Visualisation tool for peptide fractionation data in proteomics: application to OFFGEL isoelectric focussing

<p>Abstract</p> <p>Background</p> <p>OFFGEL isoelectric focussing (IEF) has become a popular tool in proteomics to fractionate peptides or proteins. As a consequence there is a need for software solutions supporting data mining, interpretation and characterisation of experimental quality.</p> <p>Results</p> <p>We can assess performance characteristics of OFFGEL IEF peptide fractionation in proteomics by generating plots of the overall fractionation patterns and the pairwise comparisons of adjacent fractions.</p> <p>Conclusions</p> <p>A visualisation tool for peptide fractionation has been developed to support the evaluation of IEF data quality and can be implemented in proteomics research.</p

Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB

Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants 1-6. In Drosophila, genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other developmental regulator genes in cells where they must stay inactive 1,7,8. Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes. Among those, Polycomb repressive complex 1 (PRC1) and the related dRing-associated factors (dRAF) complex contain an E3 ligase activity for monoubiquitination of histone H2A (refs 1-4). Here we show that the uncharacterized Drosophila PcG gene calypso encodes the ubiquitin carboxy-terminal hydrolase BAP1. Biochemically purified Calypso exists in a complex with the PcG protein ASX, and this complex, named Polycomb repressive deubiquitinase (PR-DUB), is bound at PcG target genes in Drosophila. Reconstituted recombinant Drosophila and human PR-DUB complexes remove monoubiquitin from H2A but not from H2B in nucleosomes. Drosophila mutants lacking PR-DUB show a strong increase in the levels of monoubiquitinated H2A. A mutation that disrupts the catalytic activity of Calypso, or absence of the ASX subunit abolishes H2A deubiquitination in vitro and HOX gene repression in vivo. Polycomb gene silencing may thus entail a dynamic balance between H2A ubiquitination by PRC1 and dRAF, and H2A deubiquitination by PR-DUB

Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation at Polycomb target genes

PRC2 is thought to be the histone methyltransferase (HMTase) responsible for H3-K27 trimethylation at Polycomb target genes. Here we report the biochemical purification and characterization of a distinct form of Drosophila PRC2 that contains the Polycomb group protein polycomblike (Pcl). Like PRC2, Pcl-PRC2 is an H3-K27-specific HMTase that mono-, di- and trimethylates H3-K27 in nucleosomes in vitro. Analysis of Drosophila mutants that lack Pcl unexpectedly reveals that Pcl-PRC2 is required to generate high levels of H3-K27 trimethylation at Polycomb target genes but is dispensable for the genome-wide H3-K27 mono- and dimethylation that is generated by PRC2. In Pcl mutants, Polycomb target genes become derepressed even though H3-K27 trimethylation at these genes is only reduced and not abolished, and even though targeting of the Polycomb protein complexes PhoRC and PRC1 to Polycomb response elements is not affected. Pcl-PRC2 is thus the HMTase that generates the high levels of H3-K27 trimethylation in Polycomb target genes that are needed to maintain a Polycomb-repressed chromatin state

Percentiles of fasting serum insulin, glucose, HbA1c and HOMA-IR in pre-pubertal normal weight European children from the IDEFICS cohort

OBJECTIVES: The aim of this study is to present age-and sex-specific reference values of insulin, glucose, glycosylated haemoglobin (HbA1c) and the homeostasis model assessment to quantify insulin resistance (HOMA-IR) for pre-pubertal children. METHODS: The reference population consists of 7074 normal weight 3- to 10.9-year-old pre-pubertal children from eight European countries who participated in at least one wave of the IDEFICS ('identification and prevention of dietary-and lifestyle-induced health effects in children and infants') surveys (2007-2010) and for whom standardised laboratory measurements were obtained. Percentile curves of insulin (measured by an electrochemiluminescence immunoassay), glucose, HbA1c and HOMA-IR were calculated as a function of age stratified by sex using the general additive model for location scale and shape (GAMLSS) method. RESULTS: Levels of insulin, fasting glucose and HOMA-IR continuously show an increasing trend with age, whereas HbA1c shows an upward trend only beyond the age of 8 years. Insulin and HOMA-IR values are higher in girls of all age groups, whereas glucose values are slightly higher in boys. Median serum levels of insulin range from 17.4 and 13.2 pmol l(-1) in 3-< 3.5-year-old girls and boys, respectively, to 53.5 and 43.0 pmol l(-1) in 10.5-< 11-year-old girls and boys. Median values of glucose are 4.3 and 4.5 mmol l(-1) in the youngest age group and 49.3 and 50.6 mmol l(-1) in the oldest girls and boys. For HOMA-IR, median values range from 0.5 and 0.4 in 3-< 3.5-year-old girls and boys to 1.7 and 1.4 in 10.5-< 11-year-old girls and boys, respectively. CONCLUSIONS: Our study provides the first standardised reference values for an international European children's population and provides the, up to now, largest data set of healthy pre-pubertal children to model reference percentiles for markers of insulin resistance. Our cohort shows higher values of Hb1Ac as compared with a single Swedish study while our percentiles for the other glucose metabolic markers are in good accordance with previous studies

C-reactive protein reference percentiles among pre-adolescent children in Europe based on the IDEFICS study population

OBJECTIVES: C-reactive protein (CRP) is involved in a wide range of diseases. It is a powerful marker for inflammatory processes used for diagnostic and monitoring purposes. We aimed to establish reference values as data on the distribution of serum CRP levels in young European children are scarce. SUBJECTS: Reference values of high-sensitivity CRP concentrations were calculated for 9855 children aged 2.0-10.9 years, stratified by age and sex. The children were recruited during the population-based European IDEFICS study (Identification and prevention of Dietary-and lifestyle-induced health Effects in Children and infantS) with 18 745 participants recruited from 2007 to 2010. RESULTS: In 44.1 % of the children, CRP values were below or equal the detection limit of 0.2 mg/l. Median CRP concentrations showed a slight negative age trend in boys and girls, whereas serum CRP values were slightly higher in girls than in boys across all age groups. CONCLUSIONS: Our population-based reference values of CRP may guide paediatric practice as elevated values may require further investigation or treatment. Therefore, the presented reference values represent a basis for clinical evaluation and for future research on risk assessment of diseases associated with increased CRP levels among children

Identification of acetylcholine receptor subunits differentially expressed in singly and multiply innervated fibers of extraocular muscles. Invest Ophthalmol Vis Sci 47: 3828–3834

PURPOSE. To identify the acetylcholine receptor (AChR) isoforms among the neuromuscular junctions (NMJs) of singly and multiply innervated fibers (SIFs and MIFs) of rat extraocular muscles (EOMs). METHODS. EOMs were dissected from adult rats and serially sectioned. Sections were simultaneously stained for acetylcholinesterase and with an antibody to the slow myosin heavy chain to identify NMJ topography and fiber types in the same section. Synapses and subsynaptic regions of SIFs and MIFs were isolated by laser capture microdissection and the AChR subunits identified by RT-PCR. RESULTS. The en plaque endings of SIFs expressed only the adult subunit, not the fetal ␥ subunit, of the AChR, whereas the en grappe endings of the MIFs expressed only the ␥ subunit, and not the subunit. Although the expression of the subunit was confined to the NMJ region of the SIFs, the ␥ subunit was expressed both synaptically and extrasynaptically within the MIFs. The ␥ subunit in MIFs correlated with the expression of the myogenic regulatory factor myogenin. Moreover, an unusual neuronal AChR subunit, ␣9, was found in the EOMs, but not in the limb muscles. CONCLUSIONS. The adult and fetal ␥ subunits of the AChRs are segregated into distinct synapses on distinct fiber types. The maintenance of the fetal subunit in a population of fibers is probably linked to the expression of myogenin and is a unique attribute of the EOM allotype. (Invest Ophthalmol Vis Sci

Structural basis for targeting the chromatin repressor Sfmbt to Polycomb response elements

Polycomb group (PcG) protein complexes repress developmental regulator genes by modifying their chromatin. How different PcG proteins assemble into complexes and are recruited to their target genes is poorly understood. Here, we report the crystal structure of the core of the Drosophila PcG protein complex Pleiohomeotic (Pho)-epressive complex (PhoRC), which contains the Polycomb response element (PRE)-binding protein Pho and Sfmbt. The spacer region of Pho, separated from the DNA-binding domain by a long flexible linker, forms a tight complex with the four malignant brain tumor (4MBT) domain of Sfmbt. The highly conserved spacer region of the human Pho ortholog YY1 binds three of the four human 4MBT domain proteins in an analogous manner but with lower affinity. Comparison of the Drosophila Pho: Sfmbt and human YY1: MBTD1 complex structures provides a molecular explanation for the lower affinity of YY1 for human 4MBT domain proteins. Structure-guided mutations that disrupt the interaction between Pho and Sfmbt abolish formation of a ternary Sfmbt: Pho: DNA complex in vitro and repression of developmental regulator genes in Drosophila. PRE tethering of Sfmbt by Pho is therefore essential for Polycomb repression in Drosophila. Our results support a model where DNA tethering of Sfmbt by Pho and multivalent interactions of Sfmbt with histone modifications and other PcG proteins create a hub for PcG protein complex assembly at PREs

The Nonspecific Lethal Complex is a Transcriptional Regulator in Drosophila

Here, we report the biochemical characterization of the nonspecific lethal (NSL) complex (NSL1, NSL2, NSL3, MCRS2, MBD-R2, and WDS) that associates with the histone acetyltransferase MOF in both Drosophila and mammals. Chromatin immunoprecipitation-Seq analysis revealed association of NSL1 and MCRS2 with the promoter regions of more than 4000 target genes, 70% of these being actively transcribed. This binding is functional, as depletion of MCRS2, MBD-R2, and NSL3 severely affects gene expression genome wide. The NSL complex members bind to their target promoters independently of MOF. However, depletion of MCRS2 affects MOF recruitment to promoters. NSL complex stability is interdependent and relies mainly on the presence of NSL1 and MCRS2. Tethering of NSL3 to a heterologous promoter leads to robust transcription activation and is sensitive to the levels of NSL1, MCRS2, and MOF. Taken together, we conclude that the NSL complex acts as a major transcriptional regulator in Drosophila