recombinant lsd1  (BPS Bioscience)

Journal: bioRxiv

Article Title: Disruption of ADNP-KDM1A-GTF2I complex drives neural differentiation imbalance in Helsmoortel-Van der Aa syndrome

doi: 10.1101/2025.03.06.641037

Figure Lengend Snippet: A. Complete interactome of ADNP in hNSCs derived from ChIP-MS. Nodes are colored based on their function or complex association. red: GTF2I/KDM1A complex, orange: ChAP complex, pink: EHMT1/2 complex, blue: POGZ interactors, light blue: TFIIIC complex, dark green: ATRX complex. Known physical interactions excluding ADNP were derived from Stringdb and reported. B. ChIP-seq input signal for KDM1A and GTF2I in WT hNSCs. Each row represents a 5Kb window centered on KDM1A and GTF2I peak summits, respectively. C. Distribution of peaks shared by ADNP-KDM1A-GTF2I (AKG) across different genomic features with respect to protein-coding genes. D. Heatmap of KDM1A ChIP-seq signal in ADNP WT and ADNP KO hNSCs. Each row represents a 5Kb window centered on peak summits, sorted by the KDM1A ChIP signal. Peaks are divided into ADNP-bound (top), and ADNP-free (bottom) based on effective ADNP binding in the WT line. E. Percentage of DEGs in hNSCs bound by individual AKG components or the whole complex. Significance value for each TF binding is reported. F. Heatmap representing the percentage of peaks found between pairs of ENCODE TFs, located within +2500bp and −2500bp from the TSS of protein-coding genes. A red square highlights a cluster made by the top 10,000 peaks of ADNP (“ADNP Top”) and peaks of KDM1A and GTF2I (in bold). All ADNP peaks are indicated as “ADNP All” in the bottom row of the heatmap. G. Scatterplot representing the percentage of ADNP peaks contained in ENCODE TF binding sites of H9 hESC (y-axis) and percentage of ENCODE peaks contained in CTL-FLAG iPSCs ADNP binding sites. Dots are colored by log2(%X*%Y).

Article Snippet: For KDM1A and GTF2I immunoprecipitation, 250 µL of F2V5-ADNP hNSC nuclear extract with 38 units of Benzonase (Novagen) and 1x CEF protease inhibitor (Roche) were rotated with 782 ng KDM1A antibody (Cell Signaling, 2139S) or 300 ng of GTF2I antibody (Bethyl, A301-330A) for 2 hours at 4°C in no-stick microcentrifuge tubes (Alpha Laboratories).

Techniques: Derivative Assay, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Disruption of ADNP-KDM1A-GTF2I complex drives neural differentiation imbalance in Helsmoortel-Van der Aa syndrome

doi: 10.1101/2025.03.06.641037

Figure Lengend Snippet: A. Schematic representation of the interactome of ADNP in hNSCs. Interactors are grouped by known complex identity and coloured accordingly. See also Figure S3A and Table S3 . B. Western blot analysis of immunoprecipitations with KDM1A antibody or an IgG control from hNSC nuclear extract. KDM1A (upper panel) and ADNP (lower panel) are detected by KDM1A antibody and FLAG antibody, respectively. Molecular weight (MW) markers are indicated. C. Western blot analysis of immunoprecipitations with GTF2I antibody or an IgG control from NSC nuclear extract. GTF2I (upper panel) and ADNP (lower panel) are detected by GTF2I antibody and FLAG antibody, respectively. MW markers are indicated. D. Heatmap of ADNP, KDM1A and GTF2I ChIP-seq enrichments in hNSCs. Each row represents a 5kb window centered on ADNP peak summits, sorted by the TF ChIP-seq signal. ChIP-seq input signals for each TF are shown in Figure S3B . ChIP-seq reads have been normalized by library size (1x) as reads per million coverage (RPGC). Normalized signal legend with range is reported below each heatmap. E. UpSet plot indicating the number of significant peaks shared between ADNP-KDM1A-GTF2I (AKG). For the peaks shared by all three TFs, the top DNA binding motif is shown. F. Barplot reporting the enrichment of the ADNP motif in the top 10K peaks identified for each protein (ADNP, GTF2I, KDM1A and KDM1A upon ADNP KO respectively). Motif Enrichment was calculated with Gimmemotif v0.18 upon de novo motif enrichment analysis. G. Barplot showing the portion (%) of DEGs bound by AKG (purple) or ADNP (orange) that lose KDM1A at their promoters upon ADNP KO. H. Scatter plot of H3K4me3 signal in WT (x-axis) vs KO (y-axis). Both axes report hNSC ChIP-seq reads, normalized on library size (RPGC, see Methods) and scaled on the respective input data.

Article Snippet: For KDM1A and GTF2I immunoprecipitation, 250 µL of F2V5-ADNP hNSC nuclear extract with 38 units of Benzonase (Novagen) and 1x CEF protease inhibitor (Roche) were rotated with 782 ng KDM1A antibody (Cell Signaling, 2139S) or 300 ng of GTF2I antibody (Bethyl, A301-330A) for 2 hours at 4°C in no-stick microcentrifuge tubes (Alpha Laboratories).

Techniques: Western Blot, Control, Molecular Weight, ChIP-sequencing, Binding Assay