Journal: Cell Death & Disease

Article Title: UHRF genes regulate programmed interdigital tissue regression and chondrogenesis in the embryonic limb

doi: 10.1038/s41419-019-1575-4

Figure Lengend Snippet: Expression of Uhrf1 ( a – e ) and Uhrf2 ( f – j ) in the interdigital tissue and joint forming regions of chick leg buds.Whole mount in situ hybridizations at id 5 ( a ), 4.5 ( f ), 5.5 ( b and g ), 6.5 ( c , h ), and 7.5 ( d , i ). Arrowheads in g and h show a characteristic absence of Uhrf2 transcripts in the subridge mesoderm. e and j are in situ hybridizations in vibratome tissue sections at id 7.5 to show the joint domains (arrows) of Uhrf1 ( e ), and Uhrf2 ( j ). k and l are charts showing the expression levels of Uhrf1 ( k ) and Uhrf2 ( l ) in the course of interdigit tissue remodeling, considering a value of 1 for id 5.5. Note the decreased expression of Uhrf1 between id 5.5 and 6. m BrdU incorporation to mark mitosis in the autopod at id 6. Note the almost absence of cells labeled in the interdigital tissue (ID) compared with the digit regions ( d ). n Western blotting showing the UHRF1 and UHRF2 proteins in the interdigital tissue at id 5.5 and 7.5 the stage preceding and the peak of degeneration. o – r illustrations of the most characteristic features of the third interdigit in autopods at the peak of degeneration (id 7.5). o Senescence-specific beta-galactosidase activity. Note positivity in the interdigit (ID) and in the developing joints (arrows); p interdigit after neutral red vital staining; q immunolabeling with anti-γ-H2AX showing interdigital cells undergoing DNA damage. Digit rays are labeled red with anti-Sox9; r transmission electron microscopic image to show the abundance of dark apoptotic cells in the interdigital mesoderm. *** p < 0.001. Bars = 200 µm ( a – j , m , o – q ). Bar = 15 µm ( r )

Article Snippet: The following antibodies were employed: rabbit monoclonal anti-UHRF1 (DSG8E, Cell Signaling), rabbit polyclonal anti-UHRF2 (TA337863, OriGene); mouse monoclonal anti-UHRF2 (sc-398953, Santa Cruz Biotechnology); mouse monoclonal anti-5-methylcytosine (5-mC; 33D3, Eurogentech); rabbit polyclonal anti-SOX9 (AB5535,Milipore); mouse monoclonal anti-γH2AX (JBW301, Milipore-Upstate); and mouse monoclonal anti-BdrU (BU-33, Sigma Aldrich).

Techniques: Expressing, In Situ, BrdU Incorporation Assay, Labeling, Western Blot, Activity Assay, Staining, Immunolabeling, Transmission Assay

Journal: Cell Death & Disease

Article Title: UHRF genes regulate programmed interdigital tissue regression and chondrogenesis in the embryonic limb

doi: 10.1038/s41419-019-1575-4

Figure Lengend Snippet: a – d show the expression of Uhrf1 at pc days 12 ( a ), 13 ( b ), 13.5 ( c ), and 14 ( d ). Note the presence of joint domains by pc 14 (arrows in d ). e – h show the expression of Uhrf2 at pc days 12.5 ( e ),13 ( f ), 13.5 ( g ), and 14 ( h ). Note the fading of the interdigital domains in the distal subectodermal region (arrows in e and f ). Bars = 500 µm

Article Snippet: The following antibodies were employed: rabbit monoclonal anti-UHRF1 (DSG8E, Cell Signaling), rabbit polyclonal anti-UHRF2 (TA337863, OriGene); mouse monoclonal anti-UHRF2 (sc-398953, Santa Cruz Biotechnology); mouse monoclonal anti-5-methylcytosine (5-mC; 33D3, Eurogentech); rabbit polyclonal anti-SOX9 (AB5535,Milipore); mouse monoclonal anti-γH2AX (JBW301, Milipore-Upstate); and mouse monoclonal anti-BdrU (BU-33, Sigma Aldrich).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: UHRF genes regulate programmed interdigital tissue regression and chondrogenesis in the embryonic limb

doi: 10.1038/s41419-019-1575-4

Figure Lengend Snippet: UHRF2 immunolabeling (red) of chick ( a , c , d ) and mouse ( b ) interdigital cells.( a – a ´´) UHRF2 (red) in combination with phalloidin (green) showing the occurrence of both nuclear and cytoplasmic (arrow) localization of UHRF2. ( a ) UHRF2 labeling, ( a ´) phallodin labeling showing cytoplasmic actin; ( a ´´) merged images. ( b – b ´´) UHRF2(red) in combination 5-mC immunolabeling (green) to show the presence of UHRF2 positivity in the core of 5-mC rings (arrows). ( b ) UHRF2 labeling; ( b ´) 5-mC immunolabeling; ( b ´´) merged images. ( c – c ´´) Double immunolabeling for UHRF1 (red) and 5-mC (green), to show the increased immunolabeling in cells with reduced DNA methylation (arrows). ( c ) UHRF2 labeling; ( c ´) 5-mC immunolabeling; ( c ´´) merged image. ( d – d ´´) Immunolabeling of UHRF1 (red) in combination with TUNEL (green) to show the loss of UHRF2 in apoptotic cells TUNEL-positive (arrows). ( d ) UHRF1 labeling; ( d ´) TUNEL labeling; ( d ´´) merged image. Bar = 10 µm

Article Snippet: The following antibodies were employed: rabbit monoclonal anti-UHRF1 (DSG8E, Cell Signaling), rabbit polyclonal anti-UHRF2 (TA337863, OriGene); mouse monoclonal anti-UHRF2 (sc-398953, Santa Cruz Biotechnology); mouse monoclonal anti-5-methylcytosine (5-mC; 33D3, Eurogentech); rabbit polyclonal anti-SOX9 (AB5535,Milipore); mouse monoclonal anti-γH2AX (JBW301, Milipore-Upstate); and mouse monoclonal anti-BdrU (BU-33, Sigma Aldrich).

Techniques: Immunolabeling, Labeling, DNA Methylation Assay, TUNEL Assay

Journal: Cell Death & Disease

Article Title: UHRF genes regulate programmed interdigital tissue regression and chondrogenesis in the embryonic limb

doi: 10.1038/s41419-019-1575-4

Figure Lengend Snippet: a, b Expression of Fgf8 in the limb marginal ectoderm (AER) at id 5.5 ( a ) and 7 ( b ). c q-PCR analysis of the expression decay of Fgf8 gene expression from 5.5 to 7.5 id in samples of the interdigital tissue. d , e expression of Fgf10 in the autopods at id 5.5 ( e ) and 7,5 ( f ). f q-PCR analysis of the expression decay of Fgf10 gene expression from 5.5 to 7.5 id in samples of the interdigital tissue. g – j Regulation of Uhrf1 by FGF signaling. g – i expression of Uhrf1 in control interdigit at id 7.5 ( g ); 12 h after implantation of a FGF-bead (arrow, h ); and 12 h after implantation of a SU5402 bead (arrow, h ). I q-PCR analysis of the regulation of Uhrf1 in control interdigits and 12 h after implantation of a FGF2 bead and SU5402 beads. k – n Regulation of Uhrf2 by FGF signaling. k – m expression of Uhrf2 in control interdigit at id 7.5 ( k ); 12 h after implantation of a FGF-bead (arrow, l ); and 12 h after implantation of a SU5402 bead (arrow, m ). n q-PCR analysis of the regulation of Uhrf2 in control interdigits and 12 h after implantation of a FGF2 bead and SU5402 beads. *** p < 0.001; ** p < 0.01; * p < 0.05 versus control. ### p < 0.001; ## p < 0.01 between the two treatments. Bars = 200 µm

Article Snippet: The following antibodies were employed: rabbit monoclonal anti-UHRF1 (DSG8E, Cell Signaling), rabbit polyclonal anti-UHRF2 (TA337863, OriGene); mouse monoclonal anti-UHRF2 (sc-398953, Santa Cruz Biotechnology); mouse monoclonal anti-5-methylcytosine (5-mC; 33D3, Eurogentech); rabbit polyclonal anti-SOX9 (AB5535,Milipore); mouse monoclonal anti-γH2AX (JBW301, Milipore-Upstate); and mouse monoclonal anti-BdrU (BU-33, Sigma Aldrich).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: UHRF genes regulate programmed interdigital tissue regression and chondrogenesis in the embryonic limb

doi: 10.1038/s41419-019-1575-4

Figure Lengend Snippet: a–c Limb skeletal progenitors cultured at high density (micromass) for 2 ( a ), 3, ( b ) and 4 ( c ) days stained with Alcian blue for chondrogenesis. Note the increase in the size of the cartilage nodules stained in blue. Bar = 200 µm. d q-PCR quantification of Uhrf1 gene expression in micromass cultures of 1, 2, and 4 days. Note the downregulation of Uhrf1 during the course of chondrogenic differentiation. e q-PCR quantification of Uhrf2 gene expression in micromass cultures of 1, 2, and 4. In contrast to Uhrf1 , Uhrf2 is not downregulated in the differentiating cultures. f–i evaluation of cartilage differentiation by guanidine–HCl extraction of Alcian blue dye (illustrated in the lower row of pictures) in functional experiments of Uhrf genes gain-of-function ( f and h ) and loss-of-function ( g and i ). Note the inhibition of differentiation after the overexpression of Uhrf1 ( f ) and Uhrf2 ( h ), and the increased chondrogenesis after knockdown of Uhrf1 ( g ) and Uhrf2 ( i ). *** p < 0.001; ** p < 0.01; * p < 0.05 treated versus control

Article Snippet: The following antibodies were employed: rabbit monoclonal anti-UHRF1 (DSG8E, Cell Signaling), rabbit polyclonal anti-UHRF2 (TA337863, OriGene); mouse monoclonal anti-UHRF2 (sc-398953, Santa Cruz Biotechnology); mouse monoclonal anti-5-methylcytosine (5-mC; 33D3, Eurogentech); rabbit polyclonal anti-SOX9 (AB5535,Milipore); mouse monoclonal anti-γH2AX (JBW301, Milipore-Upstate); and mouse monoclonal anti-BdrU (BU-33, Sigma Aldrich).

Techniques: Cell Culture, Staining, Expressing, Functional Assay, Inhibition, Over Expression

Journal: Cell Death & Disease

Article Title: UHRF genes regulate programmed interdigital tissue regression and chondrogenesis in the embryonic limb

doi: 10.1038/s41419-019-1575-4

Figure Lengend Snippet: a Chart showing differences in the intensity of cell death evaluated by flow cytometry between control progenitors and progenitors overexpressing (oe) or subjected to silencing (shRNAi) of Uhrf1 (white columns) and Uhrf2 (gray columns).The level of cell death in control cultures was considered 100% and is represented by the dotted line. b Graphic illustrations comparing the proportion of cells at different cell cycle stages in cultures overexpressing Uhrf1 (white columns) and after gene silencing (gray columns) versus control cultures (represented by the dotted line). c Graphic illustrations comparing the proportion of cells at different cell cycle stages in cultures overexpressing Uhrf2 (white columns) and after gene silencing (gray columns) versus control cultures (represented by the dotted line). *** p < 0.001; ** p < 0.01; * p < 0.05 treated versus control

Article Snippet: The following antibodies were employed: rabbit monoclonal anti-UHRF1 (DSG8E, Cell Signaling), rabbit polyclonal anti-UHRF2 (TA337863, OriGene); mouse monoclonal anti-UHRF2 (sc-398953, Santa Cruz Biotechnology); mouse monoclonal anti-5-methylcytosine (5-mC; 33D3, Eurogentech); rabbit polyclonal anti-SOX9 (AB5535,Milipore); mouse monoclonal anti-γH2AX (JBW301, Milipore-Upstate); and mouse monoclonal anti-BdrU (BU-33, Sigma Aldrich).

Techniques: Flow Cytometry

Journal: Cell Death & Disease

Article Title: UHRF genes regulate programmed interdigital tissue regression and chondrogenesis in the embryonic limb

doi: 10.1038/s41419-019-1575-4

Figure Lengend Snippet: Transcriptional analysis of limb progenitors subjected to gain-of-function and loss-of-function of either Uhrf1 or Uhrf2 genes

Article Snippet: The following antibodies were employed: rabbit monoclonal anti-UHRF1 (DSG8E, Cell Signaling), rabbit polyclonal anti-UHRF2 (TA337863, OriGene); mouse monoclonal anti-UHRF2 (sc-398953, Santa Cruz Biotechnology); mouse monoclonal anti-5-methylcytosine (5-mC; 33D3, Eurogentech); rabbit polyclonal anti-SOX9 (AB5535,Milipore); mouse monoclonal anti-γH2AX (JBW301, Milipore-Upstate); and mouse monoclonal anti-BdrU (BU-33, Sigma Aldrich).

Techniques: Marker

Journal: Cell Death & Disease

Article Title: UHRF genes regulate programmed interdigital tissue regression and chondrogenesis in the embryonic limb

doi: 10.1038/s41419-019-1575-4

Figure Lengend Snippet: a Chart showing differences in global methylation evaluated by ELISA between control progenitors and progenitors overexpressing (oe) or subjected to silencing (shRNAi) of Uhrf1 (white columns) and Uhrf2 (gray columns).The level of global methylation in control cultures was considered 100% and is represented by the dotted line. b Methylation level of CpG islands in the Bak1 promoter evaluated by MSRE-qPCR between control progenitors and progenitors overexpressing (oe) or subjected to silencing (shRNAi) of Uhrf1 (white columns) and Uhrf2 (gray columns). The level of CpG methylation in control cultures was considered 100% and is represented by the dotted line. *** p < 0.001; ** p < 0.01; * p < 0.05 treated versus control

Article Snippet: The following antibodies were employed: rabbit monoclonal anti-UHRF1 (DSG8E, Cell Signaling), rabbit polyclonal anti-UHRF2 (TA337863, OriGene); mouse monoclonal anti-UHRF2 (sc-398953, Santa Cruz Biotechnology); mouse monoclonal anti-5-methylcytosine (5-mC; 33D3, Eurogentech); rabbit polyclonal anti-SOX9 (AB5535,Milipore); mouse monoclonal anti-γH2AX (JBW301, Milipore-Upstate); and mouse monoclonal anti-BdrU (BU-33, Sigma Aldrich).

Techniques: Methylation, Enzyme-linked Immunosorbent Assay, CpG Methylation Assay

Journal: bioRxiv

Article Title: A marker chromosome in psychosis identifies glycine decarboxylase (GLDC) as a novel regulator of neuronal and synaptic function in the hippocampus

doi: 10.1101/2023.05.29.542745

Figure Lengend Snippet: A loxP site was placed between the Uhrf2 and Gldc genes using CRISPR/Cas-9, and trans -allelic recombination was used as described in Methods to generate duplication alleles of Gldc alone (9R fragment; duplication=3c9R, triplication=4c9R) or of all the genes in the 9LR genomic segments (9L fragment; duplication=3cL, triplication=4cL). B. Representative western blots and quantification of GLDC protein in hippocampus of mice-with 4 copies of GLDC alone (4c9R), 4 copies of all other 14 genes of the 9p24.1 CNV (4c9L) and 4 copies of the entire 9p24.1 CNV(4c9LR) compared to wildtype (WT) (n=5 for each group, One-way ANOVA followed by Bonferroni test, F(3,37)=18.77; ***p<0.001, WT vs 4c9LR (t=6.07; ***p<0.001), WT vs 4c9R (t=5.24;***p<0.001). C. GLDC enzyme activity in hippocampal tissues of wild type (WT), 4c9LR, 4c9R and 3c9R (n=3-6, One-way ANOVA followed by Bonferroni test, F(3,16)=13.53; ***p=0.0001), WT vs 4c9LR (t=5.827; **p<0.01), WT vs 4c9R (t=3.366; *p<0.05). D, E. Qualitative fluorescence microscopy images of GLDC protein colocalization with S100beta (D) or Map2 (E) in CA1, CA3 and dentate gyrus regions of hippocampus, respectively, in wildtype (WT) and 4c9R mice. F. BDNF mRNA expression is reduced in 4c9R mice compared to WT (One-way ANOVA followed by Bonferroni test; F(2,17)=7.669; **p<0.01), WT vs 4c9R (t=2.729; *p<0.05). G. miR137 mRNA expression is increased in 4c9R mice compared to WT (One-way ANOVA followed by Bonferroni test, F(2,12)=5.683; *p<0.05, WT vs 4c9R (t=3.343, *p<0.05). H. Pyroxd2 mRNA expression is increased in hippocampus of 4c9LR and 4c9R mice (One-way ANOVA followed by Bonferroni test, F(2,12)=468.4; ***p<0.0001, 4c9LR vs WT (t=24.20; ***p<0.001) and WT vs 4c9R (t=28.33; ***p<0.001). Data are mean ± SEM, (n=5).

Article Snippet: Copy number PCRs were performed using commercial kits designed to detect the Gldc and Uhrf2 genes (Mm00496811_cn GLDC (#4400291), Mm00735356_cn Uhrf2 (#4400291) and TaqManTM Copy Number Reference Assay, mouse, Tfrc (#4458366) Thermo Fisher Scientific, MA).

Techniques: CRISPR, Western Blot, Activity Assay, Fluorescence, Microscopy, Expressing

Journal: Journal of Cellular Biochemistry

Article Title: Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways

doi: 10.1002/jcb.23185

Figure Lengend Snippet: Opposite expression pattern of uhrf1 and uhrf2 during differentiation. A: Schematic outline of the multi-domain architecture of Uhrf1 in comparison to Uhrf2. An N-terminal ubiquitin-like domain (Ubl) is followed by a tandem Tudor domain (TTD), a plant homeodomain (PHD), a SET and RING associated (SRA) domain and a C-terminal really interesting new gene (RING) domain. Numbers indicate primary sequence similarities of single domains determined by BlastP search [Altschul, ]. Expression analysis of uhrf1 and uhrf2 by Real-time PCR in ESCs and somatic cells (B), during differentiation of wt J1 ESCs (C) and in various adult mouse tissues in comparison to the expression data in ESCs (D). Expression levels are relative to uhrf1 in wtJM8A (B), day 0 of differentiation (C) and to kidney (D) ( uhrf1 set to 1). Shown are means ± SD of at least two independent experiments.

Article Snippet: All Uhrf2 expression constructs were derived by PCR from mouse uhrf2 -myc cDNA (MR210744, ORIGENE).

Techniques: Expressing, Sequencing, Real-time Polymerase Chain Reaction

Journal: Journal of Cellular Biochemistry

Article Title: Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways

doi: 10.1002/jcb.23185

Figure Lengend Snippet: Cooperative binding of repressive epigenetic marks by Uhrf2. In vitro binding ratios of fluorescently labeled substrate over bound GFP fusion proteins were determined. A: Histone H3- and H4-tail binding specificities of Uhrf2. Shown are means ± SD of biological duplicates. B: Histone H3 tail binding specificity of Uhrf2, its tandem Tudor domain (TTD), its PHD domain and its TTD mutant (Y214A Y217A). Shown are means ± SEM of at least three independent experiments. C: DNA binding properties of Uhrf1, Uhrf2 and of single (SRA, TTD) and combined Uhrf2 domains (TTD–PHD–SRA). Shown are means ± SEM of three independent experiments. D: DNA binding properties of Uhrf1, Uhrf2 and Uhrf2 Y214A Y217A in combination with histone-tail peptide binding. Shown are means ± SD of three independent experiments (Uhrf1, Uhrf2) and of two independent experiments (Uhrf2 Y214A Y217A). Values were normalized to the binding ratio of each GFP fusion for unmethylated DNA without histone-tail peptide. Statistical significance of differences between the binding ratios with un- and hemimethylated DNA is indicated; * P < 0.05. E + F: H3K9me3 peptide binding by Uhrf1, Uhrf2, and Uhrf1ΔSRA with increasing concentrations of DNA substrate containing either one central hemimethylated (E) or noCpG site (F). Shown are means ± SD of biological duplicates. Values were normalized to the binding ratio of Uhrf1ΔSRA without DNA.

Article Snippet: All Uhrf2 expression constructs were derived by PCR from mouse uhrf2 -myc cDNA (MR210744, ORIGENE).

Techniques: Binding Assay, In Vitro, Labeling, Mutagenesis

Journal: Journal of Cellular Biochemistry

Article Title: Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways

doi: 10.1002/jcb.23185

Figure Lengend Snippet: Cellular localization and dynamics of Uhrf2 depend on histone H3K9 methylation. A: Confocal mid sections of fixed wt J1, TKO and Suv39h dn ESCs transiently expressing Uhrf2-GFP and RFP-PCNA and counterstained with DAPI, which preferentially highlights PH. Merged images are displayed on the right side (GFP: green; DAPI: red). Scale bar 5 µm. B: Confocal mid sections of fixed wt MEFs and Suv39h dn MEFs transiently expressing Uhrf2-GFP or Uhrf2 Y214A Y217A-GFP were immunostained for H3K9me3 and counterstained with DAPI. Merged images are displayed on the right side (GFP: green; DAPI: red). Scale bar 5 µm. C: Dynamics of Uhrf2-GFP and Uhrf2 Y214A Y217A-GFP in living MEFs determined by half nucleus FRAP analysis. GFP is shown as reference. Curves represent means ± SEM from at least 8 nuclei.

Article Snippet: All Uhrf2 expression constructs were derived by PCR from mouse uhrf2 -myc cDNA (MR210744, ORIGENE).

Techniques: Methylation, Expressing

Journal: Journal of Cellular Biochemistry

Article Title: Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways

doi: 10.1002/jcb.23185

Figure Lengend Snippet: Cooperative binding of the combined tandem Tudor–PHD domain of Uhrf2. A: Histone H3 N-terminal tail binding specificity of the TTD of Uhrf2 and of the combined TTD and PHD domain (TTD–PHD) of Uhrf1 and Uhrf2. Shown are means ± SEM from at least six independent experiments. B: Histone H3K9me3 binding of the combined TTD–PHD domains of Uhrf1 and Uhrf2, hybrid proteins (L1 and L2 specify inserted linker sequences derived from Uhrf1 and Uhrf2, respectively) and a stretch deletion Uhrf2 construct. Shown are means ± SEM from at least three independent experiments.

Article Snippet: All Uhrf2 expression constructs were derived by PCR from mouse uhrf2 -myc cDNA (MR210744, ORIGENE).

Techniques: Binding Assay, Derivative Assay, Construct

Journal: Journal of Cellular Biochemistry

Article Title: Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways

doi: 10.1002/jcb.23185

Figure Lengend Snippet: Uhrf1 and Uhrf2 are not functionally redundant in ESCs. DNA methylation analysis of wt E14 ESCs, uhrf1 −/− ESCs and of uhrf1 −/− ESCs ectopically expressing Uhrf1-GFP or Uhrf2-GFP. ESCs transiently expressing Uhrf1-GFP and Uhrf2-GFP were isolated by FACS sorting 48 h after transfection and CpG methylation levels of major satellites repeats were analysed by bisulfite treatment, PCR amplification and direct pyrosequencing. Statistical significance of differences in DNA methylation levels between uhrf1 −/− ESCs and uhrf1 −/− ESCs with ectopically expressed Uhrf1-GFP or Uhrf2-GFP are indicated; * P < 0.05. Shown are means ± SD from three independent experiments.

Article Snippet: All Uhrf2 expression constructs were derived by PCR from mouse uhrf2 -myc cDNA (MR210744, ORIGENE).

Techniques: DNA Methylation Assay, Expressing, Isolation, Transfection, CpG Methylation Assay, Amplification