A SILAC-Based Screen for Methyl-CpG Binding Proteins Identifies RBP-J as a DNA Methylation and Sequence-Specific Binding Protein

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Characterization of genome-wide p53-binding sites upon stress response

The tumor suppressor p53 is a sequence-specific transcription factor, which regulates the expression of target genes involved in different stress responses. To understand p53's essential transcriptional functions, unbiased analysis of its DNA-binding repertoire is pivotal. In a genome-wide tiling ChIP-on-chip approach, we have identified and characterized 1546 binding sites of p53 upon Actinomycin D treatment. Among those binding sites were known as well as novel p53 target sites, which included regulatory regions of potentially novel transcripts. Using this collection of genome-wide binding sites, a new high-confidence algorithm was developed, p53scan, to identify the p53 consensus-binding motif. Strikingly, this motif was present in the majority of all bound sequences with 83% of all binding sites containing the motif. In the surrounding sequences of the binding sites, several motifs for potential regulatory cobinders were identified. Finally, we show that the majority of the genome-wide p53 target sites can also be bound by overexpressed p63 and p73 in vivo, suggesting that they can possibly play an important role at p53 binding sites. This emphasizes the possible interplay of p53 and its family members in the context of target gene binding. Our study greatly expands the known, experimentally validated p53 binding site repertoire and serves as a valuable knowledgebase for future research

Variation in Structure and Process of Care in Traumatic Brain Injury: Provider Profiles of European Neurotrauma Centers Participating in the CENTER-TBI Study.

INTRODUCTION: The strength of evidence underpinning care and treatment recommendations in traumatic brain injury (TBI) is low. Comparative effectiveness research (CER) has been proposed as a framework to provide evidence for optimal care for TBI patients. The first step in CER is to map the existing variation. The aim of current study is to quantify variation in general structural and process characteristics among centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. METHODS: We designed a set of 11 provider profiling questionnaires with 321 questions about various aspects of TBI care, chosen based on literature and expert opinion. After pilot testing, questionnaires were disseminated to 71 centers from 20 countries participating in the CENTER-TBI study. Reliability of questionnaires was estimated by calculating a concordance rate among 5% duplicate questions. RESULTS: All 71 centers completed the questionnaires. Median concordance rate among duplicate questions was 0.85. The majority of centers were academic hospitals (n = 65, 92%), designated as a level I trauma center (n = 48, 68%) and situated in an urban location (n = 70, 99%). The availability of facilities for neuro-trauma care varied across centers; e.g. 40 (57%) had a dedicated neuro-intensive care unit (ICU), 36 (51%) had an in-hospital rehabilitation unit and the organization of the ICU was closed in 64% (n = 45) of the centers. In addition, we found wide variation in processes of care, such as the ICU admission policy and intracranial pressure monitoring policy among centers. CONCLUSION: Even among high-volume, specialized neurotrauma centers there is substantial variation in structures and processes of TBI care. This variation provides an opportunity to study effectiveness of specific aspects of TBI care and to identify best practices with CER approaches

Variation in general supportive and preventive intensive care management of traumatic brain injury: a survey in 66 neurotrauma centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study

Abstract Background General supportive and preventive measures in the intensive care management of traumatic brain injury (TBI) aim to prevent or limit secondary brain injury and optimize recovery. The aim of this survey was to assess and quantify variation in perceptions on intensive care unit (ICU) management of patients with TBI in European neurotrauma centers. Methods We performed a survey as part of the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. We analyzed 23 questions focused on: 1) circulatory and respiratory management; 2) fever control; 3) use of corticosteroids; 4) nutrition and glucose management; and 5) seizure prophylaxis and treatment. Results The survey was completed predominantly by intensivists (n = 33, 50%) and neurosurgeons (n = 23, 35%) from 66 centers (97% response rate). The most common cerebral perfusion pressure (CPP) target was > 60 mmHg (n = 39, 60%) and/or an individualized target (n = 25, 38%). To support CPP, crystalloid fluid loading (n = 60, 91%) was generally preferred over albumin (n = 15, 23%), and vasopressors (n = 63, 96%) over inotropes (n = 29, 44%). The most commonly reported target of partial pressure of carbon dioxide in arterial blood (PaCO2) was 36–40 mmHg (4.8–5.3 kPa) in case of controlled intracranial pressure (ICP) < 20 mmHg (n = 45, 69%) and PaCO2 target of 30–35 mmHg (4–4.7 kPa) in case of raised ICP (n = 40, 62%). Almost all respondents indicated to generally treat fever (n = 65, 98%) with paracetamol (n = 61, 92%) and/or external cooling (n = 49, 74%). Conventional glucose management (n = 43, 66%) was preferred over tight glycemic control (n = 18, 28%). More than half of the respondents indicated to aim for full caloric replacement within 7 days (n = 43, 66%) using enteral nutrition (n = 60, 92%). Indications for and duration of seizure prophylaxis varied, and levetiracetam was mostly reported as the agent of choice for both seizure prophylaxis (n = 32, 49%) and treatment (n = 40, 61%). Conclusions Practice preferences vary substantially regarding general supportive and preventive measures in TBI patients at ICUs of European neurotrauma centers. These results provide an opportunity for future comparative effectiveness research, since a more evidence-based uniformity in good practices in general ICU management could have a major impact on TBI outcome

Generation of a tagged MBD2.

<p>a) Schematic presentation of tagging approach: double Ty1 and ER epitopes are inserted at the N-terminal of human full length MBD2. b) Western blot on whole cell lysates from TTE-MBD2 MCF-7 and WT MCF-7. Antibodies against tag (Ty1) and MBD2 are used. GAPDH is shown as loading control. c) Volcano plot showing results from Mass Spectrometric Analysis of immunoprecipitation experiment. The x-axis shows the log of ratios between LFQ intensities in TTE-MBD2 against the control WT. The y-axis display −log10 of the p-value calculated by a permutation-based FDR-corrected <i>t</i> test. The black dots underline Mi2-NuRD complex components within the significantly enriched interactors (grey dots).</p

Methylation state at MBD2 binding sites.

<p>a) Boxplot displaying methylation level at TTE-MBD2 binding sites compared to random (0 = 0% methylation, 1 = 100% methylation). b) Genome wide correlation between TTE-MBD2 enrichment (green) and methylation density, calculated at 1 kb windows ranked by methylation density (dashed line). c) Screenshots from genome browser showing correlation between CpG methylation density (red track) and TTE-MBD2 peaks at KCNN2, ZNF316, and ASCL5.</p

MBD2 and Pol2 distribution at active promoters from cluster 4.

<p>a) Average profile of Pol2 (MCF-7 WT) and MBD2 (TTE-MBD2) at promoters from cluster 4, calculated 5 kb up- and downstream the TSS. Average profile of CpG density, MBD2 and methylation levels at promoters from cluster 4, calculated 5 kb up- and downstream the TSS. c) Boxplots showing RPKM values for all Ref-seq annotated expressed genes sorted and divided in 3 categories according to their transcript level, compared to RPKM values for genes annotated from cluster 4. d) Average profile of N-term Pol2 at promoters from cluster 4, calculated 5 kb up- and downstream the TSS for TTE-MBD2 and WT MCF-7. e) As for 5D average profiles of Ser5 phosphorylated Pol2 at promoters from cluster 4, calculated 5 kb up- and downstream the TSS for TTE-MBD2 and WT MCF-7. f) Average profiles of Ser2 phosphorylated Pol2 over gene bodies downstream promoters from cluster 4, calculated 2 kb up- and downstream the TSS and the TES, for TTE-MBD2 and WT MCF-7.</p

List of TTE-MBD2 specific interactors identified from Mass Spectrometry.

<p>25 most significantly enriched TTE-MBD2 interactors, identified after Ty1 immunoprecipitation on both TTE-MBD2 and WT cells followed by mass spectrometry analysis. Results from triplicate pull-downs were analyzed with MaxQuant and label-free quantitation (LFQ) intensities were used to determine statistically enriched MBD2 interactors. Immunoprecipitation from wild type (WT) MCF-7 was used as a control.</p

Genome-wide binding of TTE-MBD2.

<p>a) Heatmap displaying tag densities in TTE-MBD2 (left and right) and input (middle) at MBD2 binding sites around 5 kb up- and downstream of the center of the peaks. b) Genomic location of peaks: each category is expressed as fold over random (y-axis), the random set consists of an equal number of sites having on average same length of the peaks. c) CpG content of each category expressed as percentage of the total binding sites (y-axis). d) Screenshots from the genome browser showing example of CGI promoters (KDM2A) and exons (MZF1, ZNF837, ZNF497) binding.</p