Journal: Bone Research

Article Title: H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

doi: 10.1038/s41413-021-00148-y

Figure Lengend Snippet: Mice with Nsd1 knockout in mesenchymal progenitors showed impaired cartilage development. a mRNA levels of H3K36 methyltransferases and chondrocyte differentiation marker genes were determined by qRT-PCR in micromasses at different differentiation time points. The values are presented as the means ± SEMs, n = 4. * P < 0.05, ** P < 0.01, ns means not significant. The inset shows Alcian blue staining results of micromasses cultured for 1, 4, and 7 days with chondroprogenitor cells. Scale bar = 2 mm. b SO staining results of E11.5 limb buds. Scale bar = 100 μm. c Whole-mount in situ hybridization (WISH) results for Col2 in E12.5 embryos (top) and sections of forelimbs (bottom). The dashed purple lines show the digits already present. Scale bar (top) = 500 μm, scale bar (bottom) = 200 μm. d SO staining results of E13.5 (first line), E14.5 (second line), E15.5 (third line), and E16.5 (fourth line) femur sections from WT and Nsd1 f/f ;Prx1-Cre mice. The dashed black lines show the borders between hypertrophic chondrocytes and the primary ossification center. Scale bar = 200 μm. SO staining of P7 ( e ) and P14 ( f ) hindlimb sections from WT and Nsd1 f/f ;Prx1-Cre mice. Scale bar (top) = 200 μm. Scale bar (bottom) = 500 μm. g Whole-mount in situ hybridization (WISH) results for Col2 in E12.5 embryos (top) and sections of forelimbs (bottom). The dashed purple lines show the digits already present. Scale bar (top) = 500 μm, scale bar (bottom) = 200 μm. h SO staining results of E13.5 (top) and E15.5 (bottom) femur sections from WT, Nsd1 f/f ;Col2-Cre mice. The dashed black lines show the borders between hypertrophic chondrocytes and the primary ossification center. Scale bar = 200 μm. SO staining of P7 ( i ) and P14 ( j ) hindlimb sections from WT and Nsd1 f/f ;Col2-Cre mice. Scale bar (top) = 200 μm, Scale bar (bottom) = 500 μm

Article Snippet: Antibodies specific for the following molecules were used: NSD1 (Bioss, bs-8170R), COL2 (Abcam, ab34712), H3K36me1 (Abcam, ab9048), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), SOX9 (Millipore, AB5535), HIF1α (WB: Novus, NB100-134; IF: Bioss, bs-0737R), and Flag (Sigma, F1804).

Techniques: Knock-Out, Marker, Quantitative RT-PCR, Staining, Cell Culture, In Situ Hybridization

Journal: Bone Research

Article Title: H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

doi: 10.1038/s41413-021-00148-y

Figure Lengend Snippet: Nsd1 deficiency in mesenchymal progenitors led to skeletal growth defects in mice. Gross images of 1-month-old WT, Nsd1 f/f ;Prx1-Cre ( a ) and Nsd1 f/f ;Col2-Cre ( b ) mice. Pictures of hindlimbs (left) and quantitative statistics (right) of hindlimb length in 1-month-old WT, Nsd1 f/f ;Prx1-Cre ( c ) and Nsd1 f/f ;Col2-Cre ( d ) mice. Scale bar = 2 mm. The values are presented as the means ± SEMs, n = 6. * P < 0.05, ns means not significant. Safranin O (SO) staining results ( e , g ) and growth plate quantification data ( f , h ) of tibia sections from 1-month-old WT, Nsd1 f/f ;Prx1-Cre ( e , f ) and Nsd1 f/f ;Col2-Cre ( g , h ) mice. Scale bar (top) = 100 μm. Scale bar (bottom) = 50 μm. The values are presented as the means ± SEMs, n = 6. * P < 0.05, ** P < 0.01, ns means not significant

Article Snippet: Antibodies specific for the following molecules were used: NSD1 (Bioss, bs-8170R), COL2 (Abcam, ab34712), H3K36me1 (Abcam, ab9048), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), SOX9 (Millipore, AB5535), HIF1α (WB: Novus, NB100-134; IF: Bioss, bs-0737R), and Flag (Sigma, F1804).

Techniques: Staining

Journal: Bone Research

Article Title: H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

doi: 10.1038/s41413-021-00148-y

Figure Lengend Snippet: Mice with Nsd1 knockout in mesenchymal progenitors showed impaired fracture healing. a Radiographs of fractured femurs from WT and Nsd1 f/f ;Prx1-Cre mice at different days post fracture (dpf). b Quantitative analysis of formed calluses at different days post fracture (dpf). n = 5. Alcian blue/eosin staining ( c ) and quantitative results ( d ) of callus sections. The dashed black lines show the location of the callus. Scale bar = 500 μm. n = 5. Immunofluorescence staining ( e ) and quantitative results ( f ) of type II collagen in callus sections. The dashed white lines show the location of the callus. Scale bar= 50 µm . n = 5. g Micro-CT images of calluses in WT and Nsd1 f/f ;Prx1-Cre mice at 18 dpf. h Quantitative statistics of micro-CT results of calluses. n = 3. i Radiographs of fractured femurs in WT and Nsd1 f/f ;Col2-Cre mice at different days post fracture (dpf). j Quantitative analysis of formed calluses at different days post fracture (dpf). n = 5. Alcian blue/eosin staining ( k ) and quantitative results ( l ) of callus sections. The dashed black lines show the location of the callus. Scale bar = 500 μm. n = 5. m Micro-CT images of calluses in WT and Nsd1 f/f ;Col2-Cre mice at 21 dpf. n Quantitative statistics of micro-CT results of calluses. BV bone volume, BS bone surface, Tb.N trabecular bone number. n = 3. The values are presented as the means ± SEMs. * P < 0.05, ** P < 0.01, ns means not significant

Article Snippet: Antibodies specific for the following molecules were used: NSD1 (Bioss, bs-8170R), COL2 (Abcam, ab34712), H3K36me1 (Abcam, ab9048), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), SOX9 (Millipore, AB5535), HIF1α (WB: Novus, NB100-134; IF: Bioss, bs-0737R), and Flag (Sigma, F1804).

Techniques: Knock-Out, Staining, Immunofluorescence, Micro-CT

Journal: Bone Research

Article Title: H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

doi: 10.1038/s41413-021-00148-y

Figure Lengend Snippet: Chondroprogenitor cells with Nsd1 knockout showed impaired chondrocyte differentiation and increased proliferation. a Gross images of pellets formed by chondroprogenitor cells from neonatal mice. Scale bar = 1 mm. b HE staining (top), SO staining (middle), and Col2 in situ hybridization (bottom) results of sections from pellets formed by chondroprogenitor cells. Scale bar = 20 μm. c Alcian blue staining results of micromasses cultured for 1, 4, and 7 days with chondroprogenitor cells. Scale bar = 2 mm. d Quantitative analysis of Alcian blue staining. The values are presented as the means ± SEMs, n = 4. ** P < 0.05, ns means not significant. qRT-PCR results for Nsd1 ( e ) and chondrocyte differentiation marker genes, including Sox9 ( f ), Col2 ( g ), and Acan ( h ), in micromasses cultured for 1, 4, and 7 days with chondroprogenitor cells. The values are presented as the means ± SEMs, n = 4. * P < 0.05, ** P < 0.01, ns means not significant. i Crystal violet staining results of chondroprogenitor cells cultured for 1, 3, 5, and 7 days. Scale bar = 5 mm. j Quantification of crystal violet staining. The values are presented as the means ± SEMs, n = 6. ** P < 0.01, ns means not significant

Article Snippet: Antibodies specific for the following molecules were used: NSD1 (Bioss, bs-8170R), COL2 (Abcam, ab34712), H3K36me1 (Abcam, ab9048), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), SOX9 (Millipore, AB5535), HIF1α (WB: Novus, NB100-134; IF: Bioss, bs-0737R), and Flag (Sigma, F1804).

Techniques: Knock-Out, Staining, In Situ Hybridization, Cell Culture, Quantitative RT-PCR, Marker

Journal: Bone Research

Article Title: H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

doi: 10.1038/s41413-021-00148-y

Figure Lengend Snippet: Sox9 was regulated by NSD1 through H3K36 methylation. a Heat map of RNA-seq results for Egfp - and Cre -expressing immortalized Nsd1 f/f chondroprogenitor cells. b Pie chart showing the percentages of differentially expressed genes between Egfp and Cre samples. c Normalized reads of H3K36me2 ChIP-seq analyses in Egfp - and Cre -expressing immortalized Nsd1 f/f chondroprogenitor cells from 2 kb upstream of the TSS to 2 kb downstream of the TSS in the genome. d Venn diagram showing the numbers of genes with decreased expression in RNA-seq data (pink), genes with decreased H3K36me2 occupancy in ChIP-seq data (green), and overlapping genes (yellow). e Heat map and annotation of transcription factors from the set of overlapping genes. f Western blot analysis of SOX9 in Egfp - and Cre -expressing immortalized Nsd1 f/f chondroprogenitor cells. g Immunohistochemical assay of SOX9 in growth plate sections from P7 mice. Scale bar (left) = 100 μm, scale bar (right) = 5 μm. h H3K36me2 binding peaks on Sox9 in Egfp - and Cre -expressing immortalized Nsd1 f/f chondroprogenitor cells from the H3K36me2 ChIP-seq assay. i ChIP-PCR assay of H3K36me1 (left) and H3K36me2 (right) occupancy of Sox9 . The values are presented as the means ± SEMs, n = 3. * P < 0.05, ** P < 0.01, ns means not significant. j Alcian blue staining results of micromass culture with chondroprogenitor cells without or with Sox9 overexpression. Scale bar = 2 mm. k qRT-PCR results of Sox9 , Col2 , and Acan in micromass culture. The values are presented as the means ± SEMs, n = 4. * P < 0.05, ** P < 0.01

Article Snippet: Antibodies specific for the following molecules were used: NSD1 (Bioss, bs-8170R), COL2 (Abcam, ab34712), H3K36me1 (Abcam, ab9048), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), SOX9 (Millipore, AB5535), HIF1α (WB: Novus, NB100-134; IF: Bioss, bs-0737R), and Flag (Sigma, F1804).

Techniques: Methylation, RNA Sequencing Assay, Expressing, ChIP-sequencing, Western Blot, Immunohistochemical staining, Binding Assay, Staining, Over Expression, Quantitative RT-PCR

Journal: Bone Research

Article Title: H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

doi: 10.1038/s41413-021-00148-y

Figure Lengend Snippet: NSD1 directly regulated Hif1α . a Heat map of Hif1α and its target genes from the RNA-seq results of Egfp - and Cre -expressing immortalized Nsd1 f/f chondroprogenitor cells. b Western blot analysis of the HIF1α level in Egfp - and Cre -expressing immortalized Nsd1 f/f chondroprogenitor cells. c Immunofluorescence analysis of HIF1α in limb buds of E15.5 mice. Scale bar = 100 μm. d NSD1 binding peaks on Hif1α in ATDC5 cells from the Flag-NSD1 ChIP-seq assay. e ChIP-PCR assay of NSD1 binding on Hif1α . The values are presented as the means ± SEMs, n = 3. ** P < 0.01, ns means not significant. f Luciferase assay of the NSD1-specific binding (NSB) region in the Hif1α promoter in C3H10 cells treated with NSD1. The values are presented as the means ± SEMs, n = 3. * P < 0.05, ** P < 0.01, ns means not significant. g Model that summarizes our findings on the role of NSD1 in regulating Sox9 directly and indirectly. On the one hand, NSD1 can directly promote Sox9 expression by regulating the levels of H3K36me1/2 in the Sox9 promoter region. On the other hand, NSD1 directly binds to the promoter region of Hif1α , activating Hif1α transcription and ultimately promoting Sox9 expression

Article Snippet: Antibodies specific for the following molecules were used: NSD1 (Bioss, bs-8170R), COL2 (Abcam, ab34712), H3K36me1 (Abcam, ab9048), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), SOX9 (Millipore, AB5535), HIF1α (WB: Novus, NB100-134; IF: Bioss, bs-0737R), and Flag (Sigma, F1804).

Techniques: RNA Sequencing Assay, Expressing, Western Blot, Immunofluorescence, Binding Assay, ChIP-sequencing, Luciferase

Journal: Physiological Genomics

Article Title: Human gene copy number spectra analysis in congenital heart malformations

doi: 10.1152/physiolgenomics.00013.2012

Figure Lengend Snippet: Known CHD risk genes

Article Snippet: NSD1 , NUCLEAR RECEPTOR-BINDING Su-var, ENHANCER OF ZESTE, AND TRITHORAX , 5q35.2–5q35.3 , 176492685 , 167135 , Hs00053100_cn , S , 606681 , 15742365, 20193066.

Techniques: Retroviral, Sequencing, Virus, Reverse Transcription

Journal: Physiological Genomics

Article Title: Human gene copy number spectra analysis in congenital heart malformations

doi: 10.1152/physiolgenomics.00013.2012

Figure Lengend Snippet: Case reports of likely causal CNVs

Article Snippet: NSD1 , NUCLEAR RECEPTOR-BINDING Su-var, ENHANCER OF ZESTE, AND TRITHORAX , 5q35.2–5q35.3 , 176492685 , 167135 , Hs00053100_cn , S , 606681 , 15742365, 20193066.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Neuroinflammation regulates the balance between hippocampal neuron death and neurogenesis in an ex vivo model of thiamine deficiency

doi: 10.1186/s12974-022-02624-6

Figure Lengend Snippet: Neuroinflammation regulate the balance between hippocampal neuron death and neurogenesis in TD. NPC: neural progenitor cells; TD: thiamine deficiency; TPP thiamine pyrophosphate; H2A: histone 2A; H2B: histone 2B; H3: histone 3; H4: histone 4; K: lysine; R: arginine; P: phosphorylation; Ub: ubiquitination; Ac: acetylation; Me: methylation; H3K9me3: trimethylation in lysine 9 at histone 3; K3K4me3: trimethylation in lysine 4 at histone 3; TK: transketolase; PDHC: pyruvate dehydrogenase complex; Ogdh : oxoglutarate dehydrogenase; KGDHC: alfa-ketoglutarate dehydrogenase complex; PI3K/AKT: phosphatidylinositol 3-kinase/protein kinase B; BDNF: brain derived neurotrophic factor; H3K36me3: trimethylation in lysine 36 at histone 3; NSD1: nuclear receptor binding SET domain protein 1; SETD2: SET domain containing 2; Bmp4 : bone morphogenetic protein 4

Article Snippet: Rat-specific TaqMan Gene Expression Assays (Applied Biosystems) were used to detect Nsd1 (Nuclear Receptor Binding SET Domain Protein 1) (Rn01441947_m1), Bmp4 (Bone Morphogenetic Protein 4) (Rn00432087_m1), Hes5 (Hes Family BHLH Transcription Factor 5) (Rn00821207_g), Ogdh (oxoglutarate dehydrogenase) (Rn01443655_m1), Mmp9 (Matrix Metalloproteinase 9) (Rn00579162_m1), Dcx (Doublecortin) (Rn00570390_m1), Neurod1 (Neuronal Differentiation 1) (Rn01280117_m1), C3 (Complement C3) (Rn00566466_m1) and Lcn2 (Lipocalin 2) (Rn00590612_m1).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Methylation, Derivative Assay, Binding Assay

Journal: Physiological Genomics

Article Title: Human gene copy number spectra analysis in congenital heart malformations

doi: 10.1152/physiolgenomics.00013.2012

Figure Lengend Snippet: Known CHD risk genes

Article Snippet: , , , , , Hs00022652_cn , , , .

Techniques: Retroviral, Sequencing, Virus, Reverse Transcription

Journal: Physiological Genomics

Article Title: Human gene copy number spectra analysis in congenital heart malformations

doi: 10.1152/physiolgenomics.00013.2012

Figure Lengend Snippet: Case reports of likely causal CNVs

Article Snippet: , , , , , Hs00022652_cn , , , .

Techniques:

Journal: Science Advances

Article Title: The H3K36me2 writer-reader dependency in H3K27M-DIPG

doi: 10.1126/sciadv.abg7444

Figure Lengend Snippet: ( A ) Left: Western blot of H3K36me2, H3K36me2, histone H3, and Flag in DIPG4 (H3K27M) cells expressing doxycycline-inducible, flag-tagged H3-WT or H3K36M constructs. Doxycycline (Dox; 0, 1, or 5 ng/ml) was administrated for an induction of ectopic histones. Cell lysates were harvested 3 days after induction. Right: An MTT assay was conducted for assessing the proliferation of cells treated with respective conditions in the left panel. Cells were seeded 3 days after induction and the MTT assay was conducted 6 days after induction. ( B ) Western blot of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), H3K36me2, H3K36me3, and histone H3 in DIPG4 cells transfected with indicated siRNAs, including NSD1 and NSD2, NSD3, ASH1L, or SETD2. ( C ) Western blot of NSD1, NSD2, GAPDH, H3K36me2, and histone H3 in DIPG10 (H3-WT), DIPG6 (H3K27M), and DIPG13 (H3K27M) cells transfected with and without siRNAs against NSD1 and/or NSD2. Asterisks indicate nonspecific bands detected by an anti-NSD1 antibody (Bethyl Laboratories, no. A300-BL715). Cell lysates were harvested 3 days after transfection, and GAPDH was used as a loading control in (B) and (C). ( D ) Proliferation assays of DIPG10, DIPG6, and DIPG13 cells stably expressing control or shRNAs against NSD1 and/or NSD2. Cell numbers were counted after 2, 4, and 6 days. ( E ) Western blot of LEDGF, HDGF2, GAPDH, H3K36me2, and histone H3 in DIPG4 cells stably expressing control or shRNAs against LEDGF and/or HDGF2. ( F ) Proliferation assays of DIPG4 cells used in (E) and DIPG13 cells with the same conditions. rTTA, reverse tetracycline-controlled transactivator; EV, empty vector. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s t test.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Proteintech) rabbit polyclonal, catalog no. 25504-1-AP; HDGF2 (Proteintech) rabbit polyclonal, catalog no. 15134-1-AP; H3K27me3 (Cell Signaling Technology) rabbit monoclonal C36B11, catalog no. 9733; H3K36me2 (Cell Signaling Technology) rabbit monoclonal C75H12, catalog no. 2901; H3K36me3 (Cell Signaling Technology) rabbit monoclonal D5A7, catalog no. 4909; NSD1 (Bethyl Laboratories) rabbit monoclonal, catalog no. A300-BL715 (discontinued); NSD1 (University of California, Davis/National Institutes of Health NeuroMab Facility) mouse monoclonal, N312/10; NSD2 (Millipore) mouse monoclonal 29D1, catalog no. MABE191; NSD3 (Cell Signaling Technology) rabbit monoclonal D4N9N, catalog no. 92056; ASH1L (Bethyl Laboratories) rabbit polyclonal, catalog no. A301-748A; SETD2 (Bio-Rad) mouse monoclonal OTI1E1, catalog no. VMA00449; glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology) rabbit monoclonal D16H11, catalog no. 5174; histone H3 (Abcam) rabbit monoclonal EPR16987, catalog no. ab176842; anti-Flag (Sigma-Aldrich) mouse monoclonal M2, catalog no. F1804; and H2Av (Active Motif) rabbit polyclonal, catalog no. 39715.

Techniques: Western Blot, Expressing, Construct, MTT Assay, Transfection, Control, Stable Transfection, Plasmid Preparation

Journal: Science Advances

Article Title: The H3K36me2 writer-reader dependency in H3K27M-DIPG

doi: 10.1126/sciadv.abg7444

Figure Lengend Snippet: ( A ) Left: A Kaplan-Meier survival curve plot of mice bearing xenograft tumors with endpoints defined by a sign of distress. DIPG13 cells stably expressing a firefly luciferase and control or shRNAs against NSD1 and/or NSD2 were implanted in the cortex of NSG mice by intracranial injection. A total of 250,000 cells were implanted in each mouse ( n = 10 mice for each condition). Right: A quantification of firefly luciferase signals in mice described in the left panel. Bioluminescent imaging (BLI) data were presented at day 35 after injection before the first mouse exhibited a sign of distress. Bottom: Representative BLI images from indicated conditions. ( B ) Left: A Kaplan-Meier survival curve of mice implanted with DIPG13 cells stably expressing control or shRNAs against LEDGF and/or HDGF2 in the same experimental conditions described in (A) ( n = 10 mice for control; n = 5 for shLEDGF; n = 4 for shHDGF2; and n = 9 for shLEDGF + shHDGF2). Right: A quantification of BLI signals in mice describe in the left panel. Bottom: Representative BLI images from indicated conditions. * P < 0.05 and *** P < 0.001 by log-rank test; n.s., not significant ( P > 0.05) for Kaplan-Meier survival analysis. * P < 0.05, ** P < 0.01 and *** P < 0.001 by a nonparametric Mann-Whitney test for BLI quantifications.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Proteintech) rabbit polyclonal, catalog no. 25504-1-AP; HDGF2 (Proteintech) rabbit polyclonal, catalog no. 15134-1-AP; H3K27me3 (Cell Signaling Technology) rabbit monoclonal C36B11, catalog no. 9733; H3K36me2 (Cell Signaling Technology) rabbit monoclonal C75H12, catalog no. 2901; H3K36me3 (Cell Signaling Technology) rabbit monoclonal D5A7, catalog no. 4909; NSD1 (Bethyl Laboratories) rabbit monoclonal, catalog no. A300-BL715 (discontinued); NSD1 (University of California, Davis/National Institutes of Health NeuroMab Facility) mouse monoclonal, N312/10; NSD2 (Millipore) mouse monoclonal 29D1, catalog no. MABE191; NSD3 (Cell Signaling Technology) rabbit monoclonal D4N9N, catalog no. 92056; ASH1L (Bethyl Laboratories) rabbit polyclonal, catalog no. A301-748A; SETD2 (Bio-Rad) mouse monoclonal OTI1E1, catalog no. VMA00449; glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology) rabbit monoclonal D16H11, catalog no. 5174; histone H3 (Abcam) rabbit monoclonal EPR16987, catalog no. ab176842; anti-Flag (Sigma-Aldrich) mouse monoclonal M2, catalog no. F1804; and H2Av (Active Motif) rabbit polyclonal, catalog no. 39715.

Techniques: Stable Transfection, Expressing, Luciferase, Control, Injection, Imaging, MANN-WHITNEY