Targeting determinants of dosage compensation in Drosophila

The dosage compensation complex (DCC) in Drosophila melanogaster is responsible for up-regulating transcription from the single male X chromosome to equal the transcription from the two X chromosomes in females. Visualization of the DCC, a large ribonucleoprotein complex, on male larval polytene chromosomes reveals that the complex binds selectively to many interbands on the X chromosome. The targeting of the DCC is thought to be in part determined by DNA sequences that are enriched on the X. So far, lack of knowledge about DCC binding sites has prevented the identification of sequence determinants. Only three binding sites have been identified to date, but analysis of their DNA sequence did not allow the prediction of further binding sites. We have used chromatin immunoprecipitation to identify a number of new DCC binding fragments and characterized them in vivo by visualizing DCC binding to autosomal insertions of these fragments, and we have demonstrated that they possess a wide range of potential to recruit the DCC. By varying the in vivo concentration of the DCC, we provide evidence that this range of recruitment potential is due to differences in affinity of the complex to these sites. We were also able to establish that DCC binding to ectopic high-affinity sites can allow nearby low-affinity sites to recruit the complex. Using the sequences of the newly identified and previously characterized binding fragments, we have uncovered a number of short sequence motifs, which in combination may contribute to DCC recruitment. Our findings suggest that the DCC is recruited to the X via a number of binding sites of decreasing affinities, and that the presence of high-and moderate-affinity sites on the X may ensure that lower-affinity sites are occupied in a context-dependent manner. Our bioinformatics analysis suggests that DCC binding sites may be composed of variable combinations of degenerate motifs

Gene Expression in Embryos From Norwegian Red Bulls With High or Low Non Return Rate: An RNA-Seq Study of in vivo-Produced Single Embryos

publishedVersio

Mapping the expression of an ANK3 isoform associated with bipolar disorder in the human brain

The gene ankyrin-3 (ANK3) has been consistently associated with bipolar disorder (BD) in several genome-wide association studies (GWAS). The exact molecular mechanisms underlying this genetic association remain unknown. The discovery of a loss-of-function variant (rs41283526*G) in an alternatively spliced exon (ENSE00001786716) with a protective effect, suggested that elevated expression of this particular isoform could be a risk factor for developing the disorder. We developed a novel approach for measuring the expression level of all splice forms at a challenging genetic locus using a combination of droplet digital PCR and high-throughput sequencing of indexed PCR amplicons. The combined method was performed on a large collection of 568 postmortem brain samples of BD and SCZ cases and controls. We also studied the expression of the splice forms in a child-development cohort of 41 healthy males. We found that our approach can quantify the splice forms in brain samples, although with less precision than ddPCR. We detected highly significant differences in expression of splice forms and transcription start sites between brain regions, notably with higher expression of the BD-associated isoform in the corpus callosum compared to frontal tissue (mean fold change = 1.80, p < 1e-4). Although the patients in our sample expressed the BD-associated splice form at a similar level to controls, adolescents in our child-development cohort had a clearly higher expression level than younger children (mean fold change = 1.97, p = 5e-3). These results suggest that this ANK3 splice form may play a role in the myelin maturation of the human brain.publishedVersio

The microbiota of uterine biopsies, cytobrush and vaginal swabs at artificial insemination in Norwegian red cows

acceptedVersio

Cumulative contributions of weak DNA determinants to targeting the Drosophila dosage compensation complex

Fine-tuning of X chromosomal gene expression in Drosophila melanogaster involves the selective interaction of the Dosage Compensation Complex (DCC) with the male X chromosome, in order to increase the transcription of many genes. However, the X chromosomal DNA sequences determining DCC binding remain elusive. By adapting a ‘one-hybrid’ assay, we identified minimal DNA elements that direct the interaction of the key DCC subunit, MSL2, in cells. Strikingly, several such novel MSL2 recruitment modules have very different DNA sequences. The assay revealed a novel, 40 bp DNA element that is necessary for recruitment of DCC to an autosomal binding site in flies in the context of a longer sequence and sufficient by itself to direct recruitment if trimerized. Accordingly, recruitment of MSL2 to the single 40 bp element in cells was weak, but as a trimer approached the power of the strongest DCC recruitment site known to date, the roX1 DH site. This element is the shortest MSL2 recruitment sequence known to date. The results support a model for MSL2 recruitment according to which several different, degenerate sequence motifs of variable affinity cluster and synergise to form a high affinity site

Transcriptomics of the vaccine immune response: Priming with adjuvant modulates recall innate responses after boosting

Transcriptomic profiling of the immune response induced by vaccine adjuvants is of critical importance for the rational design of vaccination strategies. In this study, transcriptomics was employed to profile the effect of the vaccine adjuvant used for priming on the immune response following re-exposure to the vaccine antigen alone. Mice were primed with the chimeric vaccine antigen H56 of Mycobacterium tuberculosis administered alone or with the CAF01 adjuvant and boosted with the antigen alone. mRNA sequencing was performed on blood samples collected 1, 2, and 7 days after priming and after boosting. Gene expression analysis at day 2 after priming showed that the CAF01 adjuvanted vaccine induced a stronger upregulation of the innate immunity modules compared with the unadjuvanted formulation. The immunostimulant effect of the CAF01 adjuvant, used in the primary immunization, was clearly seen after a booster immunization with a low dose of antigen alone. One day after boost, we observed a strong upregulation of multiple genes in blood of mice primed with H56 + CAF01 compared with mice primed with the H56 alone. In particular, blood transcription modules related to innate immune response, such as monocyte and neutrophil recruitment, activation of antigen-presenting cells, and interferon response were activated. Seven days after boost, differential expression of innate response genes faded while a moderate differential expression of T cell activation modules was appreciable. Indeed, immunological analysis showed a higher frequency of H56-specific CD4+ T cells and germinal center B cells in draining lymph nodes, a strong H56-specific humoral response and a higher frequency of antibody-secreting cells in spleen of mice primed with H56 + CAF01. Taken together, these data indicate that the adjuvant used for priming strongly reprograms the immune response that, upon boosting, results in a stronger recall innate response essential for shaping the downstream adaptive response

The Chromosomal High-Affinity Binding Sites for the Drosophila Dosage Compensation Complex

Dosage compensation in male Drosophila relies on the X chromosome–specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC). The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called “high-affinity sites” (HAS). Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features

Monocytes accumulate in the airways of children with fatal asthma

Background: Activated T helper type 2 (Th2) cells are believed to play a pivotal role in allergic airway inflammation, but which cells attract and activate Th2 cells locally have not been fully determined. Recently, it was shown in an experimental human model of allergic rhinitis (AR) that activated monocytes rapidly accumulate in the nasal mucosa after local allergen challenge, where they promote recruitment of Th2 cells and eosinophils. Objective: To investigate whether monocytes are recruited to the lungs in paediatric asthma. Methods: Tissue samples obtained from children and adolescents with fatal asthma attack (n = 12), age-matched non-atopic controls (n = 9) and allergen-challenged AR patients (n = 8) were subjected to in situ immunostaining. Results: Monocytes, identified as CD68+S100A8/A9+ cells, were significantly increased in the lower airway mucosa and in the alveoli of fatal asthma patients compared with control individuals. Interestingly, cellular aggregates containing CD68+S100A8/A9+ monocytes obstructing the lumen of bronchioles were found in asthmatics (8 out of 12) but not in controls. Analysing tissue specimens from challenged AR patients, we confirmed that co-staining with CD68 and S100A8/A9 was a valid method to identify recently recruited monocytes. We also showed that the vast majority of accumulating monocytes both in the lungs and in the nasal mucosa expressed matrix metalloproteinase 10, suggesting that this protein may be involved in their migration within the tissue. Conclusions and clinical relevance: Monocytes accumulated in the lungs of children and adolescents with fatal asthma attack. This finding strongly suggests that monocytes are directly involved in the immunopathology of asthma and that these pro-inflammatory cells are potential targets for therapy.Peer reviewe

Global carbon budget 2019

Accurate assessment of anthropogenic carbon dioxide (CO2) emissions and their redistribution among the atmosphere, ocean, and terrestrial biosphere – the “global carbon budget” – is important to better understand the global carbon cycle, support the development of climate policies, and project future climate change. Here we describe data sets and methodology to quantify the five major components of the global carbon budget and their uncertainties. Fossil CO2 emissions (EFF) are based on energy statistics and cement production data, while emissions from land use change (ELUC), mainly deforestation, are based on land use and land use change data and bookkeeping models. Atmospheric CO2 concentration is measured directly and its growth rate (GATM) is computed from the annual changes in concentration. The ocean CO2 sink (SOCEAN) and terrestrial CO2 sink (SLAND) are estimated with global process models constrained by observations. The resulting carbon budget imbalance (BIM), the difference between the estimated total emissions and the estimated changes in the atmosphere, ocean, and terrestrial biosphere, is a measure of imperfect data and understanding of the contemporary carbon cycle. All uncertainties are reported as ±1σ. For the last decade available (2009–2018), EFF was 9.5±0.5 GtC yr−1, ELUC 1.5±0.7 GtC yr−1, GATM 4.9±0.02 GtC yr−1 (2.3±0.01 ppm yr−1), SOCEAN 2.5±0.6 GtC yr−1, and SLAND 3.2±0.6 GtC yr−1, with a budget imbalance BIM of 0.4 GtC yr−1 indicating overestimated emissions and/or underestimated sinks. For the year 2018 alone, the growth in EFF was about 2.1 % and fossil emissions increased to 10.0±0.5 GtC yr−1, reaching 10 GtC yr−1 for the first time in history, ELUC was 1.5±0.7 GtC yr−1, for total anthropogenic CO2 emissions of 11.5±0.9 GtC yr−1 (42.5±3.3 GtCO2). Also for 2018, GATM was 5.1±0.2 GtC yr−1 (2.4±0.1 ppm yr−1), SOCEAN was 2.6±0.6 GtC yr−1, and SLAND was 3.5±0.7 GtC yr−1, with a BIM of 0.3 GtC. The global atmospheric CO2 concentration reached 407.38±0.1 ppm averaged over 2018. For 2019, preliminary data for the first 6–10 months indicate a reduced growth in EFF of +0.6 % (range of −0.2 % to 1.5 %) based on national emissions projections for China, the USA, the EU, and India and projections of gross domestic product corrected for recent changes in the carbon intensity of the economy for the rest of the world. Overall, the mean and trend in the five components of the global carbon budget are consistently estimated over the period 1959–2018, but discrepancies of up to 1 GtC yr−1 persist for the representation of semi-decadal variability in CO2 fluxes. A detailed comparison among individual estimates and the introduction of a broad range of observations shows (1) no consensus in the mean and trend in land use change emissions over the last decade, (2) a persistent low agreement between the different methods on the magnitude of the land CO2 flux in the northern extra-tropics, and (3) an apparent underestimation of the CO2 variability by ocean models outside the tropics. This living data update documents changes in the methods and data sets used in this new global carbon budget and the progress in understanding of the global carbon cycle compared with previous publications of this data set (Le Quéré et al., 2018a, b, 2016, 2015a, b, 2014, 2013). The data generated by this work are available at https://doi.org/10.18160/gcp-2019 (Friedlingstein et al., 2019)