Journal: bioRxiv

Article Title: Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

doi: 10.1101/2020.04.03.003178

Figure Lengend Snippet: (a) Western blot comparison of expression of (Left) NuRD components, and (Right) CTCF and Cohesin in the cell lines used. Detailed schematic of the (b) Mbd3 , (c) Chd4 and (d) Mta2 cell lines generated. CHD4 was tagged as previously described . MTA2 was tagged in ES cells expressing the ER-MBD3-ER (estrogen receptor-MBD3-estrogen receptor) fusion protein so that nuclear localisation of MBD3 is tamoxifen-inducible . (Left) Expression of NuRD complex subunits was confirmed by western blot. Note that the stability of MTA2 and GATAD2A are both dependent upon MBD3, but that of CHD4 is not . (Right) Immunoprecipitation of either CHD4 or MTA2 confirms that the Eos-Halo-FLAG tags do not prevent association with other NuRD components, and that NuRD complex integrity is dependent upon the presence of MBD3.

Article Snippet: Immunoprecipitations were carried out using antibodies to CHD4 or MTA2, or Halo-Trap beads (ChromoTek) in the case of the CHD4-Halo line.

Techniques: Western Blot, Comparison, Expressing, Generated, Immunoprecipitation

Journal: bioRxiv

Article Title: Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

doi: 10.1101/2020.04.03.003178

Figure Lengend Snippet: (a) Schematic of holo-NuRD complex assembly with GATAD2A linking MBD3 to the CHD4 remodeller. (b) Pull-down experiments of MBP-tagged MBD with and without GATAD2A confirm that GATAD2A is required for CHD4 to interact with the deacetylase sub-complex. (c) Distribution of the four biophysical parameters described in for 20ms exposure tracking of MBD3 and CHD4 in wild-type ES cells, as well as CHD4 in the absence of either MBD3 or GATAD2A/B. The data for MTA2 in the presence and absence of nuclear localised MBD3 are also shown. The grey dotted lines indicate the upper bound of the precision limit calculated at the 95 % confidence interval for an immobilised JF 549 dye control sample. HaloTag with a nuclear localisation sequence (HaloTag-NLS) is also shown as a control for a (mostly) freely diffusing molecule , . (d) Box plot of apparent diffusion coefficients extracted from chromatin bound (C) and freely diffusing (F) CHD4 molecules in wild-type, Mbd3 knockout and GATAD2A/B knock-down ES cells (*p < 0.01, **p < 0.001, Kolmogorov-Smirnov test). The grey dotted line indicates the upper bound of the precision limit calculated at the 95 % confidence interval for an immobilised JF 549 dye control sample. (e) Cumulative distribution functions showing a higher diffusion coefficient for freely diffusing unconfined CHD4 upon removal of GATAD2A/B, and for freely diffusing MTA2 molecules upon removal of MBD3 from the nucleus. (f) Table showing the proportions (K) and estimated values of the apparent diffusion coefficients (D), as well as the number of chromatin bound and freely diffusing sub-trajectories obtained from the total number of trajectories analysed. (NB – many trajectories were discarded as they were either too short for analysis or because they had a low probability of being classified as confined or unconfined.)

Article Snippet: Immunoprecipitations were carried out using antibodies to CHD4 or MTA2, or Halo-Trap beads (ChromoTek) in the case of the CHD4-Halo line.

Techniques: Histone Deacetylase Assay, Control, Sequencing, Diffusion-based Assay, Knock-Out, Knockdown

Journal: bioRxiv

Article Title: Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

doi: 10.1101/2020.04.03.003178

Figure Lengend Snippet: (a) Segmentation of an example 20 ms trajectory into confined chromatin bound (C) (blue) and unconfined freely diffusing (F) states (orange). (b) Percentage of molecules and distribution of apparent diffusion coefficients for chromatin bound and freely diffusing CHD4 molecules. (c) (Left) Box plot of apparent diffusion coefficients for chromatin bound and freely diffusing CHD4 and MTA2 molecules in the presence and absence of MBD3 [*p < 0.01, **p < 0.001 (Kolmogorov-Smirnov test)]. The data for MBD3 is also shown as a control, and the grey dotted line indicates the upper bound of the precision limit calculated at the 95 % confidence interval for immobilised JF 549 dye molecules. (Right) Percentage of freely diffusing and chromatin bound CHD4 and MTA2 molecules in the presence and absence of MBD3 [from Gaussian fitting, *p < 0.01, **p < 0.001 (2-way ANOVA)]. [The number of cells/trajectories used in the analysis were: 30/5,557 (CHD4), 25/2,337 (CHD4-MBD3), 10/336 (MTA2), 10/652 (MTA2-MBD3) and 30/2,224 (MBD3).] (d) (Left) A plot of the confinement probability allows determination of the association T A and dissociation T D times – defined respectively as the time a trajectory spends between periods of confined or unconfined motion. (Middle) A single exponential curve of rate lambda=1/T A is then fit to the distribution of association/dissociation times. (Right) The association times extracted for CHD4 and MTA2 were then compared to those in the absence of MBD3, and to those for the MBD3 control [error bars show 95 % confidence intervals, *p < 0.01 (2-way ANOVA)]. (e) Schematic representation of a model in which MBD3-dependent assembly of the NuRD complex increases the association rate of the deacetylase sub-complex.

Article Snippet: Immunoprecipitations were carried out using antibodies to CHD4 or MTA2, or Halo-Trap beads (ChromoTek) in the case of the CHD4-Halo line.

Techniques: Diffusion-based Assay, Control, Histone Deacetylase Assay

Journal: Micromachines

Article Title: Auto-Regression Model-Based Off-Line PID Controller Tuning: An Adaptive Strategy for DC Motor Control

doi: 10.3390/mi13081264

Figure Lengend Snippet: Block diagram representation for the closed-loop system including the PID controller.

Article Snippet: The proposed method updates the PID controller gains based on the Simulink TM controller tuning toolbox.

Techniques: Blocking Assay