Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD.

To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer-promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer-promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer-promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer-promoter relationships.

The Nucleosome Remodeling and Deacetylase Complex NuRD Is Built from Preformed Catalytically Active Sub-modules.

The nucleosome remodeling deacetylase (NuRD) complex is a highly conserved regulator of chromatin structure and transcription. Structural studies have shed light on this and other chromatin modifying machines, but much less is known about how they assemble and whether stable and functional sub-modules exist that retain enzymatic activity. Purification of the endogenous Drosophila NuRD complex shows that it consists of a stable core of subunits, while others, in particular the chromatin remodeler CHD4, associate transiently. To dissect the assembly and activity of NuRD, we systematically produced all possible combinations of different components using the MultiBac system, and determined their activity and biophysical properties. We carried out single-molecule imaging of CHD4 in live mouse embryonic stem cells, in the presence and absence of one of core components (MBD3), to show how the core deacetylase and chromatin-remodeling sub-modules associate in vivo. Our experiments suggest a pathway for the assembly of NuRD via preformed and active sub-modules. These retain enzymatic activity and are present in both the nucleus and the cytosol, an outcome with important implications for understanding NuRD function.

Validation of an HPLC-UV method for sorafenib determination in human plasma and application to cancer patients in routine clinical practice.

Sorafenib, a new oral multikinase inhibitor with antiangiogenic properties, has demonstrated preclinical and clinical activity against several tumor types. The aims of this study were to validate a method for the measurement of sorafenib in plasma from cancer patients, then to test this method in clinical practice. Following liquid-liquid extraction, the compounds were separated with gradient elution (on a C18 ultrasphere ODS column using a mobile phase of acetonitrile/20 mM ammonium acetate), then detected at 255 nm. The calibration was linear in the range 0.5-20 mg/L. Intra- and inter-assay precision was lower than 7 and 10%, respectively, at 0.5, 3 and 20 mg/L. Plasma sorafenib concentrations were measured in 22 cancer patients (99 samples). The mean trough sorafenib concentration (C(min)) and concentration at peak were 4.3+/-2.5 mg/L (n=68, CV=57.5%) and 6.2+/-3.0 mg/L (n=31, CV=47.5%), respectively. Mean sorafenib C(min) in eight patients who experienced grade 3 drug-related adverse events was approximately 1.5-fold greater than that observed in the remaining patients (7.7+/-3.6 mg/L vs. 4.4+/-2.4 mg/L, P=0.0083). In conclusion, the method was successfully used in routine practice to monitor plasma concentrations of sorafenib in cancer patients. Finally, large interindividual variability and higher exposure in patients experiencing severe toxicity support the need for therapeutic drug monitoring to ensure an optimal exposure to sorafenib.