Journal: bioRxiv

Article Title: Loss of NAT10 disrupts enhancer organization via p300 mislocalization and suppresses transcription of genes necessary for metastasis progression

doi: 10.1101/2024.01.24.577116

Figure Lengend Snippet: (a) Morphology of Nat10 shRNA knockdown (top) and Nat10 KO 4T1 cells grown on different plates (bottom). Scale bar = 10 μm. (b) Hypothetical model of the interaction between NAT10 and the NUP210-bound mechanosensitive protein complex at the nuclear pore. (c) Co-IP showing the interaction of NAT10 with Myc-tagged NUP210 and SIPA1 in 4T1 cells. (d) Co-IP showing the interaction of Flag-tagged NAT10 and BRD4 isoforms in 4T1 cells. (e) and (f) Reciprocal Co-IP showing the interaction of Myc-tagged NAT10 and Flag-tagged BRD4 isoforms in human 293FT cells. (g) Proximity ligation assay showing the interactions (red dots) of NUP210 with NAT10 and SIPA1. Scale bar = 5 μm. (h) Western blot showing the level of NUP210 and ITGB1 protein in Nat10 KO 4T1 cells. (i) qRT-PCR showing the level of NUP210-dependent mechanosensitive, inflammatory response genes in Nat10 KO 4T1 cells, multiple t-test, mean ± s.e.m. (j) Immunofluorescence showing the distribution of histone H3.1/3.2 and H3K9me3 heterochromatin markers in Nat10 KO 4T1 cells. Scale bar = 10 μm. (k) Western blot showing the levels of NAT10 and associated mechanosensitive proteins in 4T1 cells grown on plates with soft (0.2kPa) and stiff (plastic dish, stiffness > GPa) matrices coated with either fibronectin or type I collagen.

Article Snippet: The antibodies used included anti-rabbit H3K27ac (0.16 μg) (Cell Signaling Technology), anti-rabbit H3K27me3 (0.51 μg) (Cell Signaling Technology), anti-rabbit H4K20ac (5 μg) (Millipore-Sigma), and anti-rabbit p300 antibody (0.57 μg) (Cell Signaling Technology), anti-rabbit Brd4 (0.5 μg) (Cell Signaling Technology), anti-rabbit Nup210 (2 μg) (Bethyl Laboratories), anti-rabbit Rrp1b (1 μg) (Millipore-Sigma), anti-rabbit HA (5 μg) (Cell Signaling Technology), anti-rabbit V5 (1:40 dilution) (Cell Signaling Technology), and anti-rabbit SIPA (10 μg) (Abcam).

Techniques: shRNA, Knockdown, Co-Immunoprecipitation Assay, Proximity Ligation Assay, Western Blot, Quantitative RT-PCR, Immunofluorescence

Journal: bioRxiv

Article Title: Loss of NAT10 disrupts enhancer organization via p300 mislocalization and suppresses transcription of genes necessary for metastasis progression

doi: 10.1101/2024.01.24.577116

Figure Lengend Snippet: (a) Co-IP showing the interaction of Flag-tagged and endogenous NAT10 with p300 in 4T1 cells. (b) Immunofluorescence showing the colocalization of NAT10 with p300 and enhancer mark H3K27ac, Scalebar = 5 μm. NAT10-Ms, mouse NAT10 antibody; NAT10-Rb, rabbit NAT10 antibody. (c) Proximity ligation assay showing the interaction of NAT10 with H3K27ac and H4K20ac marks in 4T1 cells, Scalebar = 10 μm. (d) 3D reconstruction showing NAT10 interactions with H3K27ac and H4K20ac within the nucleus of 4T1 cells, Scalebar = 2 μm. (e) Normalized ChIP-seq profile of NAT10 enrichment with active enhancer marks (H3K27ac and H4K20ac) in 4T1 cells. (f) IGV plot showing the ChIP enrichment of NAT10 with members of the nuclear pore-associated mechanosensitive protein complex at the Myc super-enhancer region. BRD4_short_V5 = V5-tagged short isoform of BRD4, RRP1B_HA = HA-tagged RRP1B, RRP1B_endo = endogenous RRP1B.

Article Snippet: The antibodies used included anti-rabbit H3K27ac (0.16 μg) (Cell Signaling Technology), anti-rabbit H3K27me3 (0.51 μg) (Cell Signaling Technology), anti-rabbit H4K20ac (5 μg) (Millipore-Sigma), and anti-rabbit p300 antibody (0.57 μg) (Cell Signaling Technology), anti-rabbit Brd4 (0.5 μg) (Cell Signaling Technology), anti-rabbit Nup210 (2 μg) (Bethyl Laboratories), anti-rabbit Rrp1b (1 μg) (Millipore-Sigma), anti-rabbit HA (5 μg) (Cell Signaling Technology), anti-rabbit V5 (1:40 dilution) (Cell Signaling Technology), and anti-rabbit SIPA (10 μg) (Abcam).

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence, Proximity Ligation Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: KDM3A and KDM3B Maintain Naïve Pluripotency Through the Regulation of Alternative Splicing

doi: 10.1101/2023.05.31.543088

Figure Lengend Snippet: Rescue of alternative splicing defects caused by loss of KDM3A/KDM3B does not depend on catalytic activity of KDM3A. (A) Schematic (top) and timeline (bottom) of KDM3A full length (FL) or KDM3A catalytic mutant (CM) overexpression rescue experiment. (B) Immunoblot for KDM3A, FLAG, and TUBLUIN (loading control) in WT ESCs or Degron clones untransduced (U), transduced with full length KDM3A (FL), or transduced with catalytic mutant KDM3A (CM). (C) Semi-quantitative PCR of splicing targets in WT ESCs and Degron clones at 4 hours of degradation, each untransduced (U), transduced with full length KDM3A (FL), or transduced with catalytic mutant KDM3A (CM). Representative IGV tracks depicting affected exon (red box) are below. (D) Electrophoresis image from 5C was quantified using Licor software, fluorescence intensity reported as arbitrary units (a.u.) and inclusion/skipped ratio calculated and represented in right panel. I=included ratio and S=Skipped ratio.

Article Snippet: Briefly, Kdm3a microhomology arms were added to the pCRIS-PITChv2-Puro-dTAG (Addgene #91793) donor vector and Kdm3b microhomology arms were cloned into the pCRIS-PITChv2-BSD-dTAG (Addgene #91792) donor vector to allow for dual selection with puromycin and blasticidin S hydrochloride.

Techniques: Alternative Splicing, Activity Assay, Mutagenesis, Over Expression, Western Blot, Control, Clone Assay, Transduction, Real-time Polymerase Chain Reaction, Electrophoresis, Software, Fluorescence

Journal: bioRxiv

Article Title: KDM3A and KDM3B Maintain Naïve Pluripotency Through the Regulation of Alternative Splicing

doi: 10.1101/2023.05.31.543088

Figure Lengend Snippet: Loss of KDM3A and KDM3B leads to differences in stable naïve state. (A) Schematic detailing derivation of naïve ESCs (serum/LIF) or ground ESCs (2i/LIF). (B) Scatterplot of splicing events called by rMATs in both WT vs DDeg+ ESCs as well as serum vs 2i/LIF ESCs measured by inclusion level difference score. Pearson correlation between the inclusion level difference of specific events called in both comparisons. Blue dots represent events chosen for further confirmation. (C) Semi-quantitative PCR of splicing targets in WT ESCs, DDeg+ clones at 4 hours of degradation, and 2i/LIF ESCs. Representative IGV tracks depicting affected exon (red box) are below. Electrophoresis image was quantified using Licor software, fluorescence intensity reported as arbitrary units (a.u.) and represented in right panel. I=included isoform and S=Skipped isoform. (D) Relative expression of c-Myc or Dazl in WT or DDeg+ ESCs in serum/LIF or 2i/LIF conditions. WT cells in serum/LIF set to 1.

Article Snippet: Briefly, Kdm3a microhomology arms were added to the pCRIS-PITChv2-Puro-dTAG (Addgene #91793) donor vector and Kdm3b microhomology arms were cloned into the pCRIS-PITChv2-BSD-dTAG (Addgene #91792) donor vector to allow for dual selection with puromycin and blasticidin S hydrochloride.

Techniques: Real-time Polymerase Chain Reaction, Clone Assay, Electrophoresis, Software, Fluorescence, Expressing

Journal: bioRxiv

Article Title: KDM3A and KDM3B Maintain Naïve Pluripotency Through the Regulation of Alternative Splicing

doi: 10.1101/2023.05.31.543088

Figure Lengend Snippet: Alternative splicing defects upon acute loss of KDM3A/KDM3B occur in signaling and chromatin related genes. (A) Venn overlap of DE Genes (blue) and aberrantly spliced genes (yellow) called from rMATs and Dexseq analysis. (B) Left panel: schematic of alternative splicing pathways, results above the dotted line are from rMATs and below are from Dexseq. Right panel: Number of significant events called in each class of alternative splicing pathway, FDR<0.1, inclusion level difference x>+/−0.5, and 15 read minimum. (C) Venn overlap of aberrantly spliced genes sorted by higher inclusion in WT (blue) or in DDeg+ (red). Gene ontology categories for aberrantly spliced genes shown, minimum p-value < 0.001. (D) IGV tracks for example genes of exons with higher percent spliced in (PSI) for WT or DDeg+ conditions. PSI value calculated in rMATs from 3 replicates in each condition. Negative value indicates event is included more in the WT condition and vice versa. The affected exon is marked in red. (E) Clustering (k-means=5) of differentially spliced events by inclusion level difference score generated in rMATs. WT vs uninduced degron (DDeg-) and WT vs induced degron (DDeg+) lines are shown. A positive number indicates greater inclusion in WT cells. Example genes from clusters and significant gene ontology terms (minimum p-value < 0.001) are in right panel. (F) ChIP-qPCR of WT (E14) and inducible KDM3A/KDM3B double degron (DDeg) ESCs uninduced or treated for 4 hours with dTAG-13. Left, H3K9me2 ChIP-qPCR. Right, H3K36me3 ChIP-qPCR. Three technical replicates performed for each condition which include alternate and constitutive spliced exons. Fold change determined by dividing alternative exon replicate over constitutive exon replicate.

Article Snippet: Briefly, Kdm3a microhomology arms were added to the pCRIS-PITChv2-Puro-dTAG (Addgene #91793) donor vector and Kdm3b microhomology arms were cloned into the pCRIS-PITChv2-BSD-dTAG (Addgene #91792) donor vector to allow for dual selection with puromycin and blasticidin S hydrochloride.

Techniques: Alternative Splicing, Generated, ChIP-qPCR

Journal: bioRxiv

Article Title: KDM3A and KDM3B Maintain Naïve Pluripotency Through the Regulation of Alternative Splicing

doi: 10.1101/2023.05.31.543088

Figure Lengend Snippet: Acute degradation of KDM3A and KDM3B leads to differential gene expression agnostic of H3K9me2 status. (A) Number of differentially expressed genes (DEG) between Double Degron (DDeg+) exposed to dTAG-13 ESCs and wildtype ESCs. Gene ontology categories for DEGs shown at minimum p-value < 0.00001. (B) Boxplot of absolute expression (TPMs) in ESCs for all annotated genes from RSEM quantification with a non-zero median TPM (All expressed genes), TPMs from up or down DEGs. Numbers indicate number of genes per condition. ****p-value < 0.0001 assessed by unpaired Welch’s t-test. (C) Boxplot of average TPMs+1 from DEGs with (H3K9me2) or without (No H3K9me2) a called consensus H3K9me2 peak ( , ). *p-value < 0.05 assessed by unpaired Welch’s t-test (D) Left: Scatterplot of log2 (DDeg+/WT) fold change of TPMs of Non-DE and DEG against H3K9me2 enrichment (normalized read counts per million and by 1kb per gene) at those genes. Right: Boxplot of total H3K9me2 enrichment at up or down DEG. (E) Four-way Venn overlap of DEG and H3K9me2 peaks in WT ESCs and KDM3A and KDM3B DKO ESCs .

Article Snippet: Briefly, Kdm3a microhomology arms were added to the pCRIS-PITChv2-Puro-dTAG (Addgene #91793) donor vector and Kdm3b microhomology arms were cloned into the pCRIS-PITChv2-BSD-dTAG (Addgene #91792) donor vector to allow for dual selection with puromycin and blasticidin S hydrochloride.

Techniques: Gene Expression, Expressing

Journal: bioRxiv

Article Title: KDM3A and KDM3B Maintain Naïve Pluripotency Through the Regulation of Alternative Splicing

doi: 10.1101/2023.05.31.543088

Figure Lengend Snippet: KDM3A and KDM3B interact with splicing machinery in ESCs (A) Immunoblot of (i) KDM3A, (ii) FLAG in ESCs with doxycycline (dox)-inducible expression of FLAG-KDM3A. Loading control = RNA polymerase II (RNAPII). OE=Overexpression (B) Same as above except in dox-inducible FLAG-KDM3B ESCs (i) KDM3B (ii) FLAG (iii) FLAG following immunoprecipitation with FLAG antibody. I=Input, UB=Unbound, E=Elute (C) Venn diagram of KDM3A and KDM3B interactome. Filtered for nuclear compartment using DAVID GO ( https://david.ncifcrf.gov/ ) analysis and frequent FLAG pulldown contaminants using “Crapome” ( https://reprint-apms.org/ ) repository at SAINT score of 0.7 . Gene Ontology (GO) enrichment of shared interactome are listed at enrichment score>2.5 and p-value<.00001. (D) String network analysis ( https://string-db.org/ ) of KDM3A and KDM3B shared interactome with high confidence interaction scores (0.7 minimum) from database and experimental sources. Node size represents fold change (FC) of KDM3A over uninduced spectral counts. FC>10 = large node, 10>FC>5 = medium node, FC<5 = small node. Inset a and b included for better view of interacting splicing machinery. A few small node protein labels are removed to improve legibility. See for all interacting proteins. (E) KDM3A and KDM3B interactors overlapped with functional categories of mRNP components from Singh et al. or categorized as other mRNA processing proteins. Shared= black, KDM3A unique=blue, and KDM3B unique=red. (F) Single slice of 1 uM confocal images of in situ Duolink ™ Proximity ligation assay (PLA) of (i) KDM3B single antibody, EFTUD2 single antibody, and KDM3B+EFTUD2 antibodies in WT ESCs or KDM3B+EFTUD2 antibodies in KDM3B KO ESCs. (ii) PLA of KDM3B and OCT4 in WT ESCs. Scale bar = 50μM with 60x objective. (iii) PLA foci and nuclei were quantified in ImageJ of one colony per field of view. The ratio of foci per nuclei is reported for each colony. (G) Immunoprecipitation (IP) with HA antibody or mouse IgG with immunoblot (IB) for KDM3A, SNRNP200, PRMT5, and EFTUD2. I=Input, U=Unbound, PE=Peptide Elute, BE=Boil Elute. Dotted line represents a cropped image.

Article Snippet: Briefly, Kdm3a microhomology arms were added to the pCRIS-PITChv2-Puro-dTAG (Addgene #91793) donor vector and Kdm3b microhomology arms were cloned into the pCRIS-PITChv2-BSD-dTAG (Addgene #91792) donor vector to allow for dual selection with puromycin and blasticidin S hydrochloride.

Techniques: Western Blot, Expressing, Control, Over Expression, Immunoprecipitation, Functional Assay, In Situ, Proximity Ligation Assay

Journal: bioRxiv

Article Title: KDM3A and KDM3B Maintain Naïve Pluripotency Through the Regulation of Alternative Splicing

doi: 10.1101/2023.05.31.543088

Figure Lengend Snippet: Inducible double knockout of KDM3A and KDM3B results in delayed global accumulation of H3K9me2. (A) Schematic of gene editing strategy to generate degron inducible KDM3A and KDM3B proteins. (B) Immunoblot for (i) KDM3A or (ii) KDM3B in WT ESCs or KDM3A/KDM3B degron ESCs. Note increased size of degron tagged protein. dTAG-13 ligand induced samples (+) treated for 24 hours. (i) Loading control= EFTUD and (ii) Loading control = TUBULIN (C) Immunoblot for KDM3A, KDM3B, and TUBULIN (loading control) in less than one hour of dTAG-13 induced degradation. (D) Immunoblot of H3K9me2, H3K9me3, and H3 (loading control) in WT ESCs and degron ESCs at indicated timepoints of degradation.

Article Snippet: Briefly, Kdm3a microhomology arms were added to the pCRIS-PITChv2-Puro-dTAG (Addgene #91793) donor vector and Kdm3b microhomology arms were cloned into the pCRIS-PITChv2-BSD-dTAG (Addgene #91792) donor vector to allow for dual selection with puromycin and blasticidin S hydrochloride.

Techniques: Double Knockout, Western Blot, Control