An atlas of endogenous DNA double-strand breaks arising during human neural cell fate determination.

Endogenous DNA double-strand breaks (DSBs) occurring in neural cells have been implicated in the pathogenesis of neurodevelopmental disorders (NDDs). Currently, a genomic map of endogenous DSBs arising during human neurogenesis is missing. Here, we applied in-suspension Breaks Labeling In Situ and Sequencing (sBLISS), RNA-Seq, and Hi-C to chart the genomic landscape of DSBs and relate it to gene expression and genome architecture in 2D cultures of human neuroepithelial stem cells (NES), neural progenitor cells (NPC), and post-mitotic neural cells (NEU). Endogenous DSBs were enriched at the promoter and along the gene body of transcriptionally active genes, at the borders of topologically associating domains (TADs), and around chromatin loop anchors. NDD risk genes harbored significantly more DSBs in comparison to other protein-coding genes, especially in NEU cells. We provide sBLISS, RNA-Seq, and Hi-C datasets for each differentiation stage, and all the scripts needed to reproduce our analyses. Our datasets and tools represent a unique resource that can be harnessed to investigate the role of genome fragility in the pathogenesis of NDDs.

GPSeq reveals the radial organization of chromatin in the cell nucleus.

With the exception of lamina-associated domains, the radial organization of chromatin in mammalian cells remains largely unexplored. Here we describe genomic loci positioning by sequencing (GPSeq), a genome-wide method for inferring distances to the nuclear lamina all along the nuclear radius. GPSeq relies on gradual restriction digestion of chromatin from the nuclear lamina toward the nucleus center, followed by sequencing of the generated cut sites. Using GPSeq, we mapped the radial organization of the human genome at 100-kb resolution, which revealed radial patterns of genomic and epigenomic features and gene expression, as well as A and B subcompartments. By combining radial information with chromosome contact frequencies measured by Hi-C, we substantially improved the accuracy of whole-genome structure modeling. Finally, we charted the radial topography of DNA double-strand breaks, germline variants and cancer mutations and found that they have distinctive radial arrangements in A and B subcompartments. We conclude that GPSeq can reveal fundamental aspects of genome architecture.

BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks.

Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.

Quantification of HER2 and estrogen receptor heterogeneity in breast cancer by single-molecule RNA fluorescence in situ hybridization.

Intra-tumor heterogeneity is a pervasive property of human cancers that poses a major clinical challenge. Here, we describe the characterization, at the transcriptional level, of the intra-tumor topography of two prominent breast cancer biomarkers and drug targets, epidermal growth factor receptor 2 (HER2) and estrogen receptor 1 (ER) in 49 archival breast cancer samples. We developed a protocol for single-molecule RNA FISH in formalin-fixed, paraffin-embedded tissue sections (FFPE-smFISH), which enabled us to simultaneously detect and perform absolute quantification of HER2 and ER mature transcripts in single cells and multiple tumor regions. We benchmarked our method with standard diagnostic techniques, demonstrating that FFPE-smFISH is able to correctly classify breast cancers into well-established molecular subgroups. By counting transcripts in thousands of single cells, we identified different expression modes and levels of inter-cellular variability. In samples expressing both HER2 and ER, many cells co-expressed both genes, although expression levels were typically uncorrelated. Finally, we applied diversity metrics from the field of ecology to assess the intra-tumor topography of HER2 and ER gene expression, revealing that the spatial distribution of these key biomarkers can vary substantially even among breast cancers of the same subtype. Our results demonstrate that FFPE-smFISH is a reliable diagnostic assay and a powerful method for quantification of intra-tumor transcriptional heterogeneity of selected biomarkers in clinical samples.

Aggregation of oligoarginines at phospholipid membranes: molecular dynamics simulations, time-dependent fluorescence shift, and biomimetic colorimetric assays.

A time-dependent fluorescence shift method, biomimetic colorimetric assays, and molecular dynamics simulations have been performed in search of explanations why arginine rich peptides with intermediate lengths of about 10 amino acids translocate well through cellular membranes, while analogous lysine rich peptides do not. First, we demonstrate that an important factor for efficient peptide adsorption, as the first prerequisite for translocation across the membrane, is the presence of negatively charged phospholipids in the bilayer. Second, we observe a strong tendency of adsorbed arginine (but not lysine) containing peptides to aggregate at the bilayer surface. We suggest that this aggregation of oligoarginines leads to partial disruption of the bilayer integrity due to the accumulated large positive charge at its surface, which increases membrane-surface interactions due to the increased effective charge of the aggregates. As a result, membrane penetration and translocation of medium length oligoarginines becomes facilitated in comparison to single arginine and very long polyarginines, as well as to lysine containing peptides.

Overcharging in biological systems: reversal of electrophoretic mobility of aqueous polyaspartate by multivalent cations.

Charge reversal as an extreme case of charge compensation is directly observed by capillary electrophoresis for a negatively charged peptide in aqueous solutions of trivalent cations. Atomistic and coarse-grained simulations provide molecular interpretation of this effect showing that it is largely of electrostatic origin with a minor contribution of chemical specificity of the salt ions.

Effect of association with sulfate on the electrophoretic mobility of polyarginine and polylysine.

Domains rich in cationic amino acids are ubiquitous in peptides with the ability to cross cell membranes, which is likely related to the binding of such polypeptides to anionic groups on the membrane surface. To shed more light on these interactions, we investigated specific interactions between basic amino acids and oligopeptides thereof and anions by means of electrophoretic experiments and molecular dynamics simulations. To this end, we measured the electrophoretic mobilities of arginine, lysine, tetraarginine, and tetralysine in sodium chloride and sodium sulfate electrolytes as a function of ionic strength. The mobility was found to be consistently lower in sodium sulfate than in sodium chloride at the same ionic strength. The decrease in mobility in sodium sulfate was greater for tetraarginine than for tetralysine and was larger for tetrapeptides compared to the corresponding free amino acids. On the basis of molecular dynamics simulations and Bjerrum theory, we rationalize these results in terms of enhanced association between the amino acid side chains and sulfate. Simulations also predict a greater affinity of sulfate to the guanidinium side chain groups of arginine than to the ammonium groups of lysine, as the planar guanidinium geometry allows simultaneous strong hydrogen bonding to two sulfate oxygens. We show that the sulfate binding to arginine, but not to lysine, is cooperative. These results are consistent with the greater decrease in the mobility of arginine compared to that of lysine upon addition of sulfate salt. The nonspecific mobility retardation by sulfate is ascribed to its electrostatic interaction with the cationic amino acid side chain groups.