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Background Lamina-associated domains (LADs) are large genomic regions that are positioned at the nuclear lamina. It has remained largely unclear what drives the positioning and demarcation of LADs. Because the insulator protein CTCF is enriched at LAD borders, it was postulated that CTCF binding could position some LAD boundaries, possibly through its function in stalling cohesin and hence preventing cohesin invading into the LAD. To test this, we mapped genome–nuclear lamina interactions in mouse embryonic stem cells after rapid depletion of CTCF and other perturbations of cohesin dynamics. Results CTCF and cohesin contribute to a sharp transition in lamina interactions at LAD borders, while LADs are maintained after depletion of these proteins, also at borders marked by CTCF. CTCF and cohesin may thus reinforce LAD borders, but do not position these. CTCF binding sites within LADs are locally detached from the lamina and enriched for accessible DNA and active histone modifications. Remarkably, despite lamina positioning being strongly correlated with genome inactivity, this DNA remains accessible after the local detachment is lost following CTCF depletion. At a chromosomal scale, cohesin depletion and cohesin stabilization by depletion of the unloading factor WAPL quantitatively affect lamina interactions, indicative of perturbed chromosomal positioning in the nucleus. Finally, while H3K27me3 is locally enriched at CTCF-marked LAD borders, we find no evidence for an interplay between CTCF and H3K27me3 on lamina interactions. Conclusions These findings illustrate that CTCF and cohesin are not primary determinants of LAD patterns. Rather, these proteins locally modulate NL interactions.

Acquired drug resistance is a major problem in the treatment of cancer. hTERT-immortalized, untransformed RPE-1 (RPE) cells can acquire resistance to taxol by derepressing the ABCB1 gene, encoding for the multidrug transporter P-gP. Here we have investigated how the ABCB1 gene is derepressed. We show that activation of the ABCB1 gene is associated with reduced DNA methylation, reduced H3K9 trimethylation and increased H3K27 acetylation at the ABCB1 promoter. In addition, we find that the ABCB1 locus has moved away from the nuclear lamina in the taxol-resistant cells. This raises the question which of these alterations were causal to derepression. Directly modifying DNA methylation or H3K27 methylation had neither significant effect on ABCB1 expression, nor did it promote drug resistance. In contrast, the disruption of Lamin B Receptor (LBR), a component of the nuclear lamina involved in genome organization, did promote the acquisition of a taxol-resistant phenotype in a subset of cells. Using CRISPRa-mediated gene activation, we could further substantiate a model in which disruption of lamina association renders the ABCB1 gene permissive to derepression. Based on these data we propose a model in which nuclear lamina dissociation of a repressed gene allows for its activation, implying that deregulation of the 3D genome topology could play an important role in tumor evolution and the acquisition of drug resistance.

The cohesin complex plays an essential role in maintaining genome organization. However, its role in gene regulation remains largely unresolved. Here, we report that the cohesin release factor WAPL creates a pool of free cohesin, in a process known as cohesin turnover, which reloads it to cell-type specific binding sites. Paradoxically, stabilization of cohesin binding, following WAPL ablation, results in depletion of cohesin from these cell-type specific regions, a loss of gene expression and differentiation. Chromosome conformation capture experiments show that cohesin turnover is important for maintaining promoter-enhancer loops. Binding of cohesin to cell-type specific sites is dependent on the pioneer transcription factors OCT4 (POU5F1) and SOX2, but not NANOG. We show the importance of cohesin turnover in controlling transcription and propose that a cycle of cohesin loading and off-loading, instead of static cohesin binding, mediates promoter and enhancer interactions critical for gene regulation.

Chromosome folding is modulated as cells progress through the cell cycle. During mitosis, condensins fold chromosomes into helical loop arrays. In interphase, the cohesin complex generates loops and topologically associating domains (TADs), while a separate process of compartmentalization drives segregation of active and inactive chromatin. We used synchronized cell cultures to determine how the mitotic chromosome conformation transforms into the interphase state. Using Hi-C, chromatin binding assays, and immunofluorescence we show that by telophase condensin-mediated loops are lost and a transient folding intermediate devoid of most loops forms. By cytokinesis, cohesin-mediated CTCF-CTCF loops and positions of TADs emerge. Compartment boundaries are also established early, but long-range compartmentalization is a slow process and proceeds for hours after cells enter G1. Our results reveal the kinetics and order of events by which the interphase chromosome state is formed and identify telophase as a critical transition between condensin and cohesin driven chromosome folding.

SUMMARYThe cohesin complex plays essential roles in sister chromatin cohesin, chromosome organization and gene expression. The role of cohesin in gene regulation is incompletely understood. Here, we report that the cohesin release factor WAPL is crucial for maintaining a pool of dynamic cohesin bound to regions that are associated with lineage specific genes in mouse embryonic stem cells. These regulatory regions are enriched for active enhancer marks and transcription factor binding sites, but largely devoid of CTCF binding sites. Stabilization of cohesin, which leads to a loss of dynamic cohesin from these regions, does not affect transcription factor binding or active enhancer marks, but does result in changes in promoter-enhancer interactions and downregulation of genes. Acute cohesin depletion can phenocopy the effect of WAPL depletion, showing that cohesin plays a crucial role in maintaining expression of lineage specific genes. The binding of dynamic cohesin to chromatin is dependent on the pluripotency transcription factor OCT4, but not NANOG. Finally, dynamic cohesin binding sites are also found in differentiated cells, suggesting that they represent a general regulatory principle. We propose that cohesin dynamically binding to regulatory sites creates a favorable spatial environment in which promoters and enhancers can communicate to ensure proper gene expression.HIGHLIGHTSThe cohesin release factor WAPL is crucial for maintaining a pluripotency-specific phenotype.Dynamic cohesin is enriched at lineage specific loci and overlaps with binding sites of pluripotency transcription factors.Expression of lineage specific genes is maintained by dynamic cohesin binding through the formation of promoter-enhancer associated self-interaction domains.CTCF-independent cohesin binding to chromatin is controlled by the pioneer factor OCT4.

Leukemia is characterized by genetic and epigenetic mutations resulting in selection of cancer cells, which are unable to differentiate. Although genetic alterations are difficult to target, the epigenome is intrinsically dynamic and readily offers new therapeutic strategies. Thus, identifying cancer-specific context-dependent targets and unraveling their biological function may open up new therapeutic perspectives. Here we identify bromodomain-containing protein 9 (BRD9) as a critical target required in acute myeloid leukemia (AML). We show that BRD9 is overexpressed in AML cells including ex vivo primary blasts compared with CD34+ cells. By targeting BRD9 expression in AML, we observed an alteration in proliferation and survival, ultimately resulting in the induction of apoptosis. Intriguingly, genome-wide profiling revealed that BRD9 binds enhancer regions in a cell type-specific manner, regulating cell type-related processes. We unveil a novel BRD9-sustained STAT5 pathway activation via regulation of SOCS3 expression levels. Our findings identify a previously undescribed BRD9-STAT5 axis as critical for leukemia maintenance, suggesting BRD9 as a potential therapeutic target.

In breast cancer, Lysine-specific demethylase-1 (LSD1) and other lysine demethylases (KDMs), such as Lysine-specific demethylase 6A also known as Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), are co-expressed and co-localize with estrogen receptors (ERs), suggesting the potential use of hybrid (epi)molecules to target histone methylation and therefore regulate/redirect hormone receptor signaling. Here, we report on the biological activity of a dual-KDM inhibitor (MC3324), obtained by coupling the chemical properties of tranylcypromine, a known LSD1 inhibitor, with the 2OG competitive moiety developed for JmjC inhibition. MC3324 displays unique features not exhibited by the single moieties and well-characterized mono-pharmacological inhibitors. Inhibiting LSD1 and UTX, MC3324 induces significant growth arrest and apoptosis in hormone-responsive breast cancer model accompanied by a robust increase in H3K4me2 and H3K27me3. MC3324 down-regulates ER&alpha, in breast cancer at both transcriptional and non-transcriptional levels, mimicking the action of a selective endocrine receptor disruptor. MC3324 alters the histone methylation of ER&alpha, regulated promoters, thereby affecting the transcription of genes involved in cell surveillance, hormone response, and death. MC3324 reduces cell proliferation in ex vivo breast cancers, as well as in breast models with acquired resistance to endocrine therapies. Similarly, MC3324 displays tumor-selective potential in vivo, in both xenograft mice and chicken embryo models, with no toxicity and good oral efficacy. This epigenetic multi-target approach is effective and may overcome potential mechanism(s) of resistance in breast cancer.

Genome-wide association studies have identified a great number of non-coding risk variants for colorectal cancer (CRC). To date, the majority of these variants have not been functionally studied. Identification of allele-specific transcription factor (TF) binding is of great importance to understand regulatory consequences of such variants. A recently developed proteome-wide analysis of disease-associated SNPs (PWAS) enables identification of TF-DNA interactions in an unbiased manner. Here we perform a large-scale PWAS study to comprehensively characterize TF-binding landscape that is associated with CRC, which identifies 731 allele-specific TF binding at 116 CRC risk loci. This screen identifies the A-allele of rs1800734 within the promoter region of MLH1 as perturbing the binding of TFAP4 and consequently increasing DCLK3 expression through a long-range interaction, which promotes cancer malignancy through enhancing expression of the genes related to epithelial-to-mesenchymal transition., Functional characterisation of non-coding risk variants for colorectal cancer is missing. Here the authors conduct a large-scale proteome-wide analysis of disease associated SNPs to characterize allele-specific transcription factor binding landscape in colorectal cancer and identify one functionally significant SNP.

Quantitative proteomics plays an important role in validation of breast-cancer-related biomarkers. In this study, we systematically compared the performance of label-free quantification (LFQ) and SILAC with shotgun and directed methods for quantifying breast-cancer-related markers in microdissected tissues. We show that LFQ leads to slightly higher coefficient of variation (CV) for protein quantification (median CV = 16.3%) than SILAC quantification (median CV = 13.7%) (P < 0.0001), but LFQmethod enables,similar to,60% more protein quantification and is also more reproducible (,similar to,20% more proteins were quantified in all replicate samples). Furthermore, we describe a method to accurately quantify multiple proteins within one pathway, that is, "focal adhesion pathway", in trace amounts of breast cancer tissues using a SILAC-based SRM assay. Using this SILAC-based SRM assay, we precisely quantified five "focal adhesion" proteins with good quantitative precision (CV range: 2.4-5.9%) in replicate whole tissue lysate samples and replicate microdissected samples (CV range: 5.8-16.1%). Our results show that in microdissected breast cancer tissues LFQ in combination with shotgun proteomics performed the best overall and is therefore suitable for both biomarker discovery and validation in these types of specimens. The SILAC-based SRM method can be used for the development of clinically relevant protein assays in tumor biopsies.

Radix Bupleuri is a traditional Chinese medicine harvested from two Bupleurum species (B. chinense and B. scorzonerifolium). It is widely used and is sourced from different regions of China. 1H NMR spectroscopy and multivariate data analysis were applied to 67 Radix Bupleuri samples to discriminate the two species, and explore the influences of habitat and culture method on the quality of Radix Bupleuri based on their metabolomics profiles. Metabolites responsible for the differences between the two species were higher levels of arginine, citric acid, sucrose, saikosaponin b1/b2 analogs, volatile oil with an (E)-2-olefin aldehyde fragment, and fatty acids in B. scoreonerifolium, and more saikosaponin a/c/d analogs in B. chinense. The variances of two cultivation areas were observed due to the higher amount of saikosaponins a/c/d in samples from Shaanxi and lipidsin samples from Shanxi. No obvious difference was detected between cultivars and wild type. 1HNMR metabolomics can simultaneously detect saikosaponins and hydrocarbon aldehydes, and also differentiate the two main saikosaponin skeletons, making it a suitable tool for the species discrimination and quality evaluation of Radix Bupleuri.

Reliable sample preparation is of utmost importance for comparative proteome analysis, particularly when investigating minute amounts of clinical specimens, such as laser capture microdissected tumor tissue. In this study, we present an optimized nanoLC-MS workflow specifically for the analysis of laser capture microdissected breast cancer tissue. Analytical performance of different laser capture microdissection (LCM) functions available on the PALM system, time dependent trypsin digestion efficiency, effect of sample preparation and digestion time on peptide modification, semi-tryptic peptides and missed cleavages were evaluated. Our results show that microdissection from uncoated glass slides results in protein degradation; that protease and phosphatase inhibitors do not result in detectable improvement in number of peptides or semi-tryptic peptides; and that digestion time longer than four hours drastically reduces the number of missed cleavages, but also increases the number of unexpectedly modified peptides. Overalkylation was the most dominant side-reaction, which significantly increased overnight (P=0.05). The latter effect could almost completely be reverted by the use of a quenching agent (P=0.001). Taken together, our results show that it is of importance to carefully control sample handling steps so that reliable protein identification and quantitation can be performed within comparative proteomics studies using LCM. This article is part of a Special Issue entitled: Proteomics: The clinical link. (C) 2012 Published by Elsevier B.V.

Treatment of uncomplicated malaria relies heavily on the sesquiterpene lactone artemisinin (ART), derived from the Chinese herb Artemisia annua. It is currently used as the first line treatment in the form of ART combination therapies in which different ART derivatives are combined with a non ART-like drug, which displays a different mechanism of action. This treatment regime is prescribed by the WHO in order to (1) effectively treat uncomplicated malaria and (2) to reduce the risk that drug resistance will develop. Considering the importance of ART in this treatment regime, various methods have been developed to increase the global production of ART. These methods include: large scale solvent extraction and purification from high yielding A. annua varieties, chemical/semi-chemical synthesis of artemisinin, and genetic engineering of unrelated organisms to produce ART in high quantities. Currently most of the commercial approaches to produce ART are based on solvent extraction and purification from the plant material. Classical extraction solvents include hexane or petroleum ether while ethanol, which is a more polar solvent, has recently been shown to be equally efficient. We developed a purification method from an ethanolic extract by using a diatomite-based purification protocol. Various volumes and ratios of solvents and diatomite were tested which resulted in ≥98% pure ART with a total recovery of around 60%. Our protocol for the isolation of ART may form a commercial scale alternative as it is both economically attractive due to the small amounts of purification materials needed and also environmentally friendlier than the methods previously described.

Introduction – Since the discovery of artemisinin in the 1970s, many techniques based on diverse chromatography techniques have been developed to detect and quantify this important antiplasmodial compound. The accurate quantification of this compound in the Artemisia annua plant material is mainly needed for breeding purposes in order to cultivate higher yielding varieties. It is also important for the quality control of herbal preparations containing A. annua plant material. Objective – To evaluate the most common validated quantification techniques (LC-MS, HPLC-ELSD and TLC) and compare the results to quantitative nuclear magnetic resonance spectroscopy (qNMR) in eight different A. annua samples collected from around the world. Methodology – The leaf material were extracted according to standard procedures and analysed with the validated quantification techniques. For the qNMR analysis we did not employ a standard curve but instead used an internal standard (maleid acid) which is not chemically related to artemisinin. Results – We found a significant difference between the results in this study. Compared with the qNMR results the HPLC-ELSD corresponded closely, followed by LC-MS. Quantitation with TLC led to an estimation range of −0.5 to +3.2 mg artemisinin/g of A. annua. Conclusion – These results imply that qNMR, with the addition of an internal standard, can be used to quantify artemisinin in A. annua samples in a rapid and reproducible manner. Copyright © 2010 John Wiley & Sons, Ltd.

The genus Artemisia consists of about 500 species, occurring throughout the world. Some very important drug leads have been discovered from this genus, notably artemisinin, the well known anti-malarial drug isolated from the Chinese herb Artemisia annua . The genus is also known for its aromatic nature and hence research has been focussed on the chemical compositions of the volatile secondary metabolites obtained from various Artemisia species. In the southern African region, A. afra is one of the most popular and commonly used herbal medicines. It is used to treat various ailments ranging from coughs and colds to malaria and diabetes. Although it is one of the most popular local herbal medicines, only limited scientific research, mainly focussing on the volatile secondary metabolites content, has been conducted on this species. The aim of this review was therefore to collect all available scientific literature published on A. afra and combine it into this paper. In this review, a general overview will be given on the morphology, taxonomy and geographical distribution of A. afra . The major focus will however be on the secondary metabolites, mainly the volatile secondary metabolites, which have been identified from this species. In addition all of the reported biological activities of the extracts derived from this species have been included as well as the literature on the pharmacology and toxicology. We aim at bringing together most of the available scientific research conducted on this species, which is currently scattered across various publications, into this review paper.

Item does not contain fulltext Ferritin heavy chain (FTH1) is a 21-kDa subunit of the ferritin complex, known for its role in iron metabolism, and which has recently been identified as a favorable prognostic protein for triple negative breast cancer (TNBC) patients. Currently, it is not well understood how FTH1 contributes to an anti-tumor response. Here, we explored whether expression and cellular compartmentalization of FTH1 correlates to an effective immune response in TNBC patients. Analysis of the tumor tissue transcriptome, complemented with in silico pathway analysis, revealed that FTH1 was an integral part of an immunomodulatory network of cytokine signaling, adaptive immunity, and cell death. These findings were confirmed using mass spectrometry (MS)-derived proteomic data, and immunohistochemical staining of tissue microarrays. We observed that FTH1 is localized in both the cytoplasm and/or nucleus of cancer cells. However, high cytoplasmic (c) FTH1 was associated with favorable prognosis (Log-rank p = 0.001), whereas nuclear (n) FTH1 staining was associated with adverse prognosis (Log-rank p = 0.019). cFTH1 staining significantly correlated with total FTH1 expression in TNBC tissue samples, as measured by MS analysis (Rs = 0.473, p = 0.0007), but nFTH1 staining did not (Rs = 0.197, p = 0.1801). Notably, IFN gamma-producing CD8+ effector T cells, but not CD4+ T cells, were preferentially enriched in tumors with high expression of cFTH1 (p = 0.02). Collectively, our data provide evidence toward new immune regulatory properties of FTH1 in TNBC, which may facilitate development of novel therapeutic targets.

Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos., Molecular Cell, 62 (6), ISSN:1097-2765, ISSN:1097-4164

Mass spectrometry (MS)-based label-free proteomics offers an unbiased approach to screen biomarkers related to disease progression and therapy-resistance of breast cancer on the global scale. However, multi-step sample preparation can introduce large variation in generated data, while inappropriate statistical methods will lead to false positive hits. All these issues have hampered the identification of reliable protein markers. A workflow, which integrates reproducible and robust sample preparation and data handling methods, is highly desirable in clinical proteomics investigations. Here we describe a label-free tissue proteomics pipeline, which encompasses laser capture microdissection (LCM) followed by nanoscale liquid chromatography and high resolution MS. This pipeline routinely identifies on average ∼10,000 peptides corresponding to ∼1,800 proteins from sub-microgram amounts of protein extracted from ∼4,000 LCM breast cancer epithelial cells. Highly reproducible abundance data were generated from different technical and biological replicates. As a proof-of-principle, comparative proteome analysis was performed on estrogen receptor α positive or negative (ER+/−) samples, and commonly known differentially expressed proteins related to ER expression in breast cancer were identified. Therefore, we show that our tissue proteomics pipeline is robust and applicable for the identification of breast cancer specific protein markers. Electronic supplementary material The online version of this article (doi:10.1007/s10911-012-9252-6) contains supplementary material, which is available to authorized users.

Ethnopharmacological relevance Artemisia afra has been used as an infusion to treat malaria throughout the southern parts of Africa, in much the same way as the antimalarial plant Artemisia annua in China. The antiplasmodial activity of purified components from an apolar fraction of Artemisia afra has been shown in the past. No data on the efficacy of the tea infusion prepared from Artemisia afra are however available. Objective To investigate the antiplasmodial activity of various extracts of Artemisia afra including an ethnopharmacological prepared sample. To identify polar metabolites in Artemisia afra and Artemisia annua and by using multivariate data analysis investigate the metabolic differences between these species. Materials and methods The antiplasmodial activity of Artemisia afra and Artemisia annua extracts were tested for activity against Plasmodiam falciparum 3D7 (chloroquine-sensitive strain) with chloroquine, quinine and artemisinin as positive controls. Hydrophilic metabolites in Artemisia afra and Artemisia annua were identified directly from the crude extracts through 1D- and 2D-NMR spectra. The NMR spectra were also used to differentiate between the two species using principal component analysis (PCA) for quality control purposes. Results The apolar fractions of both Artemisia afra and Artemisia annua showed activity against P. falciparum while activity was only found in the tea infusion of Artemisia annua . Metabolomic studies using 1D- and 2D-NMR spectroscopy identified 24 semi-polar components in Artemisia afra including three new phenylpropanoids for this species: caffeic acid, chlorogenic acid and 3,5-dicaffeoyl quinic acid. PCA analysis conducted on the samples yielded good separation between the polar extracts of Artemisia afra and Artemisia annua . Conclusion These findings shows that there are no in vitro activity in the tea infusion of Artemisia afra and lists the identified metabolites causing the metabolic differences between Artemisia afra and Artemisia annua for quality control purposes.

Ki-67 is a chromatin-associated protein with a dynamic distribution pattern throughout the cell cycle, and is thought to be involved in chromatin organization. Lack of genomic interaction maps has hampered a detailed understanding of its roles, particularly during interphase. By pA-DamID mapping in human cell lines we found that Ki-67 associates with large genomic domains that overlap mostly with late-replicating regions. Early in interphase, when Ki-67 is present in pre-nucleolar bodies, it interacts with these domains on all chromosomes. However, later in interphase, when Ki-67 is confined to nucleoli, it shows a striking shift towards small chromosomes. Nucleolar perturbations indicate that these cell cycle dynamics correspond to nucleolar maturation during interphase, and suggest that nucleolar sequestration of Ki-67 limits its interactions with larger chromosomes. Furthermore, we demonstrate that Ki-67 does not detectably control chromatin-chromatin interactions during interphase, but it competes with the nuclear lamina for interaction with late-replicating DNA, and it controls replication timing of (peri)centromeric regions. Together, these results reveal a highly dynamic choreography of genome interactions and roles for Ki-67 in heterochromatin organization.