Journal: bioRxiv

Article Title: Paraspeckle Protein NONO Regulates Active Chromatin by Allosterically Stimulating NSD1

doi: 10.1101/2025.01.21.634125

Figure Lengend Snippet: (A) Schematic illustration of annotated and putative functional domains of NSD1. NID, nuclear receptor interaction domain; PWWP, Pro-Trp-Trp-Pro domain; RBD, RNA-binding domain; PHD, plant homeodomain; AWS, associated with SET domain (also refereed as pre-SET); SET, su(var), enhancer of zeste, trithorax domain. (B) Western blot of NSD1, GAPDH, and H3K36me2 for WT or NSD1/2-dKO HEK293T cells stably rescued with WT or NSD1 mutants. (C) Top, meta-analysis profiling of genome-wide NSD1 and H3K36me2 ChIP-seq signals within −10kb of TSS to +10kb of TES. Bottom, representative track images of NSD1-WT or NSD1 ΔPWWP2 in HEK293T NSD1/2-dKO rescued background. TSS, transcription start site; TES, transcription end site. (D) Western blot of NSD1, GAPDH, and H3K36me2 for WT of NSD1/2-dKO HEK293T cells stably rescued with NSD1 or NSD1 PWWP2–4A (E) Neural Progenitor Cell (NPC) differentiation of E14-mESC cells with indicated genotypes. Top, representative images of Embryoid Bodies (EBs) undergoing NPC differentiation after 3 days of Retinoic Acid (RA) treatment. Bottom, quantifications of differentiating and non-differentiating EBs. (F) Western blot of Nestin, GAPDH, and H3K36me2 for E14-mESC cells undergoing EB and NPC differentiation.

Article Snippet: Flag-NSD1-WT, Flag-NSD1-PWWP2-4A, Flag-NONO, Flag-N-NONO (aa1-217) were cloned into pFASTBac1 (Invitrogen) and used to produce P0 polyclonal baculovirus according to Invitrogen guideline for the Bac-to-Bac system.

Techniques: Functional Assay, RNA Binding Assay, Western Blot, Stable Transfection, Genome Wide, ChIP-sequencing

Journal: bioRxiv

Article Title: Paraspeckle Protein NONO Regulates Active Chromatin by Allosterically Stimulating NSD1

doi: 10.1101/2025.01.21.634125

Figure Lengend Snippet: (A) Schematic illustration of experimental flows for proteomics analysis of NSD1-PWWP2’s interactomes. (B) Unique proteins detected by LC-MS and plotted by peptide-spectrum match (PSM) scores against percentage of coverage using DIPG13 (top) and HEK293T (bottom) cells. (C) Illustration of annotated functional domains of NONO. (D) GST pulldown assay of HA-tagged NONO using NSD1-PWWP2 as the bait. Left, pulldown of HA-tagged N-NONO or C-NONO using GST alone or GST-NSD1-PWWP2 followed by Western blot of GST and HA. Right, pulldown of HA-tagged N-NONO using GST-NSD1-PWWP2 or GST-NSD1-PWWP2-4A mutant followed by western blot of GST and HA.

Article Snippet: Flag-NSD1-WT, Flag-NSD1-PWWP2-4A, Flag-NONO, Flag-N-NONO (aa1-217) were cloned into pFASTBac1 (Invitrogen) and used to produce P0 polyclonal baculovirus according to Invitrogen guideline for the Bac-to-Bac system.

Techniques: Liquid Chromatography with Mass Spectroscopy, Functional Assay, GST Pulldown Assay, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Paraspeckle Protein NONO Regulates Active Chromatin by Allosterically Stimulating NSD1

doi: 10.1101/2025.01.21.634125

Figure Lengend Snippet: (A) Demonstration of recombinant protein expression and purification including NSD1, NSD1 PWWP2–4A , N-NONO, and recombinant di-nucleosomes by Coomassie blue staining. (B) HMT assays of full-length NSD1 or NSD1 PWWP2–4A mutant in an incremental titration of 62.5, 125, and 250nM. Top, quantifications of autoradiographic signals normalized to NSD1 alone (the second lane). Middle, representative autoradiographic images for stably incorporated [ 3 H]. Bottom, Coomassie blue staining of total nucleosomes. (C) HMT assays of 60nM full-length NSD1 with an incremental titration of N-NONO at 0, 530, 880, and 1760nM (left) or 0.25μM NSD1 PWWP2–4A mutant with an incremental titration of N-NONO at 0, 880, and 1760nM (right). Top, quantifications of autoradiographic signals normalized to NSD1 alone (the second lane). Middle, representative autoradiographic images for stably incorporated [ 3 H]. Bottom, Coomassie blue staining of total nucleosomes.

Article Snippet: Flag-NSD1-WT, Flag-NSD1-PWWP2-4A, Flag-NONO, Flag-N-NONO (aa1-217) were cloned into pFASTBac1 (Invitrogen) and used to produce P0 polyclonal baculovirus according to Invitrogen guideline for the Bac-to-Bac system.

Techniques: Recombinant, Expressing, Purification, Staining, Mutagenesis, Titration, Stable Transfection

Journal: bioRxiv

Article Title: Paraspeckle Protein NONO Regulates Active Chromatin by Allosterically Stimulating NSD1

doi: 10.1101/2025.01.21.634125

Figure Lengend Snippet: (A) Meta-analysis profiling of H3K36me2 ChIP-seq signals aligned to all genes within a window of −10kb of TSS to +10kb of TES in WT and NONO-KO E14-mESC. Representative track images are shown at the bottom. (B) Left, immunofluorescence staining of NONO in WT and NEAT1 CRISPRi HEK293T cells. Images were captured under a 63X objective and puncta of nuclear paraspeckles were highlighted by red triangles. Right, quantifications of nuclear paraspeckles present in individual WT (n=24) and NEAT1 CRISPRi (n=40) HEK293T cells. The P value is calculated by Chi-squared test. (C) Top, meta-analysis profiling of H3K36me2 ChIP-seq signals in WT and NEAT1 CRISPRi cells aligned to all genes within the same window indicated in (A). Bottom, representative track images of H3K36me2 ChIP-seq in these cells. (D) Neural Progenitor Cell (NPC) differentiation of WT, NSD1-KO, and NONO-KO E14-mESC cells. Top, representative images of Embryoid Bodies (EBs) undergoing NPC differentiation after 3 days of Retinoic Acid (RA) treatment. Bottom, quantifications of fully differentiated, partially differentiated, or non-differentiated EBs. (E) Heatmaps of differential gene expression analysis in WT, NSD1-KO, and NONO-KO cells treated with RA for 0, 3, or 6 days using RNA-seq. 252 genes associated with neural development and 102 genes associated with stem cell differentiation were presented. (F) Box plots of log2 fold changes in gene expression using the experimental conditions shown in (D). The box and whisker represent 95%, third quartile, median, first quartile, and 5% distribution of genes. P values were calculated by Wilcoxon test **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: Flag-NSD1-WT, Flag-NSD1-PWWP2-4A, Flag-NONO, Flag-N-NONO (aa1-217) were cloned into pFASTBac1 (Invitrogen) and used to produce P0 polyclonal baculovirus according to Invitrogen guideline for the Bac-to-Bac system.

Techniques: ChIP-sequencing, Immunofluorescence, Staining, Expressing, RNA Sequencing Assay, Cell Differentiation, Whisker Assay

Journal: bioRxiv

Article Title: Paraspeckle Protein NONO Regulates Active Chromatin by Allosterically Stimulating NSD1

doi: 10.1101/2025.01.21.634125

Figure Lengend Snippet: (A) Schematic illustration of annotated and putative functional domains of NSD1. NID, nuclear receptor interaction domain; PWWP, Pro-Trp-Trp-Pro domain; RBD, RNA-binding domain; PHD, plant homeodomain; AWS, associated with SET domain (also refereed as pre-SET); SET, su(var), enhancer of zeste, trithorax domain. (B) Western blot of NSD1, GAPDH, and H3K36me2 for WT or NSD1/2-dKO HEK293T cells stably rescued with WT or NSD1 mutants. (C) Top, meta-analysis profiling of genome-wide NSD1 and H3K36me2 ChIP-seq signals within −10kb of TSS to +10kb of TES. Bottom, representative track images of NSD1-WT or NSD1 ΔPWWP2 in HEK293T NSD1/2-dKO rescued background. TSS, transcription start site; TES, transcription end site. (D) Western blot of NSD1, GAPDH, and H3K36me2 for WT of NSD1/2-dKO HEK293T cells stably rescued with NSD1 or NSD1 PWWP2–4A (E) Neural Progenitor Cell (NPC) differentiation of E14-mESC cells with indicated genotypes. Top, representative images of Embryoid Bodies (EBs) undergoing NPC differentiation after 3 days of Retinoic Acid (RA) treatment. Bottom, quantifications of differentiating and non-differentiating EBs. (F) Western blot of Nestin, GAPDH, and H3K36me2 for E14-mESC cells undergoing EB and NPC differentiation.

Article Snippet: In brief, P0 polyclonal baculovirus with Flag-NSD1-WT or Flag-NSD1-PWW2-4A inserts were prepared according to Invitrogen guidelines.

Techniques: Functional Assay, RNA Binding Assay, Western Blot, Stable Transfection, Genome Wide, ChIP-sequencing

Journal: bioRxiv

Article Title: Paraspeckle Protein NONO Regulates Active Chromatin by Allosterically Stimulating NSD1

doi: 10.1101/2025.01.21.634125

Figure Lengend Snippet: (A) Schematic illustration of experimental flows for proteomics analysis of NSD1-PWWP2’s interactomes. (B) Unique proteins detected by LC-MS and plotted by peptide-spectrum match (PSM) scores against percentage of coverage using DIPG13 (top) and HEK293T (bottom) cells. (C) Illustration of annotated functional domains of NONO. (D) GST pulldown assay of HA-tagged NONO using NSD1-PWWP2 as the bait. Left, pulldown of HA-tagged N-NONO or C-NONO using GST alone or GST-NSD1-PWWP2 followed by Western blot of GST and HA. Right, pulldown of HA-tagged N-NONO using GST-NSD1-PWWP2 or GST-NSD1-PWWP2-4A mutant followed by western blot of GST and HA.

Article Snippet: In brief, P0 polyclonal baculovirus with Flag-NSD1-WT or Flag-NSD1-PWW2-4A inserts were prepared according to Invitrogen guidelines.

Techniques: Liquid Chromatography with Mass Spectroscopy, Functional Assay, GST Pulldown Assay, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Paraspeckle Protein NONO Regulates Active Chromatin by Allosterically Stimulating NSD1

doi: 10.1101/2025.01.21.634125

Figure Lengend Snippet: (A) Demonstration of recombinant protein expression and purification including NSD1, NSD1 PWWP2–4A , N-NONO, and recombinant di-nucleosomes by Coomassie blue staining. (B) HMT assays of full-length NSD1 or NSD1 PWWP2–4A mutant in an incremental titration of 62.5, 125, and 250nM. Top, quantifications of autoradiographic signals normalized to NSD1 alone (the second lane). Middle, representative autoradiographic images for stably incorporated [ 3 H]. Bottom, Coomassie blue staining of total nucleosomes. (C) HMT assays of 60nM full-length NSD1 with an incremental titration of N-NONO at 0, 530, 880, and 1760nM (left) or 0.25μM NSD1 PWWP2–4A mutant with an incremental titration of N-NONO at 0, 880, and 1760nM (right). Top, quantifications of autoradiographic signals normalized to NSD1 alone (the second lane). Middle, representative autoradiographic images for stably incorporated [ 3 H]. Bottom, Coomassie blue staining of total nucleosomes.

Article Snippet: In brief, P0 polyclonal baculovirus with Flag-NSD1-WT or Flag-NSD1-PWW2-4A inserts were prepared according to Invitrogen guidelines.

Techniques: Recombinant, Expressing, Purification, Staining, Mutagenesis, Titration, Stable Transfection

Journal: bioRxiv

Article Title: Paraspeckle Protein NONO Regulates Active Chromatin by Allosterically Stimulating NSD1

doi: 10.1101/2025.01.21.634125

Figure Lengend Snippet: (A) Meta-analysis profiling of H3K36me2 ChIP-seq signals aligned to all genes within a window of −10kb of TSS to +10kb of TES in WT and NONO-KO E14-mESC. Representative track images are shown at the bottom. (B) Left, immunofluorescence staining of NONO in WT and NEAT1 CRISPRi HEK293T cells. Images were captured under a 63X objective and puncta of nuclear paraspeckles were highlighted by red triangles. Right, quantifications of nuclear paraspeckles present in individual WT (n=24) and NEAT1 CRISPRi (n=40) HEK293T cells. The P value is calculated by Chi-squared test. (C) Top, meta-analysis profiling of H3K36me2 ChIP-seq signals in WT and NEAT1 CRISPRi cells aligned to all genes within the same window indicated in (A). Bottom, representative track images of H3K36me2 ChIP-seq in these cells. (D) Neural Progenitor Cell (NPC) differentiation of WT, NSD1-KO, and NONO-KO E14-mESC cells. Top, representative images of Embryoid Bodies (EBs) undergoing NPC differentiation after 3 days of Retinoic Acid (RA) treatment. Bottom, quantifications of fully differentiated, partially differentiated, or non-differentiated EBs. (E) Heatmaps of differential gene expression analysis in WT, NSD1-KO, and NONO-KO cells treated with RA for 0, 3, or 6 days using RNA-seq. 252 genes associated with neural development and 102 genes associated with stem cell differentiation were presented. (F) Box plots of log2 fold changes in gene expression using the experimental conditions shown in (D). The box and whisker represent 95%, third quartile, median, first quartile, and 5% distribution of genes. P values were calculated by Wilcoxon test **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: In brief, P0 polyclonal baculovirus with Flag-NSD1-WT or Flag-NSD1-PWW2-4A inserts were prepared according to Invitrogen guidelines.

Techniques: ChIP-sequencing, Immunofluorescence, Staining, Expressing, RNA Sequencing Assay, Cell Differentiation, Whisker Assay