$. We also find that the distribution of thermally supercritical cores present a range of magnetic mass-to-flux over critical mass-to-flux ratio which typically ranges between $\simeq$0.3 and 3.Comment: accepted for publication in A&

Two distinct modes for propagation of histone PTMs across the cell cycle

Epigenetic states defined by chromatin can be maintained through mitotic cell division. However, it remains unknown how histone-based information is transmitted. Here we combine nascent chromatin capture (NCC) and triple-SILAC (stable isotope labeling with amino acids in cell culture) labeling to track histone modifications and histone variants during DNA replication and across the cell cycle. We show that post-translational modifications (PTMs) are transmitted with parental histones to newly replicated DNA. Di- and trimethylation marks are diluted twofold upon DNA replication, as a consequence of new histone deposition. Importantly, within one cell cycle, all PTMs are restored. In general, new histones are modified to mirror the parental histones. However, H3K9 trimethylation (H3K9me3) and H3K27me3 are propagated by continuous modification of parental and new histones because the establishment of these marks extends over several cell generations. Together, our results reveal how histone marks propagate and demonstrate that chromatin states oscillate within the cell cycle

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.</p

The tumor suppressor CIC directly regulates MAPK pathway genes via histone deacetylation

Abstract Oligodendrogliomas are brain tumors accounting for approximately 10% of all central nervous system cancers. CIC is a transcription factor that is mutated in most patients with oligodendrogliomas; these mutations are believed to be a key oncogenic event in such cancers. Analysis of the Drosophila melanogaster ortholog of CIC, Capicua, indicates that CIC loss phenocopies activation of the EGFR/RAS/MAPK pathway, and studies in mammalian cells have demonstrated a role for CIC in repressing the transcription of the PEA3 subfamily of ETS transcription factors. Here, we address the mechanism by which CIC represses transcription and assess the functional consequences of CIC inactivation. Genome-wide binding patterns of CIC in several cell types revealed that CIC target genes were enriched for MAPK effector genes involved in cell-cycle regulation and proliferation. CIC binding to target genes was abolished by high MAPK activity, which led to their transcriptional activation. CIC interacted with the SIN3 deacetylation complex and, based on our results, we suggest that CIC functions as a transcriptional repressor through the recruitment of histone deacetylases. Independent single amino acid substitutions found in oligodendrogliomas prevented CIC from binding its target genes. Taken together, our results show that CIC is a transcriptional repressor of genes regulated by MAPK signaling, and that ablation of CIC function leads to increased histone acetylation levels and transcription at these genes, ultimately fueling mitogen-independent tumor growth. Significance: Inactivation of CIC inhibits its direct repression of MAPK pathway genes, leading to their increased expression and mitogen-independent growth. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4114/F1.large.jpg. Cancer Res; 78(15); 4114–25. ©2018 AACR.</jats:p

Ten questions to AI regarding the present and future of proteomics

The role of a scientist is at first not so different from a philosopher. They both need to question common thinking and evaluate whether reality is not as we always thought. Based on this, we need to design hypotheses, experiments, and analyses to prove our alternative vision. Artificial Intelligence (AI) is rapidly moving from an “assistant” into a proper “colleague” for literature mining, data analysis and interpretation, and literally having (almost) real scientific conversations. However, being AI based on existing information, if we rely on it excessively will we still be able to question the status quo? In this article, we are particularly interested in discussing the future of proteomics and mass spectrometry with our new electronic collaborator. We leave to the reader the judgement whether the answers we received are satisfactory or superficial. What we were mostly interested in was laying down what we think are critical questions that the proteomics community should occasionally ask to itself. Proteomics has been around for more than 30 years, but it is still missing a few critical steps to fully address its promises as being the new genomics for clinical diagnostics and fundamental science, while becoming a user-friendly tool for every lab. Will we get there with the help of AI? And will these answers change in a short period, as AI continues to advance

Extensive Characterization of Heavily Modified Histone Tails by 193 nm Ultraviolet Photodissociation Mass Spectrometry via a Middle-Down Strategy

The ability to map combinatorial patterns of post-translational modifications (PTMs) of proteins remains challenging for traditional bottom-up mass spectrometry workflows. There are also hurdles associated with top-down approaches related to limited data analysis options for heavily modified proteoforms. These shortcomings have accelerated interest in middle-down MS methods that focus on analysis of large peptides generated by specific proteases in conjunction with validated bioinformatics strategies to allow quantification of isomeric histoforms. Mapping multiple PTMs simultaneously requires the ability to obtain high sequence coverage to allow confident localization of the modifications, and 193 nm ultraviolet photodissociation (UVPD) has been shown to cause extensive fragmentation for large peptides and proteins. Histones are an ideal system to test the ability of UVPD to characterize multiple modifications, as the combinations of PTMs are the underpinning of the biological significance of histones and at the same time create an imposing challenge for characterization. The present study focuses on applying 193 nm UVPD to the identification and localization of PTMs on histones by UVPD and comparison to a popular alternative, electron-transfer dissociation (ETD), via a high-throughput middle-down LC/MS/MS strategy. Histone Coder and IsoScale, bioinformatics tools for verification of PTM assignments and quantification of histone peptides, were adapted for UVPD data and applied in the present study. In total, over 300 modified forms were identified, and the distributions of PTMs were quantified between UVPD and ETD. Significant differences in patterns of PTMs were found for histones from HeLa cells prior to and after treatment with a deacetylase inhibitor. Additional fragment ion types generated by UVPD proved essential for extensive characterization of the most heavily modified forms (&gt;5 PTMs).</p

RNA polymerase II promotes the organization of chromatin following DNA replication

Understanding how chromatin organisation is duplicated on the two daughter strands is a central question in epigenetics. In mammals, following the passage of the replisome, nucleosomes lose their defined positioning and transcription contributes to their re-organisation. However, whether transcription plays a greater role in the organization of chromatin following DNA replication remains unclear. Here we analysed protein re-association with newly replicated DNA upon inhibition of transcription using iPOND coupled to quantitative mass spectrometry. We show that nucleosome assembly and the re-establishment of most histone modifications are uncoupled from transcription. However, RNAPII acts to promote the re-association of hundreds of proteins with newly replicated chromatin via pathways that are not observed in steady-state chromatin. These include ATP-dependent remodellers, transcription factors and histone methyltransferases. We also identify a set of DNA repair factors that may handle transcription-replication conflicts during normal transcription in human non-transformed cells. Our study reveals that transcription plays a greater role in the organization of chromatin post-replication than previously anticipated.</p

Ketone Bodies Rescue Mitochondrial Dysfunction Via Epigenetic Remodeling

Ischemic cardiac disease is a major cause of mortality worldwide. However, the exact molecular processes underlying this disorder are not fully known. This study includes a comprehensive and coordinated set of in vivo and in vitro experiments using human cardiac specimens from patients with postischemic heart failure (HF) and healthy control subjects, a murine model of HF, and cellular systems. These approaches identified for the first time a specific pattern of maladaptive chromatin remodeling, namely a double methylation of histone 3 at lysine 27 and a single methylation at lysine 36 (H3_K27me2K36me1) consistently induced by ischemic injury in all these settings: human HF; murine HF; and in vitro models. Mechanistically, this work demonstrates that this histone modification mediates the ischemia-induced transcriptional repression of PPARG coactivator 1α (PGC1α), master regulator of mitochondrial function and biogenesis. Intriguingly, both the augmented H3_K27me2K36me1 and the mitochondrial dysfunction ensued by PGC1α down-regulation were significantly attenuated by the treatment with β-hydroxybutyrate, the most abundant ketone body in humans, revealing a novel pathway coupling metabolism to gene expression. Taken together, these findings establish maladaptive chromatin remodeling as a key mechanism in postischemic heart injury, functionally modulated by ketone bodies

PROSER1 modulates DNA demethylation through dual mechanisms to prevent syndromic developmental malformations

The link between DNA methylation and neurodevelopmental disorders is well established. However, how DNA methylation is fine-tuned—ensuring precise gene expression and developmental fidelity—remains poorly understood. PROSER1, a known TET2 interactor, was recently linked to a severe neurodevelopmental disorder. Here, we demonstrate that PROSER1 interacts with all TET enzymes and stabilizes chromatin-bound TETOGT–PROSER1–DBHS (TOPD) complexes, which regulate DNAdemethylation and developmental gene expression. Surprisingly, we found that PROSER1 also sequesters TET enzymes, preventing widespread demethylation and transposable element derepression. Our findings identify PROSER1 as a key factor that both positively and negatively regulates DNA demethylation essential for mammalian neurodevelopment

PROSER1 Modulates DNA Demethylation through Dual Mechanisms to Prevent Syndromic Developmental Malformations

The link between DNA methylation and neurodevelopmental disorders is well established. However, how DNA methylation is fine-tuned – ensuring precise gene expression and developmental fidelity – remains poorly understood. PROSER1, a known TET2 interactor, was recently linked to a severe neurodevelopmental disorder. Here, we demonstrate that PROSER1 interacts with all TET enzymes and stabilizes chromatin-bound TET-OGT-PROSER1-DBHS (TOPD) complexes, which regulate DNA demethylation and developmental gene expression. Surprisingly, we find that PROSER1 also sequesters TET enzymes, preventing widespread demethylation and transposable element de-repression. Our findings identify PROSER1 as a key factor which both positively and negatively regulates DNA demethylation essential for mammalian neurodevelopment