ATP-dependent Remodelling of Linker Histone-Containing Nucleosomal Fibres

Eukaryotic genomes are condensed into a multilevel structure called chromatin which serves to organize and package the DNA, but at the same time needs to be flexible to permit regulated access to the stored information. ATP-dependent chromatin remodelling factors largely contribute to this dynamic nature of chromatin by catalysing processes such as the disruption of histone-DNA contacts, nucleosome repositioning and histone exchange. ATP-dependent remodelling has been well documented on a mononucleosomal level, but little is known about its regulation in a more physiological chromatin environment, where neighbouring nucleosomes and linker histones might interfere with the remodelling reaction. If and to what extent remodelling can work on chromatin bound by linker histones remains controversial, in spite of their high abundance and their strong influence on chromatin folding. We therefore investigated chromatin remodelling in the presence of linker histones H1 or H5 using regularly spaced, oligonucleosomal substrates reconstituted from purified components. Surprisingly, we found that both the remodelling complex ACF – consisting of the ATPase ISWI and the regulatory subunit ACF1 – and ISWI alone were able to catalyse the repositioning of entire chromatosomes (nucleosomes + H1). Linker histones inhibited their remodelling activity by only about 50%. In contrast, the related ATPase CHD1 remodelled chromatin only in the absence of linker histones, suggesting that linker histones allow remodelling by selected factors only. In addition, our data indicate that repositioning in the presence of H1 might be unidirectional. ACF1 is abundant during early Drosophila development, when H1 gradually replaces its early placeholder HMG-D. HMG-D binds to chromatin less tightly than H1 and unlike the latter, did not affect the remodelling activity of ACF in our assay. H1 was able to displace HMG-D from and bind to our reconstituted arrays without the help of cofactors. Strikingly, both H1 and HMG-D are more abundant in embryonic nuclei of acf1 null flies compared to the wild-type, raising the possibility that an ACF1-containing complex controls linker histone incorporation

High-resolution Xist binding maps reveal 2-step spreading during X-inactivation

The Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation (XCI), the process by which mammals compensate for unequal numbers of sex chromosomes1-3. During XCI, Xist coats the future inactive X (Xi)4 and recruits Polycomb Repressive Complex 2 (PRC2) to the X-inactivation center (Xic)5. How Xist spreads silencing on a 150 Mb scale is unclear. Here we generate high-resolution maps of Xist binding on the X chromosome across a developmental time course using CHART-seq. In female cells undergoing XCI de novo, Xist follows a two-step mechanism, initially targeting gene-rich islands before spreading to intervening gene-poor domains. Xist is depleted from genes that escape XCI but may concentrate near escapee boundaries. Xist binding is linearly proportional to PRC2 density and H3 lysine 27 trimethylation (H3K27me3), suggesting co-migration of Xist and PRC2. Interestingly, when the Xi is acutely stripped off Xist in post-XCI cells, Xist recovers quickly within both gene-rich and -poor domains on a time-scale of hours instead of days, suggesting a previously primed Xi chromatin state. We conclude that Xist spreading takes distinct stage-specific forms: During initial establishment, Xist follows a two-step mechanism, but during maintenance, Xist spreads rapidly to both gene-rich and -poor regions

Multiplexed Exchange-PAINT imaging reveals ligand-dependent EGFR and Met interactions in the plasma membrane

Signal transduction by receptor tyrosine kinases (RTKs) involves complex ligand- and time-dependent changes in conformation and modification state. High resolution structures are available for individual receptors dimers, but less is known about receptor clusters that form in plasma membranes composed of many different RTKs with the potential to interact. We report the use of multiplexed super-resolution imaging (Exchange-PAINT) followed by mean-shift clustering and random forest analysis to measure the precise distributions of five receptor tyrosine kinases (RTKs) from the ErbB, IGF-1R and Met families in breast cancer cells. We find that these receptors are intermixed nonhomogenously on the plasma membrane. Stimulation by EGF does not appear to induce a change in the density of EGFR in local clusters but instead results in formation of EGFR-Met and EGFR-ErbB3 associations; non-canonical EGFR-Met interactions are implicated in resistance to anti-cancer drugs but have not been previously detected in drug-naïve cells

The CMS Phase-1 pixel detector upgrade

The CMS detector at the CERN LHC features a silicon pixel detector as its innermost subdetector. The original CMS pixel detector has been replaced with an upgraded pixel system (CMS Phase-1 pixel detector) in the extended year-end technical stop of the LHC in 2016/2017. The upgraded CMS pixel detector is designed to cope with the higher instantaneous luminosities that have been achieved by the LHC after the upgrades to the accelerator during the first long shutdown in 2013–2014. Compared to the original pixel detector, the upgraded detector has a better tracking performance and lower mass with four barrel layers and three endcap disks on each side to provide hit coverage up to an absolute value of pseudorapidity of 2.5. This paper describes the design and construction of the CMS Phase-1 pixel detector as well as its performance from commissioning to early operation in collision data-taking.Peer reviewe

Calcite-accumulating large sulfur bacteria of the genus Achromatium in Sippewissett Salt Marsh

Large sulfur bacteria of the genus Achromatium are exceptional among Bacteria and Archaea as they can accumulate high amounts of internal calcite. Although known for more than 100 years, they remain uncultured, and only freshwater populations have been studied so far. Here we investigate a marine population of calcite-accumulating bacteria that is primarily found at the sediment surface of tide pools in a salt marsh, where high sulfide concentrations meet oversaturated oxygen concentrations during the day. Dynamic sulfur cycling by phototrophic sulfide-oxidizing and heterotrophic sulfate-reducing bacteria co-occurring in these sediments creates a highly sulfidic environment that we propose induces behavioral differences in the Achromatium population compared with reported migration patterns in a low-sulfide environment. Fluctuating intracellular calcium/sulfur ratios at different depths and times of day indicate a biochemical reaction of the salt marsh Achromatium to diurnal changes in sedimentary redox conditions. We correlate this calcite dynamic with new evidence regarding its formation/mobilization and suggest general implications as well as a possible biological function of calcite accumulation in large bacteria in the sediment environment that is governed by gradients. Finally, we propose a new taxonomic classification of the salt marsh Achromatium based on their adaptation to a significantly different habitat than their freshwater relatives, as indicated by their differential behavior as well as phylogenetic distance on 16S ribosomal RNA gene level. In future studies, whole-genome characterization and additional ecophysiological factors could further support the distinctive position of salt marsh Achromatium

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

The Drosophila MSL complex activates the transcription of target genes

The mechanism through which gene expression originating from the single male or the two female X chromosomes in Drosophila is adjusted to autosomal gene expression has remained controversial. According to the prevalent model, transcription of the male X is increased twofold by the male-specific-lethal (MSL) complex. However, a significant body of data supports an alternative model, whereby compensation involves a global repression of autosomal gene expression in males by sequestration and neutralization of an activator onto the X chromosome. In order to rigorously discriminate between these models we identified direct target genes for the MSL complex and quantified transcription in absolute terms after knockdown of MSL2. The results unequivocally document an approximate twofold activation of target genes by the MSL complex

Impact of geography and surgical approach on recurrence in global pilonidal sinus disease.

Pilonidal sinus disease (PSD) is increasing globally. A recent meta-analysis and merged-data analysis showed that recurrence rates in PSD depend essentially on follow-up time and specific surgical procedures. However, the global distribution of surgical approaches and respective recurrence rates have never been studied in PSD. We aimed at studying the impact of geographic distribution of surgical approaches to treat PSD and subsequent geography-specific recurrence rates. We searched relevant databases as described previously. Recurrence rates were then associated with reported follow-up times and geographic origin. We simulated individual patients to enable analogy across data. Globally, recurrence rates range from 0.3% for Limberg/Dufourmentel approaches (95% CI 0.2-0.4) and flaps (95% CI 0.1-0.5) and up to 6.3% for incision (95% CI 3.2-9.3) at 12 months. Recurrence rates range from 0.3% for Karydakis/Bascom approaches (95% CI 0.0-0.8) up to 67.2% for incision (95% CI 7.5-100) in the USA, and 0.0% for primary asymmetric closure in Germany (95% CI 0.0-0.0). Our analysis shows that recurrence rates in PSD not only depend on therapeutic approaches and follow-up time but also on geography. Primary asymmetric closure and various flap techniques remain superior regardless of the geographical region. Some approaches have extraordinarily good outcomes in specific countries