EMBO/soda-nel huggingface dataset:
The NuRD complex suppresses transcriptional noise during lineage commitment (2019)
Figure 1

Figure 1. Biochemical characterisation of epitope-tagged MTA proteins

Schematic of MTA proteins with different protein domains indicated as coloured boxes, and the C-terminal epitope tags indicated. The model below shows the MTA- and HDAC-containing subunit joined to the CHD4-containing subunit through the MBD protein.

Tagged MTA proteins in heterozygously targeted cell lines were immunoprecipitated and subsequent western blots probed with antibodies indicated at right. Solid black triangles indicate the locations of untagged proteins and open triangles show the position of epitope-tagged proteins in the anti-Mta1, Mta2 or Mta3 panels; and a grey triangle shows the location of IgG bands in the anti-Mbd3 panel. 10% of the input was loaded into "Input" lanes. Molecular weights in kDa are shown at left. (Full western blot images are available in Mendeley Data.)

Proteins co-purifying with Mta1, Mta2 or Mta3 in IP-mass spectrometry experiments in wild type cells (top) or Mbd3-null cells (bottom). Proteins showing significant enrichment with the bait protein are located outside the dotted lines. For all panels the protein being immunoprecipitated is indicated in red, the other MTA proteins in purple, and other NuRD components in blue. Each IP/Mass spec experiment set comprised three independent immunoprecipitations from one nuclear extract preparation.

Relative enrichment of indicated proteins in MTA pulldowns from wild type (WT) or Mbd3-null (Mbd3KO) ES cells, normalised to 2x MTA proteins. NuRD components comprising the remodelling subunit are labelled in blue, those comprising the deacetylase subunit in red. As MTA proteins are the bait in these experiments, they may be isolated more efficiently than their interaction partners. Error bars represent standard deviation from three (Mta1 and Mta2) or six (Mta3) replicate pulldowns.
The NuRD complex suppresses transcriptional noise during lineage commitment (2019)
Figure 2

Figure. 2. Mta proteins show similar chromatin binding patterns

ChIP-seq peaks identified for Mta1 and Mta2 (left), Mta1 and Mta3 (middle) or Mta2 and Mta3 (right) are plotted by enrichment for each protein. Peaks called for both proteins are indicated in the overlap.

Overlap of peaks identified by ChIP-seq for each MTA protein in wild type ES cells. Total peak numbers are indicated below each protein name. Each ChIP-seq dataset was made from biological triplicates.

Comparison of Mta1 and Mta2 peaks with Chd4 peaks, as in Panel (B).

ChIP-seq enrichment for indicated proteins or histone modifications is plotted across different subsets of Mta-bound sites. Mta1/2/3 refers to peaks identified in all three ChIP-seq datasets, whereas "Mta1 Only", "Mta2 Only" or "Mta3 Only" refer to peaks only called for that protein.

Average enrichment of density plots in (D) are plotted for different subsets of Mta ChIP-seq peaks.
The NuRD complex suppresses transcriptional noise during lineage commitment (2019)
Figure 3

Figure 3. Mta1/Mta2/Mta3 triple null ES cells represent a complete NuRD KO

Western blots of wild type (Control), double or triple-null ES cell nuclear extract was probed with antibodies indicated at right. LaminB1 acts as a loading control. Approximate sizes are indicated in kDa.

Phase-contrast images of wild type or Mta123∆ ES cells in 2i+LIF conditions. Scale bars represent 100µm.

Western blots of anti-Chd4 immunoprecipitation of nuclear extract from indicated cell lines (top) probed with antibodies indicated at right. For the Gatad2b and Mbd3 blots, arrowheads indicate target proteins, while an asterisk marks a non-specific IgG bands. 10% of the input was loaded into "Input" lanes. Approximate sizes are indicated in kDa.

Western blot of a time course of Mta3 deletion in Mta1∆Mta2∆Mta3Flox/Flox: Cre-ER (Mta12∆3F/F) or Control (Mta12∆3F/F without Cre-ER) ES cells probed for indicated NuRD component proteins or RNA Polymerase II as a loading control (αRNAPII). The time course is indicated at the top as Days + tamoxifen.

Western blot showing rescue of Mta123∆ ES cells by ectopic expression of Mta1, Mta2 or Mta3 from a transgene (TG). Total RNA Polymerase II acts as a loading control (αRNAPII). For all western blots molecular weight is indicated at left in kDa.

Model of NuRD complex structure. Upon loss of Mbd3 most of the NuRD complex falls apart into the Chd4-containing remodeller subcomplex and the MTA-containing deacetylase subcomplex, but some intact Mbd2-NuRD still remains. Upon loss of all three MTA proteins no intact NuRD remains, both Mbd3 and Gatad2a/b become unstable and neither of the intact subcomplexes remain.
The NuRD complex suppresses transcriptional noise during lineage commitment (2019)
Figure 4

Figure 4. Mta proteins act redundantly to control gene expression

Comparison of gene expression in Mta123∆ ES cells and wild type (WT) ES cells. Each circle represents a gene: red indicates spike-in controls, blue indicates genes that are not differentially expressed to a significant degree, and green indicates differentially expressed genes (2404 increased, 1293 decreased) defined with an adjusted p-value < 0.05 and a log2 fold-change > 1. N=3 for each genotype.

Fold change in gene expression is plotted for different subsets of genes. All genes are plotted in black, while subsets of genes located nearest to ChIP-seq peaks for the indicated proteins +Chd4 and which show significant changes in expression compared to wild type cells are plotted in dashed coloured lines as indicated. The number of genes in each of the Mta categories are: all genes (32271), all differentially expressed (n=3701), Mta1+2+3 (n=1738), Mta1+2 (n=1460), Mta1 (n=1020) and Mta2 (n=924).

Principal component analysis of RNAseq data from ES cell lines of indicated genotypes in either self-renewing conditions (2i) or after 48 hours in the absence of two inhibitors and LIF (Diff48). Each point represents a biological replicate.

GO term enrichment for genes differentially expressed in Mta123∆ ES cells (left) or Mbd3∆ ES cells (right) compared to wild type cells in 2iL conditions. For each comparison the top five most significant gene ontology terms are plotted by log10 of the Benjamini-adjusted p-value. The significant GO terms and p-values were calculated using David v.6.8

Genes associated with indicated GO terms plotted by fold change in expression in Mta123∆ ES cells (x-axis) or Mbd3∆ ES cells (y-axis). Genes are coloured if they are differentially expressed (log2 fold-change > 1 and adjusted p-value < 0.05) in either comparison as indicated. The dotted lines show the fold-change cut-off of 2. GO-terms were identified using David v.6.8 (Huang da et al., 2009a) using a Benjamini score with a cutoff of 0.05.

Genes associated with core NuRD peaks, with an MTA-only peak, with a Chd4 peak and an MTA-peak but not an Mbd3 peak (Chd4+Mta-Mbd3 genes), or with an MTA-peak but not a Chd4 peak (MTA-only genes) were divided into quartiles based upon expression levels in wild type cells. Change in gene expression is plotted on the x-axis and calculated significance of the expression change on the y-axis. Those genes significantly misexpressed (|log2FC|>1; p≤0.05) are indicated in red, genes showing no significant change are plotted in black. The percentage of genes within each quartile which is significantly misexpressed is indicated at the top of each plot.
The NuRD complex suppresses transcriptional noise during lineage commitment (2019)
Figure 5

Figure 5. NuRD activity maintains an appropriate ES cell differentiation trajectory

Phase contrast pictures of ES cells of indicated genotypes after 5 days in neuroectoderm differentiation conditions (N2B27). White arrowheads in the Mbd3∆ panel indicate pockets of ES-like cells. Scale bars represent 100 µm.

Expression of indicated genes in the neuroectoderm differentiation time course was measured by RT-qPCR in Control (green) Mta123∆ (magenta) or Mbd3∆ (blue) ES cells. N ≥ 3 biological replicates, error bars indicate SEM.

Phase contrast images of embryoid body outgrowths of indicated genotypes after 12 days in differentiation conditions (N2B27 + 10% serum). Scale bars represent 100µm.

Expression of indicated genes in embryoid body cultures over time was measured by RT-qPCR as in Panel (B).

Comparison of expression data for wild type, Mbd3∆ or Mta123∆ ES cells in self-renewing (2i) conditions or after 48 hours in differentiation conditions (48h N2B27) with mouse embryonic single cell RNA-seq data from (Mohammed et al., 2017). Larger shading encloses biological replicates, smaller circles represent individual cells. PC4 vs PC1 is plotted in Fig. EV5F and a loading plot in EV5G.
The NuRD complex suppresses transcriptional noise during lineage commitment (2019)
Figure 6

Figure 6. NuRD-null ES cells cannot contribute to normal embryonic development

Composite images of representative chimaeric embryos made with control (Mta1Flox/∆Mta2+/+Mta3Flox/Flox) or Mta123∆ ES cells. ES-derived cells express the Kusabira Orange fluorescent marker. Sox2 indicates epiblast cells and Cdx2 is expressed in trophectoderm cells. Arrows indicate examples of K-Orange expressing cells in the mutant embryos. Scale bars = 20µm.

(Top) Number of K-Orange expressing cells observed in chimaeric embryos obtained using control ES cells, Mta123∆ ES cells, or Mta123∆ ES cells in which Mta2 was reintroduced on a constitutively expressed transgene (Mta123∆+Mta2TG). P-values calculated using a two-tailed t-test. (Middle) Mean number of K-Orange cells per embryo separated by Sox2 expression. P-values calculated using a two-tailed t-test. (Bottom) Number of K-Orange and Caspase-3 positive cells per embryo. P-values calculated using a one-tailed t-test: *P < 0.05, ****P < 0.0001, "ns" = not significant.

Model of NuRD function during differentiation of pluripotent cells. NuRD facilitates lineage commitment of ES cells after exit from pluripotency (red arrows), allowing cells to form differentiated cell types (Top). In the absence of Mbd3, residual NuRD activity ensures cells retain the appropriate differentiation trajectory, but the cells are unable to reach a differentiated cell fate (dotted red arrows; Middle). In the absence of all three MTA proteins there is no residual NuRD activity and ES cells are unable to either achieve appropriate lineage commitment, or to maintain the proper differentiation trajectory (dotted arrows, Bottom).

Expanded View Figures
Feedback regulation of heat shock factor 1 (Hsf1) activity by Hsp70-mediated trimer unzipping and dissociation from DNA (2020)
Figure 1

Figure 1: Hsf1 binds with high affinity but highly dynamic to heat shock elements (HSEs) and is dissociated from DNA by Hsc70 and DnaJB1.

A Domain structure of mammalian Hsf1. Intrinsically disordered regions are indicated with cylinders of smaller diameter. DBD, DNA binding domain; HR-AB, heptad repeat regions A and B; RD, regulatory domain; HR-C, heptad repeat region C; TAD, transactivation domain.

B Equilibrium titration of Hsf1 binding to Alexa Fluor® 488-labeled HSE-DNA. Fluorescence polarization plotted versus the Hsf1 trimer concentration. KD = 4.7 ± 1.9 nM (n = 4).

C Hsf1 rapidly switches between different HSE-containing double stranded DNA oligonucleotides. Trimeric Hsf1 was pre-incubated with Alexa Fluor® 488-labeled HSE-DNA in MgCl2-free buffer. At timepoint 0 (arrow) buffer, 4 mM MgCl2, 2 mM ATP+4 mM MgCl2, control DNA (DNACtrl at 10-fold molar excess over HSE-DNA), and HSE-containing DNA was added as indicated and fluorescence polarization followed over time. Shown is one of three identical experiments.

D Trimeric Hsf1 were bound to HSE-DNA and the indicated components added at timepoint 0. A representative experiment is shown.

E Hsp70 dissociates Hsf1 from DNA with similar rates as Hsc70. Rates were determined as shown in Fig EV1E. Shown are mean ± SD (n = 3). Significance was tested with Student's T-test.

F-I The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on temperature (for 25°C n = 6, for 37°C n = 5, T-test, p = 0.011) (F), concentration of Hsc70 and DnaJB1 (n = 3) (G), concentration of ATP (n = 3) (H), and concentration of the nucleotide exchange factor Apg2 (HSPA4) (n = 3, ANOVA, **, p < 0.01; ****, p < 0.0001) (I). Shown are mean ± SD.
Feedback regulation of heat shock factor 1 (Hsf1) activity by Hsp70-mediated trimer unzipping and dissociation from DNA (2020)
Figure 2

Figure 2: Hsc70 and DnaJB1 disso­ciate Hsf1 from HSE-DNA by mono­merization of the Hsf1 trimer.

A,B Standard Hsc70/DnaJB1-mediated dissociation reaction of Hsf1 from Alexa Fluor® 488-labeled HSE-DNA similar to Fig 1D monitored by fluorescence polarization (A). The same reaction mixture was incubated for different time intervals as indicated by the arrows below the x-axis, the reaction stopped by addition of blue-native loading buffer and stored on ice until separation by blue-native PAGE, blotted onto a PVDF membrane and detected with an Hsf1-specific antiserum (B). Lanes 1, purified Hsf1 trimer (T); 2, purified Hsf1 monomer (M); 3-11, samples from the Hsc70/DnaJB1-mediated Hsf1 dissociation reaction (0 to 80 min); 12-15, Dissociation reaction incubated for 80 min missing individual components as indicated. HO, higher order oligomers; T, trimer; D, dimer; M, mono­mer.

C Quantification of the amounts of Hsf1 species of the blot shown in (B) and five similar blots. For each lane the intensities of the two areas indicated by the brackets to the right were integrated; upper bracket, DNA bound timers and higher order oligomers (Hsf1b); lower bracket, monomers and Hsf1-species possibly bound by Hsc70 or DnaJB1 or both dissociated from DNA (Hsf1d). Shown are means ± SD (n = 3). Inset, deoligomerization rate as determined by fitting an exponential decay function to the data. Shown are means ± SD (n = 3)
Feedback regulation of heat shock factor 1 (Hsf1) activity by Hsp70-mediated trimer unzipping and dissociation from DNA (2020)
Figure 4

Figure 4: An Hsc70 binding site close to the trimerization domain of Hsf1 is necessary for Hsc70 mediated dissociation of Hsf1 from DNA.

A Hsp70 binding score as predicted by a published algorithm (Rüdiger et al, 1997). The score value is attributed to the center of the sequence window of 13 residues. Values below -5 are considered to be good Hsp70 binding sites. Small horizontal gray and black lines underneath the prediction curve labeled with HX to the right represent the segments protected from hydrogen exchange.

B KD values for the indicated peptides as determined by fluorescence polarization. Peptides N74-Q86-C (NMYGFRKVVHIEQC), P223-H235-C (PKYSRQFSLEHVHC) and A343-R352-C (ASVTALTDARC) represent the most hydrophobic part of HX protected segments 78-98, 229-252, and 347-375, respectively; N200-K208-C (NRILGVKRKC), N200-K208-C_SSS (NRSSGSKRKC), G204-N214-C (GVKRKIPLMLNC), and G204-N214-C_SSS (GVKRKSPSMSNC) represent the two potential Hsc70 binding sites in segment 202-213 in original sequence or with hydrophobic residues replaced by serine; S461-Q471-C (SGKQLVHYTAQC) and Y468-S480-C (YTAQPLFLLDPGSC) represent the two potential Hsc70 binding sites in segment 442-474. Shown are means ± SD (n = 3).

C Hsf1(1-408) with a deleted TAD is dissociated from HSE-DNA by Hsc70, DnaJB1 and ATP with similar rates as Hsf1wt. Fluorescence polarization assays containing Alexa Fluor® 488-HSE-DNA bound Hsf1(1-408) trimers and the indicated components. One representative experiment of three is shown.

D The HR-B proximal Hsc70 binding site is essential for Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1. One representative experiment of three is shown.
Feedback regulation of heat shock factor 1 (Hsf1) activity by Hsp70-mediated trimer unzipping and dissociation from DNA (2020)
Figure 5

Figure 5: Hsc70/DnaJB1 monomerize Hsf1 by cycles of successive entropic pulling.

A The rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1 depends on the number of HR-B proximal Hsc70 binding sites in the Hsf1 trimer. Hsf1wt (wt) and Hsf1∆(202-213) (∆) monomers were mixed in the indicated ratios and heat shocked at 42°C for 10 min to form mixtures of different homo- and heterotrimers as indicated with the cartoons (the red line and the arrow head indicate the Hsc70 binding site). Numbers indicate the relative fraction of the different species. For fit of the data see Fig EV2.

B Moving the Hsc70 binding site away from the trimerization domain reduces the rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA-bound Hsf1. Hsf1_t10/20/30, region 202-213 moved by 10, 20, or 30 residues towards the C-terminus.

C Anti-FLAG antibodies are not able to dissociate HSE-DNA-bound Hsf1. Red lines in the cartoon indicate the Hsc70 binding site; blue lines and arrow head indicate the inserted FLAG epitope DYKDDDDK. Hsf1_i201/211/221, FLAG-epitope inserted after residue 201, 211 or 221. Anti-FLAG antibodies were split in halfmers by incubation with 2 mM DTT (see Appendix Fig S3).

D Anti-FLAG antibody halfmers bound to FLAG-epitope containing Hsf1 variants. Hsf1wt and FLAG-insertion variants were analyzed by blue-native gel electrophoresis in the absence or presence of DTT-treated anti-FLAG antibodies as indicated and Hsf1 detected by immunoblotting; t-bound, FLAG antibody-bound Hsf1 trimers; m-bound, FLAG antibody bound Hsf1 monomer.

E Predicted Hsc70 binding sites in the trimerization domain of Hsf1. Hsp70 score of the trimerization region residues 130-216 of Hsf1wt (black); Hsf1-I190S,I194S, magenta; Hsf1-I202S,L203S,V205S, blue; and Hsf1-I209S,L211S,L213S, green. Values below -5 are considered good Hsc70 binding sites. Above the graph is the trimeric homology model of the trimerization domain residues 130-203 with side chains of hydrophobic and charged residues in space filling representation in atom colors with carbon in black except for Ile190 and Ile194 where carbon is shown in green. Above the model is the corresponding sequence. Positively charged residues, blue; negatively charged residues, red. Lines below the sequence indicate two distinct regions with net charge +1 and +5.

F Trimer-to-monomer ratio of freshly purified Hsf1wt and Hsf1-I190S,I194S in the absence of heat shock, determined by gel filtration and BN-Gel (see Appendix Fig S4). Hsf1wt, mean ± SD (n = 3).

G Rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1wt and Hsf1-I190S,I194S; mean ± SD (n=3); **, P < 0.01; (paired t-test).
Feedback regulation of heat shock factor 1 (Hsf1) activity by Hsp70-mediated trimer unzipping and dissociation from DNA (2020)
Figure 6

Figure 6: Compromising Hsc70 binding potentiates expression of a heat shock reporter in HSF1-/- mouse embryonic fibroblasts (MEF).

A Schematics of the used system. MEFs, in which HSF1 on chromosome 8 was deleted, were stably transfected with mTag-BFP expressed under the control of the HSPA6 promoter and subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP, which is translated from an IRES element.

B Exemplary flow cytometry data plotting BFP fluorescence versus GFP fluorescence, indicating the gates used for analysis. Numbers indicate the fraction (%) of cells (forward scatter singlets) in each gate. Color indicates the cell density from dark blue (low) to red (high).

C-H Geometric mean of BFP (C, E) and GFP fluorescence (D, F, H) of cells grown at 37°C (non-stress, NS) or incubated for 1 h at 43°C with a recovery period of 3 h at 37°C (heat shocked, HS) in the GFPhigh (C, D), GFPlow (E, F), and GFPlow-BFPhigh (H) gates. (G) Geometric mean of BFP fluorescence weighted by the relative number of cells in the GFPlow-BFPhigh gate (fluorescence * fraction forward scatter singlets). Note that the GFPlow gate also comprises the GFPlow-BFPhigh gate. Hsf1-202SSS, Hsf1-I202S,L203S,V205S; Hsf1-209SSS, Hsf1-I209S,L211S,L213S; Hsf1-427SSSS, Hsf1-M427S,L429S,L432S,L436S; Hsf1-465SSS, Hsf1-L465S,V466S,Y468S; Hsf1-473SSSS, Hsf1-L473S,F474S,L475S,L476S; Geometric mean ± SD of up to 6 independent experiments; ANOVA Sidak's multiple comparison, *, p < 0.05; **, p < 0.01; ****, p < 0.0001.
Feedback regulation of heat shock factor 1 (Hsf1) activity by Hsp70-mediated trimer unzipping and dissociation from DNA (2020)
Figure 7

Figure 7: Compromising Hsc70 binding potentiates expression of a heat shock reporter under non-stress and heat shock conditions in HeLa cells.

A Schematics of the used system. HeLa cells were stably transfected with mTag-BFP expressed under the control of the HSPA6 promoter and subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP, which is translated from an IRES element.

B Exemplary flow cytometry data plotting BFP fluorescence versus GFP fluorescence, indicating the gates used for analysis. Numbers indicate the fraction (%) of cells (forward scatter singlets) in each gate. Color indicates the cell density from dark blue (low) to red (high). NS, cells grown at 37°C; HS, cells heat shocked at 43°C for 1 h with a recovery period of 3 h at 37°C.

C-H Geometric mean of BFP (C, E) and GFP fluorescence (D, F, H) in the GFPhigh (C, D), GFPlow (E, F), and GFPlow-BFPhigh43 (H) gates. Of note, the number of data points show the number of experiments in which a significant number of cells was found in this gate and missing bars indicate that cells did not fall within this gate in more than two out of four experiments. (G) Geometric mean of BFP fluorescence weighted by the relative number of cells in the GFPlow-BFPhigh43 gate (fluorescence * fraction forward scatter singlets). Note that the GFPlow gate also comprises the GFPlow-BFPhigh37 and that the GFPlow-BFPhigh37 gate also comprises the GFPlow-BFPhigh43 gate. Hsf1-202SSS, Hsf1-I202S,L203S,V205S; Hsf1-209SSS, Hsf1-I209S,L211S,L213S; Hsf1-427SSSS, Hsf1-M427S,L429S,L432S,L436S; Hsf1-465SSS, Hsf1-L465S,V466S,Y468S; Hsf1-473SSSS, Hsf1-L473S,F474S,L475S,L476S; Geometric mean ± SD of 4 independent experiments; ANOVA Sidak's multiple comparison, *, p < 0.05; **, p < 0.01; ****, p < 0.0001.