Emerging single-cell epigenomic methods are being developed with the exciting potential to transform our knowledge of gene regulation. Here we review available techniques and future possibilities, ...arguing that the full potential of single-cell epigenetic studies will be realized through parallel profiling of genomic, transcriptional, and epigenetic information.
Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a single-cell method ...for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.
Total Synthesis of (−)-Nakadomarin A Clark, J. Stephen; Xu, Chao
Angewandte Chemie (International ed.),
March 18, 2016, Volume:
55, Issue:
13
Journal Article
Peer reviewed
Open access
A highly efficient 12‐step synthesis of the marine alkaloid (−)‐nakadomarin A has been accomplished. The key advanced intermediate, a tetracyclic ketone derivative, was constructed in just seven ...steps using a sequence that includes an asymmetric Pauson–Khand reaction, an Overman rearrangement reaction, a ring‐closing metathesis reaction, and an amination reaction. Late introduction of the furan ring during the synthesis of (−)‐nakadomarin A means that the key tetracyclic ketone derivative has the potential to serve as an advanced intermediate for the synthesis of related marine alkaloids.
The later the better: A highly efficient 12‐step synthesis of (−)‐nakadomarin A is reported. The key tetracyclic ketone intermediate was constructed from a simple enyne in seven steps. As the furan is introduced at a later synthetic stage, this intermediate may have potential in the syntheses of related marine alkaloids such as manzamine A and ircinal A.
Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes
. Global ...epigenetic reprogramming accompanies these changes
, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.
The I–K fragment (C31–C49) of the ciguatoxin CTX3C has been synthesized from a simple chiral pool derived tetrahydropyranyl alcohol. An efficient gold-catalyzed cyclization reaction of a γ′-hydroxy ...ynone has been used to accomplish efficient closure of ring K under mild conditions. The resulting vinylogous ester has been elaborated to give a complete tricyclic fragment bearing the dimethyl-substituted side chain required for assembly of the LM spirocyclic acetal portion of the target.
Summary Background High quantities of tumour-infiltrating lymphocytes (TILs) in primary HER2-positive breast cancer are associated with improved prognosis and response to therapy. We aimed to ...investigate the prognostic role of host antitumour immunity as represented by baseline quantities of TILs in patients with advanced HER2-positive breast cancer treated with either pertuzumab or placebo in addition to trastuzumab and docetaxel. Methods CLEOPATRA was a randomised phase 3 study comparing the addition of either pertuzumab or placebo to first-line therapy with trastuzumab and docetaxel for patients with locally recurrent, unresectable, or metastatic HER2-positive breast cancer. We assessed the quantity of stromal TILs in prospectively collected tumour samples and investigated their association with progression-free survival, overall survival, clinicopathological characteristics, and pertuzumab treatment. We estimated hazard ratios (HR) and 95% CIs with multivariate Cox regression models fitting stromal TILs as a continuous variable (per 10% increment). The CLEOPATRA trial is registered with ClinicalTrials.gov , number NCT00567190. Findings Tumour samples from 678 (84%) of 808 participants were evaluable for TILs, including 519 (77%) archival samples, 155 (23%) freshly obtained samples (collected 45 days or fewer before randomisation), and four samples of unknown archival status. Median follow-up was 50 months (IQR 41–54) for progression-free survival and 51 months (IQR 46–57) for overall survival. 519 progression-free survival events occurred and 358 patients died. The median TIL value was 10% (IQR 5–30). Freshly obtained tumour samples had significantly lower TIL values than did archival samples (10·00% 95% CI 5·00–20·00 vs 15·00% 5·00–35·00; p=0·00036). We detected no significant association between TIL values and progression-free survival (adjusted HR 0·95, 95% CI 0·90–1·00, p=0·063). However, for overall survival, each 10% increase in stromal TILs was significantly associated with longer overall survival (adjusted HR 0·89, 95% CI 0·83–0·96, p=0·0014). The treatment effect of pertuzumab did not differ significantly by stromal TIL value for either progression-free survival (pinteraction =0·23) or overall survival (pinteraction =0·21). Interpretation In patients with advanced HER2-positive breast cancer treated with docetaxel, trastuzumab, and pertuzumab or placebo, higher TIL values are significantly associated with improved overall survival, suggesting that the effect of antitumour immunity extends to the advanced setting. Future clinical studies in this cancer subtype should consider TILs as a stratification factor and investigate whether therapies that can augment immunity could potentially further improve survival. Funding F Hoffmann-La Roche–Genentech and the Breast Cancer Research Foundation.
The integrity of chromatin, which provides a dynamic template for all DNA-related processes in eukaryotes, is maintained through replication-dependent and -independent assembly pathways. To address ...the role of histone deposition in the absence of DNA replication, we deleted the H3.3 chaperone Hira in developing mouse oocytes. We show that chromatin of non-replicative developing oocytes is dynamic and that lack of continuous H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and a severe fertility phenotype. On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation. Our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non-replicative system in vivo.
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•Histone H3/H4 replacement is continuous and mediated by Hira during mouse oogenesis•Loss of Hira results in chromatin abnormalities and extensive oocyte loss•Hira depletion reduces histone load, which prevents normal transcriptional regulation•Hira-mediated histone replacement is required for normal 5mC deposition in oocytes
To address the extent to which basic cellular processes depend on replication-independent chromatin assembly in vivo, Nashun et al. deleted histone H3.3 chaperone Hira during mouse oogenesis. Their results demonstrate a critical relationship between continuing histone replacement, chromatin homeostasis, transcriptional regulation, and de novo DNA methylation.
The complete carbon framework of the macrocyclic marine natural product amphidinolide F has been prepared by a convergent synthetic route in which three fragments of similar size and complexity have ...been coupled. Key features of the syntheses of the fragments include the stereoselective construction of the tetrahydrofuran in the C1–C9 fragment by oxonium ylide (free or metal-bound) formation and rearrangement triggered by the direct generation of a rhodium carbenoid from 1-sulfonyl-1,2,3-triazole, the highly diastereoselective aldol reaction between a boron enolate and an aldehyde with 1,4-control to prepare the C10–C17 fragment, and the formation of the tetrahydrofuran in the C18–C29 fragment by intramolecular nucleophilic ring opening of an epoxide with a hydroxyl group under acidic conditions.
Exploration of an ambitious new strategy for the total synthesis of the cytotoxic marine natural product amphidinolide F is described, which features fabrication of the core structure from four ...readily accessible fragments and macrocycle construction through C9–C10 bond formation by intramolecular Stille coupling between an alkenyl iodide and alkenyl stannane. Efficient stereoselective synthesis of each of the four building-blocks and subsequent coupling of them to produce the requisite cyclization precursor has been accomplished, but suitable conditions for high-yielding palladium-mediated closure of the macrocycle to produce the fully protected amphidinolide F ring system have yet to be identified.
DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular ...differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.
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