De novo motif identification improves the accuracy of predicting transcription factor binding sites in ChIP-Seq data analysis

Dramatic progress in the development of next-generation sequencing technologies has enabled accurate genome-wide characterization of the binding sites of DNA-associated proteins. This technique, baptized as ChIP-Seq, uses a combination of chromatin immunoprecipitation and massively parallel DNA sequencing. Other published tools that predict binding sites from ChIP-Seq data use only positional information of mapped reads. In contrast, our algorithm MICSA (Motif Identification for ChIP-Seq Analysis) combines this source of positional information with information on motif occurrences to better predict binding sites of transcription factors (TFs). We proved the greater accuracy of MICSA with respect to several other tools by running them on datasets for the TFs NRSF, GABP, STAT1 and CTCF. We also applied MICSA on a dataset for the oncogenic TF EWS-FLI1. We discovered >2000 binding sites and two functionally different binding motifs. We observed that EWS-FLI1 can activate gene transcription when (i) its binding site is located in close proximity to the gene transcription start site (up to ∼150 kb), and (ii) it contains a microsatellite sequence. Furthermore, we observed that sites without microsatellites can also induce regulation of gene expression—positively as often as negatively—and at much larger distances (up to ∼1 Mb)

Comparison of Audiovisual and Paper-Based Materials for 1-Time Informed Consent for Research in Prison: A Randomized Clinical Trial.

Importance Few studies are available on informed consent (IC) among detained persons, even with ethics being a critical aspect of prison research. In IC research, audiovisual material seems to improve understanding and satisfaction compared with conventional paper-based material, but findings remain unclear. Objective To compare audiovisual and paper-based materials for 1-time general IC for research in prisons. Design, Setting, and Participants This cross-sectional randomized clinical trial was conducted in 2 corrections facilities in Switzerland (an adult prison and a juvenile detention center). The study was conducted from December 14, 2019, to December 2, 2020, in the adult prison and from January 15, 2020, to September 9, 2021, in the juvenile detention center. In the adult prison, study participation was offered to detained persons visiting the medical unit (response rate, 84.7%). In the juvenile detention center, all newly incarcerated adolescents were invited to participate (response rate, 98.0%). Interventions Participants were randomized to receive paper-based conventional material or to watch a 4-minute video. Materials included the same legal information, as required by the Swiss Federal Act on Research Involving Human Beings. Main Outcomes and Measures The main outcome was acceptance to sign the IC form. Secondary outcomes included understanding, evaluation, and time to read or watch the IC material. Results The study included 190 adults (mean [SD] age, 35.0 [11.8] years; 190 [100%] male) and 100 adolescents (mean [SD] age, 16.0 [1.1] years; 83 [83.0%] male). In the adult prison, no significant differences were found between groups in acceptance to sign the IC form (77 [81.1%] for paper-based material and 81 [85.3%] for audiovisual material; P = .39) and to evaluate it (mean [SD] correct responses, 5.09 [1.13] for paper-based material and 5.01 [1.07] for audiovisual material; P = .81). Understanding was significantly higher in the audiovisual material group (mean [SD] correct responses, 5.09 [1.84]) compared with the paper-based material group (mean [SD] correct responses, 4.61 [1.70]; P = .04). In the juvenile detention center, individuals in the audiovisual material group were more likely to sign the IC form (44 [89.8%]) than the paper-based material group (35 [68.6%], P = .006). No significant difference was found between groups for understanding and evaluation. Adults took a mean (SD) of 5 (2) minutes to read the paper material, and adolescents took 7 (3) minutes. Conclusions and Relevance Given the small benefit of audiovisual material, these findings suggest that giving detained adults and prison health care staff a choice regarding IC material is best. For adolescents, audiovisual material should be provided. Future studies should focus on increasing understanding of the IC process. Trial Registration ClinicalTrials.gov Identifier: NCT05505058

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

Conserved interactions of the splicing factor Ntr1/Spp382 with proteins involved in DNA double-strand break repair and telomere metabolism

The ligation of DNA double-strand breaks in the process of non-homologous end-joining (NHEJ) is accomplished by a heterodimeric enzyme complex consisting of DNA ligase IV and an associated non-catalytic factor. This DNA ligase also accounts for the fatal joining of unprotected telomere ends. Hence, its activity must be tightly controlled. Here, we describe interactions of the DNA ligase IV-associated proteins Lif1p and XRCC4 of yeast and human with the putatively orthologous G-patch proteins Ntr1p/Spp382p and NTR1/TFIP11 that have recently been implicated in mRNA splicing. These conserved interactions occupy the DNA ligase IV-binding sites of Lif1p and XRCC4, thus preventing the formation of an active enzyme complex. Consistently, an excess of Ntr1p in yeast reduces NHEJ efficiency in a plasmid ligation assay as well as in a chromosomal double-strand break repair (DSBR) assay. Both yeast and human NTR1 also interact with PinX1, another G-patch protein that has dual functions in the regulation of telomerase activity and telomere stability, and in RNA processing. Like PinX1, NTR1 localizes to telomeres and associates with nucleoli in yeast and human cells, suggesting a function in localized control of DSBR

Structural and functional characterization of interactions involving the Tfb1 subunit of TFIIH and the NER factor Rad2

The general transcription factor IIH (TFIIH) plays crucial roles in transcription as part of the pre-initiation complex (PIC) and in DNA repair as part of the nucleotide excision repair (NER) machinery. During NER, TFIIH recruits the 3′-endonuclease Rad2 to damaged DNA. In this manuscript, we functionally and structurally characterized the interaction between the Tfb1 subunit of TFIIH and Rad2. We show that deletion of either the PH domain of Tfb1 (Tfb1PH) or several segments of the Rad2 spacer region yield yeast with enhanced sensitivity to UV irradiation. Isothermal titration calorimetry studies demonstrate that two acidic segments of the Rad2 spacer bind to Tfb1PH with nanomolar affinity. Structure determination of a Rad2–Tfb1PH complex indicates that Rad2 binds to TFIIH using a similar motif as TFIIEα uses to bind TFIIH in the PIC. Together, these results provide a mechanistic bridge between the role of TFIIH in transcription and DNA repair

An international working group consensus report for the prioritization of molecular biomarkers for Ewing sarcoma

The advent of dose intensified interval compressed therapy has improved event-free survival for patients with localized Ewing sarcoma (EwS) to 78% at 5 years. However, nearly a quarter of patients with localized tumors and 60-80% of patients with metastatic tumors suffer relapse and die of disease. In addition, those who survive are often left with debilitating late effects. Clinical features aside from stage have proven inadequate to meaningfully classify patients for risk-stratified therapy. Therefore, there is a critical need to develop approaches to risk stratify patients with EwS based on molecular features. Over the past decade, new technology has enabled the study of multiple molecular biomarkers in EwS. Preliminary evidence requiring validation supports copy number changes, and loss of function mutations in tumor suppressor genes as biomarkers of outcome in EwS. Initial studies of circulating tumor DNA demonstrated that diagnostic ctDNA burden and ctDNA clearance during induction are also associated with outcome. In addition, fusion partner should be a pre-requisite for enrollment on EwS clinical trials, and the fusion type and structure require further study to determine prognostic impact. These emerging biomarkers represent a new horizon in our understanding of disease risk and will enable future efforts to develop risk-adapted treatment

Dynamic Interaction of TTDA with TFIIH Is Stabilized by Nucleotide Excision Repair in Living Cells

Transcription/repair factor IIH (TFIIH) is essential for RNA polymerase II transcription and nucleotide excision repair (NER). This multi-subunit complex consists of ten polypeptides, including the recently identified small 8-kDa trichothiodystrophy group A (TTDA)/ hTFB5 protein. Patients belonging to the rare neurodevelopmental repair syndrome TTD-A carry inactivating mutations in the TTDA/hTFB5 gene. One of these mutations completely inactivates the protein, whereas other TFIIH genes only tolerate point mutations that do not compromise the essential role in transcription. Nevertheless, the severe NER-deficiency in TTD-A suggests that the TTDA protein is critical for repair. Using a fluorescently tagged and biologically active version of TTDA, we have investigated the involvement of TTDA in repair and transcription in living cells. Under non-challenging conditions, TTDA is present in two distinct kinetic pools: one bound to TFIIH, and a free fraction that shuttles between the cytoplasm and nucleus. After induction of NER-specific DNA lesions, the equilibrium between these two pools dramatically shifts towards a more stable association of TTDA to TFIIH. Modulating transcriptional activity in cells did not induce a similar shift in this equilibrium. Surprisingly, DNA conformations that only provoke an abortive-type of NER reaction do not result into a more stable incorporation of TTDA into TFIIH. These findings identify TTDA as the first TFIIH subunit with a primarily NER-dedicated role in vivo and indicate that its interaction with TFIIH reflects productive NER

Drug sensitivity testing on patient-derived sarcoma cells predicts patient response to treatment and identifies c-Sarc inhibitors as active drugs for translocation sarcomas

BACKGROUND: Heterogeneity and low incidence comprise the biggest challenge in sarcoma diagnosis and treatment. Chemotherapy, although efficient for some sarcoma subtypes, generally results in poor clinical responses and is mostly recommended for advanced disease. Specific genomic aberrations have been identified in some sarcoma subtypes but few of them can be targeted with approved drugs. METHODS: We cultured and characterised patient-derived sarcoma cells and evaluated their sensitivity to 525 anti-cancer agents including both approved and non-approved drugs. In total, 14 sarcomas and 5 healthy mesenchymal primary cell cultures were studied. The sarcoma biopsies and derived cells were characterised by gene panel sequencing, cancer driver gene expression and by detecting specific fusion oncoproteins in situ in sarcomas with translocations. RESULTS: Soft tissue sarcoma cultures were established from patient biopsies with a success rate of 58%. The genomic profile and drug sensitivity testing on these samples helped to identify targeted inhibitors active on sarcomas. The cSrc inhibitor Dasatinib was identified as an active drug in sarcomas carrying chromosomal translocations. The drug sensitivity of the patient sarcoma cells ex vivo correlated with the response to the former treatment of the patient. CONCLUSIONS: Our results show that patient-derived sarcoma cells cultured in vitro are relevant and practical models for genotypic and phenotypic screens aiming to identify efficient drugs to treat sarcoma patients with poor treatment options.Peer reviewe

The First European Interdisciplinary Ewing Sarcoma Research Summit

The European Network for Cancer Research in Children and Adolescents (ENCCA) provides an interaction platform for stakeholders in research and care of children with cancer. Among ENCCA objectives is the establishment of biology-based prioritization mechanisms for the selection of innovative targets, drugs, and prognostic markers for validation in clinical trials. Specifically for sarcomas, there is a burning need for novel treatment options, since current chemotherapeutic treatment protocols have met their limits. This is most obvious for metastatic Ewing sarcoma (ES), where long term survival rates are still below 20%. Despite significant progress in our understanding of ES biology, clinical translation of promising laboratory results has not yet taken place due to fragmentation of research and lack of an institutionalized discussion forum. To fill this gap, ENCCA assembled 30 European expert scientists and five North American opinion leaders in December 2011 to exchange thoughts and discuss the state of the art in ES research and latest results from the bench, and to propose biological studies and novel promising therapeutics for the upcoming European EWING2008 and EWING2012 clinical trials

IGF1 Is a Common Target Gene of Ewing's Sarcoma Fusion Proteins in Mesenchymal Progenitor Cells

The EWS-FLI-1 fusion protein is associated with 85-90% of Ewing's sarcoma family tumors (ESFT), the remaining 10-15% of cases expressing chimeric genes encoding EWS or FUS fused to one of several ets transcription factor family members, including ERG-1, FEV, ETV1 and ETV6. ESFT are dependent on insulin-like growth factor-1 (IGF-1) for growth and survival and recent evidence suggests that mesenchymal progenitor/stem cells constitute a candidate ESFT origin. To address the functional relatedness between ESFT-associated fusion proteins, we compared mouse progenitor cell (MPC) permissiveness for EWS-FLI-1, EWS-ERG and FUS-ERG expression and assessed the corresponding expression profile changes. Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3-5 days. Only 14% and 4% of the total number of genes that were respectively induced and repressed in MPCs by the three fusion proteins were shared. However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression. Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis