Xenopus HJURP and condensin II are required for CENP-A assembly

Chromatin structure imposed by condensin II at centromeres enables xHJURP-mediated incorporation of CENP-A

Analysis of Protein Turnover by Quantitative SNAP-Based Pulse-Chase Imaging

Assessment of protein dynamics in living cells is crucial for understanding their biological properties and functions. The SNAP-tag, a self labeling suicide enzyme, presents a tool with unique features that can be adopted for determining protein dynamics in living cells. Here we present detailed protocols for the use of SNAP in fluorescent pulse-chase and quench-chase-pulse experiments. These time-slicing methods provide powerful tools to assay and quantify the fate and turnover rate of proteins of different ages. We cover advantages and pitfalls of SNAP-tagging in fixed- and live-cell studies and evaluate the recently developed fast-acting SNAPf variant. In addition, to facilitate the analysis of protein turnover datasets, we present an automated algorithm for spot recognition and quantification.FCT doctoral fellowships: (SFRH/BD/74284/2010, SFRH/BD/33567/2008); FCT grants: (BIA-BCM/100557/2008, BIA-PRO/100537/2008); Fundação Calouste Gulbenkian; European Commission FP7 programme; EMBO installation grant

BMC Cancer

Background: The exhaustive collection of new sarcoma cases and their second histologic review offer a unique opportunity to study their incidence and time trends in France according to the major subtypes. Methods: Data were collected from population-based cancer registries covering 22% of the French population. Crude and world age-standardized incidence rates (ASR) were estimated according to anatomic, histological and genetic groups, age and sex over the 2010–2013 period. Results: Time trends in incidence were calculated by the annual percent change over the 2000–2013 period. During the most recent period (2010–2013), 3942 patients with sarcoma were included. The ASR of soft-tissue and bone sarcomas, and gastro-intestinal stromal tumors (GIST) were 2.1, 1.0 and 0.6, respectively. For the four most frequent histological subtypes (unclassified, leiomyosarcoma, GIST and liposarcoma), the ASR ranged from 0.4 to 0.7. ASRs were 1.9 for complex genomic and 1.3 for recurrent translocation sarcomas. The time-trend analysis showed a significant increase of sarcoma incidence rate between 2000 and 2005, which stabilized thereafter. Incidence rates increased for four histological subtypes (GIST, chondrosarcoma, myxofibrosarcoma, solitary fibrous tumors) and decreased for three (leiomyosarcomas, Kaposi sarcoma and fibrosarcoma). Conclusion: To our knowledge, this study is the first to investigate sarcoma incidence based on a systematic pathological review of these cancers and on the updated sarcoma classifications. Due to the paucity of literature on sarcomas, future studies using data from population-based cancer registries should consider a standardized inclusion criterion presented in our study to better describe and compare data between countries

Transdimensional inference of archeomagnetic intensity change

One of the main goals of archeomagnetism is to document the secular changes of Earth's magnetic field by laboratory analysis of the magnetization carried by archeological artefacts. Typical techniques for creating a time-dependent model assume a prescribed temporal discretisation which, when coupled with sparse data coverage, require strong regularisation generally applied over the entire time series in order to ensure smoothness. Such techniques make it difficult to characterise uncertainty and frequency content, and robustly detect rapid changes. Key to proper modelling (and physical understanding) is a method that places a minimum level of regularisation on any fit to the data. Here we apply a transdimensional Bayesian technique based on piecewise linear interpolation to sparse archeointensity datasets, in which the temporal complexity of the model is not set a priori, but is self-selected by the data. The method produces two key outputs: (i) a posterior distribution of intensity as a function of time, a useful tool for archeomagnetic dating, whose statistics are smooth but formally unregularised; (ii) by including the data ages in the model of unknown parameters, the method also produces posterior age statistics of each individual contributing datum. We test the technique using synthetic datasets and confirm agreement of our method with an integrated likelihood approach. We then apply the method to three archeomagnetic datasets all reduced to a single location: one temporally well-sampled within 700km from Paris (here referred to as Paris700), one that is temporally sparse centred on Hawaii, and a third (from Lübeck, Germany and Paris700) that has additional ordering constraints on age from stratification. Compared with other methods, our average posterior distributions largely agree, however our credible intervals appear to much better reflect the uncertainty during periods of sparse data coverage. Because each ensemble member of the posterior distribution is piecewise linear, we only fit oscillations when required by the data. As an example, we show that an oscillatory signal, associated with temporally-localised intensity maxima reported for a sparse Hawaiian dataset, is not required by the data. However, we do recover the previously reported oscillation of period 260 yrs for the Paris700 dataset and compute the probability distribution of the period of oscillation. We further demonstrate that such an oscillation is unresolved when accounting for age uncertainty by using a fixed age and with an artificially inflated error budget on intensity

Enhancer regions show high histone H3.3 turnover that changes during differentiation

The organization of DNA into chromatin is dynamic; nucleosomes are frequently displaced to facilitate the ability of regulatory proteins to access specific DNA elements. To gain insight into nucleosome dynamics, and to follow how dynamics change during differentiation, we used a technique called time-ChIP to quantitatively assess histone H3.3 turnover genome-wide during differentiation of mouse ESCs. We found that, without prior assumptions, high turnover could be used to identify regions involved in gene regulation. High turnover was seen at enhancers, as observed previously, with particularly high turnover at super-enhancers. In contrast, regions associated with the repressive Polycomb-Group showed low turnover in ESCs. Turnover correlated with DNA accessibility. Upon differentiation, numerous changes in H3.3 turnover rates were observed, the majority of which occurred at enhancers. Thus, time-ChIP measurement of histone turnover shows that active enhancers are unusually dynamic in ESCs and changes in highly dynamic nucleosomes predominate at enhancers during differentiation. DOI: http://dx.doi.org/10.7554/eLife.15316.00

Genetic Variation Stimulated by Epigenetic Modification

Homologous recombination is essential for maintaining genomic integrity. A common repair mechanism, it uses a homologous or homeologous donor as a template for repair of a damaged target gene. Such repair must be regulated, both to identify appropriate donors for repair, and to avoid excess or inappropriate recombination. We show that modifications of donor chromatin structure can promote homology-directed repair. These experiments demonstrate that either the activator VP16 or the histone chaperone, HIRA, accelerated gene conversion approximately 10-fold when tethered within the donor array for Ig gene conversion in the chicken B cell line DT40. VP16 greatly increased levels of acetylated histones H3 and H4, while tethered HIRA did not affect histone acetylation, but caused an increase in local nucleosome density and levels of histone H3.3. Thus, epigenetic modification can stimulate genetic variation. The evidence that distinct activating modifications can promote similar functional outcomes suggests that a variety of chromatin changes may regulate homologous recombination, and that disregulation of epigenetic marks may have deleterious genetic consequences

The Oncogenic EWS-FLI1 Protein Binds In Vivo GGAA Microsatellite Sequences with Potential Transcriptional Activation Function

The fusion between EWS and ETS family members is a key oncogenic event in Ewing tumors and important EWS-FLI1 target genes have been identified. However, until now, the search for EWS-FLI1 targets has been limited to promoter regions and no genome-wide comprehensive analysis of in vivo EWS-FLI1 binding sites has been undertaken. Using a ChIP-Seq approach to investigate EWS-FLI1-bound DNA sequences in two Ewing cell lines, we show that this chimeric transcription factor preferentially binds two types of sequences including consensus ETS motifs and microsatellite sequences. Most bound sites are found outside promoter regions. Microsatellites containing more than 9 GGAA repeats are very significantly enriched in EWS-FLI1 immunoprecipitates. Moreover, in reporter gene experiments, the transcription activation is highly dependent upon the number of repeats that are included in the construct. Importantly, in vivo EWS-FLI1-bound microsatellites are significantly associated with EWS-FLI1-driven gene activation. Put together, these results point out the likely contribution of microsatellite elements to long-distance transcription regulation and to oncogenesis

Recent Progress in the Use of Glucagon and Glucagon Receptor Antagonists in the Treatment of Diabetes Mellitus

Glucagon is an important pancreatic hormone, released into blood circulation by alpha cells of the islet of Langerhans. Glucagon induces gluconeogenesis and glycogenolysis in hepatocytes, leading to an increase in hepatic glucose production and subsequently hyperglycemia in susceptible individuals. Hyperglucagonemia is a constant feature in patients with T2DM. A number of bioactive agents that can block glucagon receptor have been identified. These glucagon receptor antagonists can reduce the hyperglycemia associated with exogenous glucagon administration in normal as well as diabetic subjects. Glucagon receptor antagonists include isoserine and beta-alanine derivatives, bicyclic 19-residue peptide BI-32169, Des-His1-[Glu9] glucagon amide and related compounds, 5-hydroxyalkyl-4-phenylpyridines, N-[3-cano-6- (1,1 dimethylpropyl)-4,5,6,7-tetrahydro-1-benzothien-2-yl]-2-ethylbutamide, Skyrin and NNC 250926. The absorption, dosage, catabolism, excretion and medicinal chemistry of these agents are the subject of this review. It emphasizes the role of glucagon in glucose homeostasis and how it could be applied as a novel tool for the management of diabetes mellitus by blocking its receptors with either monoclonal antibodies, peptide and non-peptide antagonists or gene knockout techniques

Spt6 is a maintenance factor for centromeric CENP-A

Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. Centromeric transcription is required for stable incorporation of the centromere-specific histone dCENP-A in M/G1 phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes. Here we demonstrate that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and dCENP-A mutants carrying phosphomimetic residues alleviate this association. Retention of phosphomimetic dCENP-A mutants is reduced relative to wildtype, while non-phosphorylatable dCENP-A retention is increased and accumulates at the centromere. We conclude that Spt6 acts as a conserved CENP-A maintenance factor that ensures long-term stability of epigenetic centromere identity during transcription-mediated chromatin remodeling