Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a EZH2 ChIP peak profiles of gene Otx2 for WT, Mtf2 null, and Jarid2 null cells. Peaks were RPKM normalized and scaled across backgrounds. b Phase-contrast of Embryoid Bodies from different background and time point of differentiation. Each differentiation time point and background were performed in replicates. c , d Single-cell UMAPs of 4949 cells over all backgrounds and time points. e Single-cell UMAP of cells colored by predicted clusters. f – i Feature maps of selected lineage genes, color intensity based on normalized expression of individual gene.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Expressing

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a Heatmap of differentially expressed genes comparing different mutants against WT in undifferentiated mESCs. Z-score normalized counts (row scaling) are shown in heatmap. b Venn diagram depicting overlap of a number of genes which were up/downregulated in Mtf2 null and Jarid2 null cells, with PRC2 targets (as determined from EZH2 ChIP targets; next panel). Significance of overlap calculated using hyper-geometrical test. c Heatmap of EZH2 binding peaks (RPKM normalized) for upregulated genes (from Mtf2 and Jarid2 null ESCs) for different genetic backgrounds. Heatmap depicts a window of +/−400 bp from the transcription start site (TSS) of the genes. d Dot plot showing the enriched Gene Ontology terms for the differentially expressed genes in different genetic backgrounds.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a ChIP peak profiles of histone mark H3K27me3 and H3K4me3 for selected upregulated ( Mtf2 null) lineage transcription factors for WT and Mtf2 null cells at pluripotent stage. ChIP profiles were RPKM normalized and scaled between two merged profiles per histone ChIP. b Boxplots depicting the RPKM values of promoter (as defined by +/−500 bp from TSS) H3K27me3, H3K4me3, and EZH2 for all PRC2 targets and the upregulated genes in Mtf2 null and Jarid2 null cells. Asterisk(*) represents a Two-sample Kolmogorov–Smirnov test p -value <0.05. n = 2878 for all PRC2 targets, n = 242 for upregulated Mtf2 null genes and n = 58 for upregulated Jarid2 null genes. Whisker ends of boxplot represent the maximum (top) and minimum values, respectively. Top and bottom of boxplots represent 75th and 25th percentile values, respectively, and finally, median values are shown as colored lines within the boxplots. c Transcription factor motif activity that explains part of the variance in transcript levels, based on motifs in the promoters (as defined by +/−500 bp from TSS) of all upregulated PRC2-bound genes (“Methods”; upregulated genes in all four cell lines, cf. Fig. ). Motifs are shown in aggregated z-scores. d – f Heatmaps showing the mRNA fold change, H3K27me3 and H3K4me3 levels for a set of transcription factors (left, identified in panel c ) and signaling factors (right, identified from Mtf2 and Jarid2 null DEGs list). Genes shown are all PRC2 targets. g Barplot depicting the GATA2 ChIP recovery relative to input. Control is a gene desert region (“Methods”). Dots in bars represent 4 replicates per sample.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Whisker Assay, Activity Assay, Control

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a Schematic of directed differentiation for monolayer cells from different genetic backgrounds (WT, Jarid2 null, and Mtf2 null). b Line plots showing the expression (normalized counts from RNA-seq) of selected temporally regulated genes during early lineage specification processes for different genetic backgrounds and differentiation directions. c , d Heatmap of differentially expressed genes across all time points and between genetic backgrounds. Data was k-means clustered and the normalized counts were shown. e , f Dot plots showing the enrichment of biological pathways for each cluster in ( c , d ), selected by p -value and gene-ratios of the terms. Top selected pathways were picked for each cluster and shown here. g , h Barplots of of H3K27me3 and H3K4me3 ChIP for selected targets ( Eomes and Gata6) . Each bar represents the average of the percentage of input recovered in the ChIP experiment of replicate experiments. Mtf2 WT and Jarid2 WT represent the background-matched wild-type lines of, respectively, Mtf2 null and Jarid2 null cells. Each dot represents a qPCR technical replicate for the sample.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Expressing, RNA Sequencing

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: Model of the role of PRC2 in the exit of pluripotency. PRC2 contributes to a repressive threshold for activation of key regulators of differentiation. This affects the exit of pluripotency and the differentiation to lineages of the three germ layers. One example is the upregulation of GATA2 upon MTF2 loss which results in increasing expression of other factors such as WNT7B and IRX3, as part of a feedforward loop that regulates the exit of pluripotency.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Activation Assay, Expressing

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Schematic representation of the recruitment of PRC2.1 and PRC2.2. MTF2 binds to DNA, while the EED subunit of core PRC2 (orange) binds to H3K27me3 as part of an allosteric feedback loop. The EZH2 subunit of core PRC2 catalyses H3K27 methylation. The PRC2.2 complex contains JARID2 but not MTF2. Both contain the core PRC2 subunits, however the interactions of the PRC2.1 and PRC2.2-specific subunits with chromatin are different. The arrow from JARID2 to DNA is dashed as DNA binding has been shown in vitro but not in vivo b) PRC2.1 (MTF2) and PRC2.2 (JARID2) co-localize to all EZH2 targets. c-f) Heatmap and rpkm quantification (boxplots) of PRC2 subunits and the catalytic product H3K27me3. EZH2 recruitment is heavily affected by the absence of MTF2, while JARID2 and H3K27me3 absence have minor effects (c). The effect of MTF2 and JARID2 on EZH2 recruitment is reflected on H3K27me3 deposition (d). MTF2 is marginally affected by H3K27me3 removal, but its binding is reduced to approximately half the WT level in the absence of JARID2 (e). JARID2 recruitment is strongly reduced in the absence of either H3K27me3 or MTF2 (f). ChIP profiles are highly reproducible g) Genome browser examples of PRC2 binding to classical Polycomb targets. Box plots represent median and interquartile range (IQR; whiskers, 1.5 IQR).

Article Snippet: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

Techniques: Methylation, Binding Assay, In Vitro, In Vivo

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a-d) Heatmap of WT ChIP-seq signal on the indicated peak set. H3K27me3-negative JARID2 peaks were excluded from further analysis. e) Venn diagram showing the overlap of peaks called for the ChIP-Seq of each protein independently. f) Mass spectrometry quantification of PRC2 subunits in the different cell lines. Detection of JARID2 and MTF2 in the respective mutants (asterisks) is due to value imputation in Perseus. g) Western blot validation of EED226 depletion of H3K27me3, for the ChIP shown in Figs 1 and 2. h) Scatterplot of peak RPKM showing high reproducibility of ChIP replicates.

Article Snippet: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

Techniques: ChIP-sequencing, Mass Spectrometry, Western Blot

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Clustering of all PRC2 targets using ChIPseq data in multiple PRC2 mutants. Cluster 1-4 are unmethylated CpG islands (strong BioCap) signal, showing bivalent marks in WT (H3K4me3 and H3K27me3). These regions display heavy reduction of EZH2 recruitment in the MTF2 mutant, milder effects of H3K27me3 absence (EED226 treatment), and little or no effect of JARID2 absence. The intensity of MTF2 binding depends on both H3K27me3 and JARID2 but binding is still clearly detectable even in the absence of PRC2 core ( Eed -/- ) indicating a primary binding to DNA, reinforced by other mechanisms, such as JARID2-mediated recruitment, which in turn also depends on both H3K27me3 and MTF2. Cluster 5 and 6 have lower BioCap and H3K4me3 signal, and, while still affected by the absence of MTF2, this has a much less marked effect on recruitment of both EZH2 and JARID2, and on H3K27me3 deposition. b) WT-normalized, input-subtracted RPKM quantification of signal shown in (a). c) Quantification of GCG trinucleotides matching DNA shape requirement for MTF recruitments as defined in . Cluster 1-4 are strongly enriched in shape-matching GCGs, indicating potential for strong DNA-mediated MTF2 recruitment. d) Enrichment of anatomical terms in the genes associated with peaks in the six clusters. Enrichment within PRC2 targets. Cluster 4 show strong enrichment for CNS structures, cluster 5 and 6 for limb and branchial arches tissues and mesenchyme. See for the full overview.

Article Snippet: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

Techniques: Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Heatmap showing the cluster specific effect of H3K27me3 depletion on the binding of EZH2. WT and MTF2 GT/GT show mild reduction of EZH2 binding when treated with EED226 inhibitor, while the treatment is highly synergistic with the depletion of JARID2. b) Bootstrapping-based RPKM quantification (methods) of the signal in (a). Each coloured dot represent the median of one round of bootstrapping, grey bar represent 99.9% confidence interval for the mean of bootstrapped values in each condition and cluster. c) Treatment with EED226 further affected MTF2 recruitment in Jarid2 -/- and JARID2 recruitment in Mtf2 GT/GT , with the former leading to recruitment patter closely resembling the Eed -/- line (cf. ), highlighting the recruitment differences between cluster 1-4 and 5-6. d) Bootstrapping-based RPKM quantification (methods) of the signal in (c) similar as in 3b. e) Genome browser view of example Polycomb targets. For each genotype two tracks are overlaid: the darker colour represent EED226 treated samples, the lighter colour untreated cells.

Article Snippet: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Heatmap showing EZH2, MTF2 and JARID2 binding in the absence of H3K27me3 in PRC2 and PRC1 mutant lines. In the absence of H3K27me3, JARID2 and RING1A/B mutant phenocopy each other with regard to EZH2 and MTF2 binding, suggesting JARID2 and RING1B act in the same PRC2 recruitment mechanism. JARID2 recruitment is also strongly affected by the absence of RING1A/B, in line with the JARID2-mediated PRC2 recruitment via binding to PRC1-deposited H2AK119ub. b-d) Average plot of the ChIP signal shown in (a), for EZH2 (b) MTF2 (c) and JARID2 (d) centred on called peaks. Lower panels represent the same data with cropped y axis, for better visualization. e) Heatmap showing Ring1b binding in the discussed conditions. Ring1b is only mildly affected by removing H3K27me3 using EED226 (~40%). Binding is further attenuated in MTF2 and JARID2 mutant ESCs. f) Average plot of the ChIP signal shown in (e), centred on called peaks. g) Examples of loci of the data as shown in (e).

Article Snippet: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

Techniques: Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Heatmap of ChIP-seq signal for EZH2 in multiple conditions including Mtf2 cells with double inhibition (d.i.) using EED226 (to remove H3K27me3) and MG132 (to remove H2AK119ub). b) Example loci of the data shown in (a). c) Average profiles of the ChIP signal shown in (a), centred on called peaks. Lower panels represent the same data with cropped y axis, for better visualization.

Article Snippet: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

Techniques: ChIP-sequencing, Inhibition

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) On PRC2.1 main targets (clusters 1-4) relatively little MTF2 binding is sufficient to kick start the EED positive feedback loop which heavily relies on JARID2. As primary recruitment is mediated to a large extent via MTF2, such a loop can still exist in the absence JARID2. In the absence of H3K27me3, an alternative route can take over that requires JARID2 binding to H2AK119ub. b) On PRC2.2/PRC1 targets (clusters 5-6), instead, Polycomb binding is initiated by PRC1 that, upon H2AK119ub deposition, is followed by JARID2-containing PRC2.2. These regions also see the presence of MTF2 in physiological conditions, but this is the result of indirect recruitment via the PRC2 core binding to PRC2.2-initiated H3K27me3 deposition.

Article Snippet: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

Techniques: Binding Assay

Journal: European Journal of Human Genetics

Article Title: An MTF1 binding site disrupted by a homozygous variant in the promoter of ATP7B likely causes Wilson Disease

doi: 10.1038/s41431-018-0221-4

Figure Lengend Snippet: Genomic context of the candidate causative variant. a UCSC Genome Browser (hg19 minus strand) view of the region near the candidate causative variant positioned 676 bp upstream of the canonical ATP7B translation start site (chr13:g.52,586,149A>G, cyan highlight and asterisk). The variant lies in a 100-way vertebrate alignment conserved element and region of high cross-species sequence conservation as computed by PhastCons and PhyloP [30]. The chromatin surrounding chr13:g.52,586,149T>C is hypersensitive to DNaseI (and, therefore, open and accessible) in HepG2 cells. The sequence cloned into the luciferase reporter vector is indicated by the black bar. b Genotype verification for chr13:g.52,586,149T>C (red asterisk) by Sanger sequencing. Representative chromatograms show the indicated number (n=x/y) of amplification products containing the reference (T) or alternate (C) allele for each individual. Box indicates a predicted binding site for MTF1. c The candidate causative variant (bolded, pink) in the Wilson Disease (WD) patient disrupts a key base in the MTF1 position weight matrix. This exact base (boxed) is unaltered in all primates and in dozens of other mammals, even as distant as Tasmanian devil (Supplementary Fig. 1b) (color figure online).

Article Snippet: Polyclonal rabbit anti-MTF1 antibody (Proteintech, Rosemont, IL, USA: 25383-1-AP) was used for chromatin immunoprecipitation.

Techniques: Variant Assay, Sequencing, Clone Assay, Luciferase, Plasmid Preparation, Amplification, Binding Assay

Journal: European Journal of Human Genetics

Article Title: An MTF1 binding site disrupted by a homozygous variant in the promoter of ATP7B likely causes Wilson Disease

doi: 10.1038/s41431-018-0221-4

Figure Lengend Snippet: Functional interrogation of chr13:g.52,586,149T>C. a Luciferase reporter assays in HepG2 cells were performed to quantify differences in transactivation by a 379 bp fragment of the ATP7B promoter with the chr13:g.52,586,149T (Ref) or chr13:g.52,586,149C (Alt) allele, in the presence (+) or absence (−) of MTF1 overexpression (OE). Both with and without MTF1 OE, the reference allele drove significantly higher expression compared to the alternate allele (comparison 1 and 2, respectively). Both alleles experienced dramatic increases in activity with MTF1 OE (comparisons 3 and 4), but the reference allele yielded a greater increase in expression than did the alternate allele in this context (comparison 5). Bars represent mean ± SD. Two-tailed p-values from unpaired t-tests for each comparison are shown above the associated bracket. b ChIP-qPCR was performed to determine the extent of MTF1 binding at the SNV locus (ATP7B) compared to a computationally predicted negative control (Neg Ctrl, in an intron of WDPCP) and to a previously published [32] experimentally validated locus (Pos Ctrl, in the 5′ UTR of SELENOH). Enrichment of DNA immunoprecipitated by MTF1-specific vs. non-specific isotype-matched control antibodies was measured in technical triplicate by quantitative PCR analysis. Bars represent mean ± SD. One-tailed p-values from unpaired t-tests for each comparison are shown above the associated bracket. c Proposed model for disease-causing mechanism of chr13:g.52,586,149T>C: (1) Excess intracellular accumulation of copper, Cu2+, increases (2) expression or nuclear translocation of MTF1 [12, 13]. In wildtype individuals, MTF1 then binds at chr13:g.52,586,149T (3) to recruit transcriptional machinery for upregulating ATP7B expression [34] and eliminating copper through serum ceruloplasmin and, ultimately, through the bile. We propose that the Wilson Disease (WD) patient’s homozygous single nucleotide promoter variant chr13:g.52,586,149T>C exhibits reduced affinity to MTF1. (4) The result is an insufficient ATP7B transcriptional response, consequent copper accumulation, and symptoms characteristic of Wilson Disease

Article Snippet: Polyclonal rabbit anti-MTF1 antibody (Proteintech, Rosemont, IL, USA: 25383-1-AP) was used for chromatin immunoprecipitation.

Techniques: Functional Assay, Luciferase, Over Expression, Expressing, Comparison, Activity Assay, Two Tailed Test, ChIP-qPCR, Binding Assay, Negative Control, Immunoprecipitation, Control, Real-time Polymerase Chain Reaction, One-tailed Test, Translocation Assay, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation

doi: 10.1016/j.jbc.2021.101009

Figure Lengend Snippet: APOL1 has a leucine zipper domain at the C terminus. A , top , domain structure of APOL1 showing putative transmembrane domains (PTM 0–PTM 4; see also Table S1 ) and topology based on in silico prediction. Bottom , the C-terminal 30 amino acid residues of APOL1 constitutes a leucine zipper domain, which contains both a leucine (371, 378, 385, 392) and a hydrophobic residue (368, 375, 382, 389) every seventh amino acid. Within this heptad repeat pattern (positions a–g), the hydrophobic residue occupies position a and the leucine residue occupies position d (indicated by an asterisk ). Hydrophobic residues are shown in black , charged residues in magenta , and uncharged polar residues in green . B , DrawCoil 1.0 helical wheel diagram of the APOL1 C-terminal leucine zipper domain. The a and d -position residues are expected to constitute the hydrophobic face of an amphipathic alpha-helix that is suited to form a leucine zipper type coiled-coil homodimer. Leucine zipper dimers are stabilized by the packing of d -position leucine side chains of one helix into the hole formed by four hydrophobic residues on the opposing helix, known as “knobs-into-holes” packing. Charged residues at the exterior can further stabilize dimer formation via electrostatic interactions.

Article Snippet: Recombinant APOL1 was expressed from the pNIC28 vector (Addgene) containing an N-terminal 6× His-tagged G0 APOL1 coding sequence (aa 28–398).

Techniques: In Silico

Journal: The Journal of Biological Chemistry

Article Title: Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation

doi: 10.1016/j.jbc.2021.101009

Figure Lengend Snippet: Functional effects of sequential alanine substitutions in the C-terminal leucine zipper domain of APOL1. A , leucine zipper domain amino acid sequences of G0 APOL1 and the purified APOL1 mutants, in which the indicated d -position leucines ( shaded gray ) were replaced by alanines, either entirely (LZA) or sequentially (LZA1–6). B – D , trypanosome lysis assays. Plotted is mean trypanosome survival ±SD of three replicate wells, after 24 h incubation with the indicated concentrations of G0 APOL1 or APOL1 LZA mutants. B , APOL1 LZA (L371A/L378A/L385A/L392A) was nontrypanolytic. C and D , we observed a positive correlation between the number of substituted alanines and loss of trypanolytic activity, with substitutions at the N-terminal end of the leucine zipper domain (LZA1–3) being more detrimental than those at the C-terminal end (LZA4–6). Of note, the single substitution of the most N-terminal leucine (LZA1) was enough to reduce trypanolytic activity compared with APOL1 G0, whereas substitution of only the most C-terminal leucine (LZA4) had no effect. The data plotted are from a single representative experiment (of three independent experiments), wherein all APOL1 LZA1 to 6 proteins were tested in the same 96-well plate.

Article Snippet: Recombinant APOL1 was expressed from the pNIC28 vector (Addgene) containing an N-terminal 6× His-tagged G0 APOL1 coding sequence (aa 28–398).

Techniques: Functional Assay, Purification, Lysis, Incubation, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation

doi: 10.1016/j.jbc.2021.101009

Figure Lengend Snippet: APOL1 LZA lacks channel forming activity. A , schematic of the planar lipid bilayer system. B , effect of APOL1 G0 on bilayer current as the voltage was alternated between ±20 mV. The cis side was first adjusted to pH 5.6 and then APOL1 G0 was added. The current magnitude increased slightly and then the pH was neutralized, resulting in a large increase in the current. C , a constant voltage of +20 mV was applied, and the current was recorded. With symmetric pH 7.2 conditions, 150 ng of APOL1 LZA was added to the cis side of the bilayer, which was then acidified to pH 5.6. This was followed by a slight increase in the current ( inset , expanded 4-pA current scale), indicating protein insertion into the bilayer. When the cis side was then neutralized to pH 7.2, there was a brief increase in the current magnitude and noise, but this soon subsided and the current returned to near zero. In contrast, addition of the same concentration of APOL1 G0 to the trans side resulted in the usual increase in current magnitude following trans acidification and reneutralization. The data shown are representative of at least three independent experiments.

Article Snippet: Recombinant APOL1 was expressed from the pNIC28 vector (Addgene) containing an N-terminal 6× His-tagged G0 APOL1 coding sequence (aa 28–398).

Techniques: Activity Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation

doi: 10.1016/j.jbc.2021.101009

Figure Lengend Snippet: Targeted cysteine substitution and the effect of conjugation with MTSET. A , the C-terminal amino acid sequences of APOL1 A375C and K373C proteins compared with APOL1 G0. B , helical wheel projections showing the location of the A375C ( a -position) and K373C ( f -position) within the putative leucine zipper dimer. Conjugation of positively charged MTSET (278 Da) to A375C at the dimer interface is expected to prevent dimer formation owing to steric interference and charge repulsion, whereas conjugation to the peripheral K373C ( f -position) is expected to allow dimer formation. C , i n vitro 24-h trypanolytic assay. Plotted is mean trypanosome survival ±SD APOL1 A375C and K373C proteins showed no loss of lytic activity compared with APOL1 G0. D and E , effect of preincubation with MTSET on channel formation by APOL1 K373C and A375C proteins in planar lipid bilayers. The data shown are representative of three independent experiments. D , the current was recorded as the voltage was held at +20 mV. MTSET, 1 mM, was preadded to the cis chamber and then ~175 ng/4 nM APOL1 A375C ( a -position) was added to both the cis and the trans chambers. After a 10-min reaction time, the cis chamber was acidified for ~30 s and then neutralized, but there was little evidence of channel opening. ( Inset : above, a slight increase in current at pH 5.6 indicates protein insertion into the bilayer.) Conversely, when the trans chamber (which contained APOL1 A375C but no MTSET) was then acidified and neutralized, the usual increase in channel conductance occurred. E , the above experiment was similarly performed with APOL1 K373C ( f -position) and a 250,000-fold molar excess of MTSET. The current was recorded ( upper trace ) as the voltage was manipulated ( lower trace ). Channel formation occurred normally upon acidification and neutralization (a downward deflection in the current indicates channel opening at −20 mV; the delayed current response to acid and base additions was due to a stirring delay). MTSET, [2-(trimethylammonium)ethyl] methanethiosulfonate.

Article Snippet: Recombinant APOL1 was expressed from the pNIC28 vector (Addgene) containing an N-terminal 6× His-tagged G0 APOL1 coding sequence (aa 28–398).

Techniques: Conjugation Assay, Activity Assay, Neutralization

Journal: The Journal of Biological Chemistry

Article Title: Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation

doi: 10.1016/j.jbc.2021.101009

Figure Lengend Snippet: APOL1 A375C forms pH-dependent disulfide dimers. A , C-terminal amino acid sequence alignment of APOL1 A363C ( purple ), A375C ( red ) and LZA A375C ( pink ) mutant proteins compared with APOL1 G0. Note that, unlike the A375C substitution ( a -position), the A363C substitution is outside of the leucine zipper domain. B , helical wheel projection of the leucine zipper dimer interface shows the proximity of A375C residues on opposed helices, which could allow for the formation of a disulfide bond between monomers. C , trypanosome survival (mean ± SD) is plotted after 24 h incubation with the indicated concentrations of APOL1 mutant proteins and compared with APOL1 G0. D and E , the indicated APOL1 mutant proteins were incubated in chamber buffer under the following pH conditions: (1) 2 h at pH 7.2; (2) 1 h at pH 5.6, followed by 1 h at pH 7.2; or (3) 2 h at pH 5.6. The reactions were then separated by nonreducing SDS-PAGE alongside an equivalent concentration of an untreated control protein (c), and the gel was silver stained. Note that, in addition to the APOL1 monomer band (~43 kDa, m), an APOL1 A375C ~90-kDa dimer band (d) formed specifically when the protein was exposed to first acid then neutral conditions.

Article Snippet: Recombinant APOL1 was expressed from the pNIC28 vector (Addgene) containing an N-terminal 6× His-tagged G0 APOL1 coding sequence (aa 28–398).

Techniques: Sequencing, Mutagenesis, Incubation, SDS Page, Concentration Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation

doi: 10.1016/j.jbc.2021.101009

Figure Lengend Snippet: Trypanolytic activity of cysteine-substituted and MPB-conjugated APOL1. A , possible APOL1 topology based on in silico prediction. Lumenal/extracellular ( red ), cytoplasmic ( blue ), and putative transmembrane (PTM, black ). Trypanosome lysis assays of 35 APOL1 cysteine-substituted proteins ( B – E ), or the same cysteine-substituted proteins following conjugation with MPB and purification using monomeric-avidin ( F – I ). APOL1 proteins with cysteine substitutions in the N terminus ( B and F ), MID ( C and G ), PTM 3 ( D and H ), and PTM 4/PLR plus ZIP ( E and I ) regions were assayed. Plotted is mean trypanosome survival ±SD of triplicate measurements, after incubation with the 800 ng/ml of the indicated protein. The symbol color indicates the tentative location of the substituted cysteine (extracellular, transmembrane, or cytoplasmic) as indicated in ( A ). The average lytic activity of G0 (N = 10) is shown for reference ( green ). Cysteine-substituted proteins that were inactive in the absence of MPB conjugation were not tested after conjugation. In this case, trypanosome numbers in the absence of MPB conjugation are repeated in the right-hand panel for clarity ( grey symbols ). MPB, N -(3-maleimidylpropionyl)biocytin.

Article Snippet: Recombinant APOL1 was expressed from the pNIC28 vector (Addgene) containing an N-terminal 6× His-tagged G0 APOL1 coding sequence (aa 28–398).

Techniques: Activity Assay, In Silico, Lysis, Conjugation Assay, Purification, Avidin-Biotin Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation

doi: 10.1016/j.jbc.2021.101009

Figure Lengend Snippet: Working model of APOL1 G0 channel topology and its interrogation by substituted cysteine accessibility method. A , Protter diagram of APOL1 topology incorporating in silico ( Table S1 ) and MPB modification results ( Fig. 6 ). Note that the structure of the N terminus has been recently solved whereas loop structures are unknown. Filled circles indicate Cys substitutions or Cys-MPB modifications that increased trypanosome survival to greater than 50% ( purple ), or had no effect on trypanosome lysis ( green ) when incubated with trypanosomes at 800 ng/ml for 24 h. Note that PTM 0 was excluded owing to both a marginal in silico prediction and a lack of functional inhibition when MBP was conjugated to residues in this region. B , to test the 4-TM model of the APOL1 channel, the cis versus trans accessibility of an MPB-conjugated residue can be probed by SCAM. First, a purified (and trypanolytic) MPB-conjugated APOL1 protein is allowed to form pH-dependent channels in planar lipid bilayers under standard conditions. Streptavidin (a 53-kDa tetramer) is then added to either the cis or the trans side. If the streptavidin can bind to the Cys-MPB residue it may affect some measurable property of channel function ( e.g. , channel conductance or pH gating), owing to steric effects on the channel’s three-dimensional structure. The MPB-conjugated residue is thus mapped to either the cis or the trans side of the channel based on its accessibility to cis versus trans streptavidin. Alternatively, if streptavidin addition to both sides has no effect on channel properties (either because MPB is not accessible or because binding to MPB has no measurable effect) no conclusion can be made regarding the position of Cys-MPB. In the hypothetical example shown, red MPB is conjugated to a cysteine residue that is accessible to streptavidin binding on the cis , but not the trans , side. Some of the additional positions that were tested on the hypothetical trans side, blue MPB are also shown. MPB, N -(3-maleimidylpropionyl)biocytin.

Article Snippet: Recombinant APOL1 was expressed from the pNIC28 vector (Addgene) containing an N-terminal 6× His-tagged G0 APOL1 coding sequence (aa 28–398).

Techniques: In Silico, Modification, Lysis, Incubation, Functional Assay, Inhibition, Purification, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation

doi: 10.1016/j.jbc.2021.101009

Figure Lengend Snippet: Representative effects of streptavidin addition on the conductance formed by APOL1 Cys-MPB proteins in planar lipid bilayers. In each case the voltage was set by the experimenter ( lower trace ) as the current was recorded ( upper trace ). A , starting with symmetric pH 7.2 conditions the cis side was first adjusted to pH 5.6 and then 9.3 nM APOL1 R305C-MPB was added. After allowing for bilayer insertion, the cis side was adjusted to pH 7.2 resulting in the usual conductance increase. Streptavidin (94 nM) was added to first the trans and then the cis sides. In neither case was there an appreciable effect on the conductance, meaning no conclusion can be made about the position of R305C-MPB. B , with cis pH 5.6, trans pH 7.2 conditions 9.3 nM APOL1 V244C-MPB was added and then allowed to insert into the bilayer. The cis chamber was then adjusted to pH 7.2 with KOH, resulting in the expected conductance increase. During the break in the record the cis side was perfused with chamber buffer (pH 7.2) to remove free biotinylated protein. Streptavidin (94 nM) was added first to the trans side, which had no effect on the conductance, and then to the cis side, which caused a substantial decrease in conductance, indicating that V244C-MPB is accessible to cis but not trans streptavidin. C , before the start of the record, a conductance was obtained with 9.3 nM APOL1 K373C-MPB in the usual way, and cis side was perfused with chamber buffer (pH 7.2). Streptavidin (94 nM) was then added to the trans side as indicated, which had no effect on conductance. The cis side was then acidified (pH 5.6) and neutralized (pH 7.2) to demonstrate normal pH gating. After cis addition of 94 nM streptavidin there was a significant decrease in conductance, indicating that the modified leucine zipper domain K373C-MPB is accessible to cis , but not trans , streptavidin. In all cases the data shown are representative of three independent experiments. MPB, N -(3-maleimidylpropionyl)biocytin.

Article Snippet: Recombinant APOL1 was expressed from the pNIC28 vector (Addgene) containing an N-terminal 6× His-tagged G0 APOL1 coding sequence (aa 28–398).

Techniques: Modification

Journal: The Journal of Biological Chemistry

Article Title: Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation

doi: 10.1016/j.jbc.2021.101009

Figure Lengend Snippet: The conductance formed by APOL1 G278C-MPB is affected by trans streptavidin. A , before the start of the record, APOL1 G278C-MPB (6 nM) was added to the membrane ( cis pH 5.6, trans pH 7.5) until a detectable conductance was obtained. The cis pH was then adjusted to pH 7.5 to allow for channel opening. The voltage was alternated between ±50 mV ( lower trace ) and the current was recorded as shown ( upper trace ). As is typical of APOL1, the current magnitude was greater at negative voltage than at positive voltage. This remained true after addition of streptavidin (94 nM) to the cis side as indicated. However, after streptavidin (94 nM) was added to the trans side, the voltage effect on the current was reversed: the current magnitude became greater at positive than at negative voltage. The data shown are representative of three independent experiments. B , Protter diagram showing proposed membrane topology of an APOL1 monomer subunit in the active open cation channel state. The structure of the N terminus has been recently solved whereas loop structures are unknown. Filled circles indicate the position of Cys-MPB modifications that yielded “functional” (<50% trypanosome survival, green ), or “nonfunctional” (>50% trypanosome survival, purple ) APOL1 proteins when incubated with trypanosomes at 800 ng/ml for 24 h. Asterisks mark the positions of MPB-modified cysteines that were tested for accessibility to both cis and trans streptavidin. The blue asterisk marks G278C-MPB, which affected function when incubated with trans streptavidin; the red asterisks mark residues A108C-MPB, V244C-MPB, and K373C-MPB, which affected function when incubated with cis streptavidin, and the green asterisks mark residues that did not affect function when incubated with either cis or trans streptavidin. Green residues without asterisks were not tested with streptavidin. MPB, N -(3-maleimidylpropionyl)biocytin.

Article Snippet: Recombinant APOL1 was expressed from the pNIC28 vector (Addgene) containing an N-terminal 6× His-tagged G0 APOL1 coding sequence (aa 28–398).

Techniques: Functional Assay, Incubation, Modification

Journal: The Journal of Biological Chemistry

Article Title: Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation

doi: 10.1016/j.jbc.2021.101009

Figure Lengend Snippet: Model of the open APOL1 cation channel. The channel is shown as a dimer wherein the pore-lining region of the two monomers are brought together by a coiled-coil interdigitation between the C-terminal leucine zipper domains. The leucine zipper could also allow for the formation of trimer or tetramer channels (see section). Each subunit contributes four transmembrane domains to the channel structure, with TM 1 and TM 2 constituting the membrane insertion domain and TM 4 constituting the pore-lining region (PLR). The positions of Cys-MPB residues that were mapped by substituted cysteine accessibility method analysis are indicated: A108C, V244C, G278C, and K373C. Also shown are the pore-lining residues responsible for pH gating and cation selectivity: D348, Y351, and E355 . Structure of the N terminus has been recently solved whereas loop structures are unknown.

Article Snippet: Recombinant APOL1 was expressed from the pNIC28 vector (Addgene) containing an N-terminal 6× His-tagged G0 APOL1 coding sequence (aa 28–398).

Techniques: