Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multifunctional regulator of self-renewal

The gene regulatory network (GRN) that supports neural stem cell (NS cell) self-renewal has so far been poorly characterized. Knowledge of the central transcription factors (TFs), the noncoding gene regulatory regions that they bind to, and the genes whose expression they modulate will be crucial in unlocking the full therapeutic potential of these cells. Here, we use DNase-seq in combination with analysis of histone modifications to identify multiple classes of epigenetically and functionally distinct cis-regulatory elements (CREs). Through motif analysis and ChIP-seq, we identify several of the crucial TF regulators of NS cells. At the core of the network are TFs of the basic helix-loop-helix (bHLH), nuclear factor I (NFI), SOX, and FOX families, with CREs often densely bound by several of these different TFs. We use machine learning to highlight several crucial regulatory features of the network that underpin NS cell self-renewal and multipotency. We validate our predictions by functional analysis of the bHLH TF OLIG2. This TF makes an important contribution to NS cell self-renewal by concurrently activating pro-proliferation genes and preventing the untimely activation of genes promoting neuronal differentiation and stem cell quiescence.Welcome Trust grants: (WT095908, WT098051), FEBS Long-Term Fellowship, Medical Research Council Grant-in-Aid (U117570528)

Automated entire thrombus density measurements for robust and comprehensive thrombus characterization in patients with acute ischemic stroke

Background and Purpose: In acute ischemic stroke (AIS) management, CT-based thrombus density has been associated with treatment success. However, currently used thrombus measurements are prone to inter-observer variability and oversimplify the heterogeneous thrombus composition. Our aim was first to introduce an automated method to assess the entire thrombus density and then to compare the measured entire thrombus density with respect to current standard manual measurements. Materials and Method: In 135 AIS patients, the density distribution of the entire thrombus was determined. Density distributions were described usingmedians, interquartile ranges (IQR), kurtosis, and skewedness. Differences between themedian of entire thrombusmeasurements and commonly applied manualmeasurements using 3 regions of interest were determined using linear regression. Results: Density distributions varied considerably with medians ranging from 20.0 to 62.8 HU and IQRs ranging from 9.3 to 55.8 HU. The average median of the thrombus density distributions (43.5 ± 10.2 HU) was lower than the manual assessment (49.6 ± 8.0 HU) (p<0.05). The difference between manual measurements and median density of entire thrombus decreased with increasing density (r = 0.64; p<0.05), revealing relatively higher manual measurements for low density thrombi such that manual density measurement tend overestimates the real thrombus density. Conclusions: Automatic measurements of the full thrombus expose a wide variety of thrombi density distribution, which is not grasped with currently used manual measurement. Furthermore, d

Two-year clinical follow-up of the Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in The Netherlands (MR CLEAN): Design and statistical analysis plan of the extended follow-up study

Background: MR CLEAN was the first randomized trial to demonstrate the short-term clinical effectiveness of endovascular treatment in patients with acute ischemic stroke caused by large vessel occlusion in the anterior circulation. Several other trials confirmed that endovascular treatment improves clinical outcome at three months. However, limited data are available on long-term clinical outcome. We aimed to estimate the effect of endovascular treatment on functional outcome at two-year follow-up in patients with acute ischemic stroke. Secondly, we aimed to assess the effect of endovascular treatment on major vascular events and mortality during two years of follow-up. Methods: MR CLEAN is a multicenter clinical trial with randomized treatment allocation, open-label treatment, and blinded endpoint evaluation. Patients included were 18 years or older with acute ischemic stroke caused by a proven anterior proximal artery occlusion who could be treated within six hours after stroke onset. The intervention contrast was endovascular treatment and usual care versus no endovascular treatment and usual care. The current study extended the follow-up duration from three months to two years. The primary outcome is the score on the modified Rankin scale at two years. Secondary outcomes include all-cause mortality and the occurrence of major vascular events within two years of follow-up. Discussion: The results of our study provide information on the long-term clinical effectiveness of endovascular treatment, which may have implications for individual treatment decisions and estimates of cost-effectiveness. Trial registration:NTR1804. Registered on 7 May 2009; ISRCTN10888758. Registered on 24 July 2012 (main MR CLEAN trial); NTR5073. Registered on 26 February 2015 (extended follow-up study)

Transcription-coupled DNA–protein crosslink repair by CSB and CRL4^CSA-mediated degradation

DNA–protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based processes such as replication, which is counteracted through proteolysis-mediated DPC removal by spartan (SPRTN) or the proteasome. However, whether DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here we show that DPCs severely impede RNA polymerase II-mediated transcription and are preferentially repaired in active genes by transcription-coupled DPC (TC-DPC) repair. TC-DPC repair is initiated by recruiting the transcription-coupled nucleotide excision repair (TC-NER) factors CSB and CSA to DPC-stalled RNA polymerase II. CSA and CSB are indispensable for TC-DPC repair; however, the downstream TC-NER factors UVSSA and XPA are not, a result indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independently of SPRTN but is mediated by the ubiquitin ligase CRL4CSA and the proteasome. Thus, DPCs in genes are preferentially repaired in a transcription-coupled manner to facilitate unperturbed transcription.</p

An Oct4-Centered Protein Interaction Network in Embryonic Stem Cells

Transcription factors, such as Oct4, are critical for establishing and maintaining pluripotent cell identity. Whereas the genomic locations of several pluripotency transcription factors have been reported, the spectrum of their interaction partners is underexplored. Here, we use an improved affinity protocol to purify Oct4-interacting proteins from mouse embryonic stem cells (ESCs). Subsequent purification of Oct4 partners Sall4, Tcfcp2l1, Dax1, and Esrrb resulted in an Oct4 interactome of 166 proteins, including transcription factors and chromatin-modifying complexes with documented roles in self-renewal, but also many factors not previously associated with the ESC network. We find that Esrrb associated with the basal transcription machinery and also detect interactions between transcription factors and components of the TGF-β, Notch, and Wnt signaling pathways. Acute depletion of Oct4 reduced binding of Tcfcp2l1, Dax1, and Esrrb to several target genes. In conclusion, our purification protocol allowed us to bring greater definition to the circuitry controlling pluripotent cell identity