Journal: bioRxiv

Article Title: NPM1 mediates genome-nucleolus interactions and the establishment of their repressive chromatin states

doi: 10.1101/2024.07.03.601885

Figure Lengend Snippet: a. Representation of the DNA-FISH probes targeting NADs at chromosomes 1, 2, 5, and 19, the latter containing rRNA genes at the 5’, close to the centromeric region. The corresponding NADs profiles from Nucleolar-DamID are shown. iNAD: genomic domains not interacting with nucleoli. b-d. Representative immuno-FISH images of NADs at chromosome 2 in ESCs depleted via siRNA of NPM1 (siNPM1, b ), Nucleolin (siNCL, c ), and Fibrillarin (siFBL, d ). NPM1 and Nucleolin serve as nucleolar markers. e. Quantifications of immuno-FISH analyses showing the percentage of cells displaying NADs contacting nucleoli in ESCs depleted of NPM1, Nucleolin, or Fibrillarin. Error bars represent s.d. Statistical significance ( P -values) from three independent experiments was calculated using the Mann-Whitney test (** < 0.01; *** < 0.001; ns: non-significant). f. Scheme representing the strategy to measure genomic contacts with nucleoli by Nucleolar-DamID coupled to DpnII digestion and qPCR. g. Relative resistance to DpnII digestion in ESCs expressing H2B-Dam-NoLS-DD with siRNA-Control siRNA- NPM1 . Expression of H2B-Dam-NoLS-DD was induced by treatment with doxycycline and Shield for 15 hours. Data are from two independent experiments and values obtained in each experiment are shown as black or blue circles, respectively. The mean values are shown. h. Scheme representing the detachment of NADs from nucleoli upon loss of NPM1.

Article Snippet: ESCs were co-transfected with a plasmid expressing Cas9 protein and the gRNA sequence targeting the NPM1 locus (pX330 Mouse 5’ Npm1 gRNA, a gift from Mark Groudine, Addgene plasmid #127900; http://n2t.net/addgene:127900 ; RRID:Addgene_127900) and the HDR donor plasmid derived from Mouse 5’ Npm1-AID-GFP-PuroR plasmid (a gift from Mark Groudine, Addgene plasmid #127899 ; http://n2t.net/addgene:127899 ; RRID:Addgene_127899) in which AID-GFP sequences were replaced with FLAG-HA or FKBP sequence (synthesized from IDT).

Techniques: MANN-WHITNEY, Expressing, Control

Journal: bioRxiv

Article Title: NPM1 mediates genome-nucleolus interactions and the establishment of their repressive chromatin states

doi: 10.1101/2024.07.03.601885

Figure Lengend Snippet: a. IGV track displaying NPM1 genome occupancy and NAD profiles from Nucleolar-DamID of chromosome 5 in ESCs. b. Genomic annotations of NPM1-bound regions in ESCs. Promoter corresponds to ± 3kb regions from transcription start sites. c. NPM1 distribution on NADs and LAD-only sequences in ESCs. d. Volcano plot showing fold change (log 2 values) in transcript levels of ESCs+siRNA- NPM1 vs. ESC+siRNA-Control. Gene expression values of three replicates were averaged and selected for 1.5-fold changes and P < 0.05. e. Top 10 gene ontology (GO) terms as determined using DAVID for genes upregulated and downregulated upon NPM1-KD. f. Pie chart showing the number of genes located at NADs that are significantly up- or downregulated in ESC upon NPM1-KD.

Article Snippet: ESCs were co-transfected with a plasmid expressing Cas9 protein and the gRNA sequence targeting the NPM1 locus (pX330 Mouse 5’ Npm1 gRNA, a gift from Mark Groudine, Addgene plasmid #127900; http://n2t.net/addgene:127900 ; RRID:Addgene_127900) and the HDR donor plasmid derived from Mouse 5’ Npm1-AID-GFP-PuroR plasmid (a gift from Mark Groudine, Addgene plasmid #127899 ; http://n2t.net/addgene:127899 ; RRID:Addgene_127899) in which AID-GFP sequences were replaced with FLAG-HA or FKBP sequence (synthesized from IDT).

Techniques: Control, Gene Expression

Journal: bioRxiv

Article Title: NPM1 mediates genome-nucleolus interactions and the establishment of their repressive chromatin states

doi: 10.1101/2024.07.03.601885

Figure Lengend Snippet: a. Representative immunofluorescence images showing H3K9me2 distribution in ESCs treated with siRNA-Control or siRNA- NPM1 . The magnification on the right shows the distribution of H3K9me2 around nucleoli, forming a ring-like shape, that is destroyed in the absence of NPM1. UBF serves as nucleolar marker. The corresponding DAPI staining is shown in Extended Data Fig. 3f . b. Quantification of cells showing the perinucleolar H3K9me2 ring in ESCs upon depletion of G9a, SUV39H1/2, or SETDB1. Data are from three independent experiments. Error bars represent s.d. Statistical significance ( P -values) was calculated using the Mann-Whitney test (*** < 0.001). c. Representative immunofluorescence images showing H3K9me2 distribution in ESCs upon depletion of G9a, SUV39H1/2, or SETDB1 via siRNA. UBF serves as nucleolar marker. d. Quantification of cells showing the perinucleolar H3K9me2 ring in ESCs upon depletion of NPM1, Nucleolin (NCL), or Fibrillarin (FBL). Data are from three independent experiments. Error bars represent s.d. Statistical significance ( P -values) was calculated using Mann-Whitney test (**** < 0.0001; ns: non-significant). e. Western blot showing global H3K9me2 levels in ESCs upon NPM1-KD. f. Tracks displaying H3K9me2 occupancy in ESCs treated with siRNA-Control or siRNA-NPM1 of chromosome 6 and 9 in ESCs. Lower panels show the corresponding NAD profile from Nucleolar-DamID . g. Quantifications of H3K9me2 levels at NAD-only, NAD/LAD, and LAD-only regions at 25% top H3K9me2 enriched regions in parental ESCs. Values from ESCs treated with siRNA-Control or siRNA-NPM1 are shown as average RPKM of a 10kb bin size region. Error bars represent s.d. Statistical significance ( P -values) was calculated using the paired two-tailed t test (** < 0.01; ****<0.0001; ns: non-significant). h. Pie charts showing the number of genes located at the 25% top H3K9me2 enriched regions in parental ESCs and at NAD-only or NAD/LAD regions associated with NPM1.

Article Snippet: ESCs were co-transfected with a plasmid expressing Cas9 protein and the gRNA sequence targeting the NPM1 locus (pX330 Mouse 5’ Npm1 gRNA, a gift from Mark Groudine, Addgene plasmid #127900; http://n2t.net/addgene:127900 ; RRID:Addgene_127900) and the HDR donor plasmid derived from Mouse 5’ Npm1-AID-GFP-PuroR plasmid (a gift from Mark Groudine, Addgene plasmid #127899 ; http://n2t.net/addgene:127899 ; RRID:Addgene_127899) in which AID-GFP sequences were replaced with FLAG-HA or FKBP sequence (synthesized from IDT).

Techniques: Immunofluorescence, Control, Marker, Staining, MANN-WHITNEY, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: NPM1 mediates genome-nucleolus interactions and the establishment of their repressive chromatin states

doi: 10.1101/2024.07.03.601885

Figure Lengend Snippet: a. Volcano plot showing log 2 fold changes of NPM1-immunoprecipitated peptides in ESCs relative to IgG control. Data are from three independent experiments. b. KEGG pathways and molecular functions of NPM1-interacting proteins detected by String analyses. c. HA-immunoprecipitation of HEK293T cells transfected with plasmid expressing HA-G9a. Signals were detected using antibodies against G9a and NPM1, respectively. d. Volcano plot showing fold change (log 2 values) in transcript levels of ESCs+siRNA- G9a vs. ESC+siRNA-Control. Gene expression values of three replicates were averaged and selected for 1.5-fold changes and P < 0.05. e. Left panel. Venn diagram showing the number of G9a-regulated genes that are located at NADs and bound by NPM1. Right panel shows their changes in gene expression upon G9a-KD. f. Pie chart showing the numbers of NPM1-bound genes located at the top 25% of H3K9me2 enriched chromatin regions that are regulated by G9a. g. Scheme representing NMP1-mediated NAD association with nucleoli followed by NPM1-mediated recruitment of G9a to establish H3K9me2.

Article Snippet: ESCs were co-transfected with a plasmid expressing Cas9 protein and the gRNA sequence targeting the NPM1 locus (pX330 Mouse 5’ Npm1 gRNA, a gift from Mark Groudine, Addgene plasmid #127900; http://n2t.net/addgene:127900 ; RRID:Addgene_127900) and the HDR donor plasmid derived from Mouse 5’ Npm1-AID-GFP-PuroR plasmid (a gift from Mark Groudine, Addgene plasmid #127899 ; http://n2t.net/addgene:127899 ; RRID:Addgene_127899) in which AID-GFP sequences were replaced with FLAG-HA or FKBP sequence (synthesized from IDT).

Techniques: Immunoprecipitation, Control, Transfection, Plasmid Preparation, Expressing, Gene Expression

Journal: Npj viruses

Article Title: Influenza A virus NS1 suppresses nuclear speckles promoted gene expression by inhibition of transcription.

doi: 10.1038/s44298-025-00124-x

Figure Lengend Snippet: Fig. 3 | NS1 fusion constructs. A Schematic representation of the NS1 constructs used to guide NS1 to different subcellular sites. B Western blot to prove expression of the constructs described above. GFP11: the 11th domain of GFP superfolder protein; aa: amino acid, numbers describe the sequence of the indicated protein that was fused to NS1; H2B: his- tone 2B; SRRM1: human serine and arginine repe- titive matrix protein 1; SC35: human Serine/ arginine-rich splicing factor SC35; PSPC1: human paraspeckle component 1; TFIP11: human tuftelin interacting p;rotein 11; GAPDH: calatytic domain of GAPDH; NS1-gfp11wt: wild type NS1 fused with GFP11; lifeAct: yeast lifeAct peptide that mediates binding to actin filaments; GGGS: (Gly)3Ser-pep- tide linker to enable proper secondary structure formation of the domains; aa: amino acid position. Each NS1 in these constructs is mutated at positions 38 and 41 (R38A, K41A) to inactivate the internal nuclear localisation site.

Article Snippet: Cells, plasmids, viruses, chemicals and antibodies, siRNAs Reporter plasmids CMV-LUC2CP/ARE and CMV-LUC2CP/intron/ARE (GideonDreyfuss, addgene #62857 and #62858), pQCXIP-GFP1-10 (gift of npj Viruses | (2025) 3:46 8 Yutaka Hata, addgene #68715), and pCW57.1-luc (gift from Stephen Tapscott, addgene #99283), pSBtet rtTA G72V SE Δsplice GP Luc (gift of AlexanderMcLellan addgene #194328), SB100X in pCAGglobin pA (gift of Mark Groudine, addgene #127909) were obtained from Addgene. pSV40luc. pCMV-luc and pCMV-RlucpA-IRES-FlucpA were described earlier4. mCherry2-SRRM1 was used as a source for the nuclear speckle localising domain of SRRM1 and was obtained from Addgene (#54563). pEGFPCyclin L1was used as a source for the Cyclin L1 nuclear speckle localisation domain (gift of W. Becker, Aachen) HEK293 (transfections), A549 (RNASeq, infections), or MDCK (split gfp assays) cells were used throughout this study.

Techniques: Construct, Western Blot, Expressing, Sequencing, Binding Assay

Journal: Npj viruses

Article Title: Influenza A virus NS1 suppresses nuclear speckles promoted gene expression by inhibition of transcription.

doi: 10.1038/s44298-025-00124-x

Figure Lengend Snippet: Fig. 4 | Fluorescence microscopical analysis shows the subcellular localisations of NS1 fusion- proteins (as described in Fig. 2). NS1 fusion pro- teins were visualised in vivo using the split-GFP assay. SRRM1: Nuclear speckle localising domain (NSLD) of human serine and arginine repetitive matrix protein 1; SC35: NSLD of human Serine/ arginine-rich splicing factor SC35; PSPC1: NSLD of human paraspeckle component 1; lifeAct: yeast lifeAct peptide that mediates binding to actin fila- ments; 2HB: histone 2B; TFIP11: NSLD of human tuftelin interacting protein 11. A representative picture is shown for each transfected plasmid.

Article Snippet: Cells, plasmids, viruses, chemicals and antibodies, siRNAs Reporter plasmids CMV-LUC2CP/ARE and CMV-LUC2CP/intron/ARE (GideonDreyfuss, addgene #62857 and #62858), pQCXIP-GFP1-10 (gift of npj Viruses | (2025) 3:46 8 Yutaka Hata, addgene #68715), and pCW57.1-luc (gift from Stephen Tapscott, addgene #99283), pSBtet rtTA G72V SE Δsplice GP Luc (gift of AlexanderMcLellan addgene #194328), SB100X in pCAGglobin pA (gift of Mark Groudine, addgene #127909) were obtained from Addgene. pSV40luc. pCMV-luc and pCMV-RlucpA-IRES-FlucpA were described earlier4. mCherry2-SRRM1 was used as a source for the nuclear speckle localising domain of SRRM1 and was obtained from Addgene (#54563). pEGFPCyclin L1was used as a source for the Cyclin L1 nuclear speckle localisation domain (gift of W. Becker, Aachen) HEK293 (transfections), A549 (RNASeq, infections), or MDCK (split gfp assays) cells were used throughout this study.

Techniques: Fluorescence, In Vivo, Split GFP Assay, Binding Assay, Transfection, Plasmid Preparation

Journal: Npj viruses

Article Title: Influenza A virus NS1 suppresses nuclear speckles promoted gene expression by inhibition of transcription.

doi: 10.1038/s44298-025-00124-x

Figure Lengend Snippet: Fig. 6 | Expression of the NS1 effector domain is sufficient for the deregulation of nuclear speckle-localised reporter gene expression. NS1 N-terminal and C-terminal deletion mutants reveal that the region between amino acids (aa) 78 and 200 is sufficient for NS1-mediated transcriptional inhibition when it is guided to NSP. The number indicates the aa region of NS1 that was fused to the speckle localising domain of SRRM1 and co-transfected with a reporter gene construct. The reporter gene activity measured from extracts of GAPDH (instead of a NS1 fusion) transfected cells were arbitrarily set 1. The experiments were repeated five times. *p < 0.05 compared to GAPDH control

Article Snippet: Cells, plasmids, viruses, chemicals and antibodies, siRNAs Reporter plasmids CMV-LUC2CP/ARE and CMV-LUC2CP/intron/ARE (GideonDreyfuss, addgene #62857 and #62858), pQCXIP-GFP1-10 (gift of npj Viruses | (2025) 3:46 8 Yutaka Hata, addgene #68715), and pCW57.1-luc (gift from Stephen Tapscott, addgene #99283), pSBtet rtTA G72V SE Δsplice GP Luc (gift of AlexanderMcLellan addgene #194328), SB100X in pCAGglobin pA (gift of Mark Groudine, addgene #127909) were obtained from Addgene. pSV40luc. pCMV-luc and pCMV-RlucpA-IRES-FlucpA were described earlier4. mCherry2-SRRM1 was used as a source for the nuclear speckle localising domain of SRRM1 and was obtained from Addgene (#54563). pEGFPCyclin L1was used as a source for the Cyclin L1 nuclear speckle localisation domain (gift of W. Becker, Aachen) HEK293 (transfections), A549 (RNASeq, infections), or MDCK (split gfp assays) cells were used throughout this study.

Techniques: Expressing, Gene Expression, Inhibition, Transfection, Construct, Activity Assay, Control