Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-1 was located on the upstream of miR-23a∼27a∼24-2 cluster and activated its expression during erythropoiesis. ( A ) Q-PCR analysis of the primary transcript of the miR-23a∼27a∼24-2 cluster in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( B ) Q-PCR analysis of the mature transcripts of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( C ) Northern blot analysis of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation. U6 snRNA was used as a loading control. ( D ) Q-PCR analysis of mature miR-27a and miR-24 expression in CD34+ HPCs undergoing day 4, 7, 11, 15 and 18 of E culture. ( E ) Representation of the human −603 bp miR-23a∼27a∼24 cluster promoter fragments. ( F ) ChIP-PCR and ChIP-qPCR analysis of the GATA-1 hit on the −557 site at the miR-23a∼27a∼24 cluster promoter in K562s. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01. ( G ) Immunoblot analysis of GATA-1 expression in K562s treated with siRNAs specific to GATA-1 (si_GATA-1) for 48 h or K562s transfected with a construct overexpressing GATA-1 (over_GATA-1) for 48 h, respectively. ( H ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (G). ( I ) Immunoblot analysis of GATA-1 expression in K562s undergoing erythroid differentiation. ( J ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( K ) Functional activity of GATA-1 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Northern Blot, Control, ChIP-qPCR, Standard Deviation, Western Blot, Transfection, Construct, Functional Assay, Activity Assay, Mutagenesis, Luciferase

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: MiR-27a and miR-24 promoted erythroid differentiation in CD34+ HPCs. ( A ) Monitoring of the GFP + population (left panel) and the CD235a stained GFP + fraction (medium panel) of Lenti-miRNA-transduced CD34+ HPCs on day 15 of E culture. The morphology (May-Grunwald Giemsa staining) of CD34+ HPCs derivatives on day 15 is shown in the right panel. A 400X magnification of a representative field is shown. ( B ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. Percentages of basophilic (Bas), polychromatophilic (Pol), orthochromatic (Ort) erythroblasts and erythrocytes (Ery) were determined by May-Grunwald/Giemsa staining of cytospin preparations. ( C ) Q-PCR analysis of gamma-globin mRNA expression in CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. ( D ) A comparison of the erythroid colony-forming capacity (CFU-E, BFU-E) of CD34+ HPCs transduced with Lenti-miRNAs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) FACS monitoring of CD34+ HPCs transduced with Zip-miRNA or Zip-GFP as described in (A). ( F ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Zip-27a, Zip-24 or Zip-GFP as described in (B). ( G ) Detection of gamma-globin mRNA level in CD34+ HPCs transduced with Zip-miRNA. ( H ) Colony-forming assay of CD34+ HPCs transduced with Zip-miRNA as described in (D).

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Staining, Transduction, Expressing, Comparison, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-2 was post-transcriptionally regulated by miR-27a and miR-24 during erythropoiesis. ( A ) A computer prediction of conserved and mutated binding sites within the 3′ UTR of GATA-2 mRNA for miR-27a and miR-24. ( B ) Relative luciferase activity of the indicated GATA-2 reporter constructs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( C ) Immunoblot analysis of GATA-2 in K562s transfected with scramble or miRNA mimics (miR-27a, miR-24) or inhibitors (Anti-27a, Anti-24). ( D ) Immunoblot analysis of GATA-2 in CD34+ HPCs transduced with Lenti-GFP control and lentivirus expressing miR-27a or miR-24 (Lenti-27a or Lenti-24). ( E, F ) ‘Rescue’ assays for miRNAs and GATA-2 during erythroid differentiation. Immunoblot analysis of GATA-2 in K562s treated with scramble or Anti-27a/24 (E) for 24 h. These cells were subsequently treated for another 24 h with control siRNAs or siRNAs specific to GATA-2 and were then treated with hemin treatment for 0, 48 and 72 h. (F) FACS analysis of K562s stained for CD71 and CD235a expression after 48 h of hemin induction as described earlier in the text.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Standard Deviation, Western Blot, Transfection, Transduction, Control, Expressing, Staining

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: The GATA switch regulated miR-27a and miR-24 expression. ( A ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1, GATA-2 and Pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( B ) Immunoblot analysis of GATA-1 and GATA-2 expression in K562s undergoing erythroid differentiation for 0, 24, 48 and 72 h. ( C ) Functional activity of GATA-1 and GATA-2 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( D ) Immunoblot analysis of GATA-2 in K562s treated with siRNAs specific to GATA-2 (si_GATA-2) for 48 h or K562s transfected with a construct overexpressing GATA-2 (over_GATA-2) for 48 h, respectively. ( E ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (D). ( F ) ChIP-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with control siRNAs or siRNAs specific to GATA-1/GATA-2 or K562s transfected with empty pcDNA3.1 vectors or pcDNA3.1_GATA-1/GATA-2. ( G ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in CD34+ HPCs of E culture. ( H ) Q-PCR analysis of Pri-27a-24 abundance in CD34+ HPCs of E culture (Top panel). Error bars represent the standard deviation obtained from three independent experiments. An immunoblot analysis of GATA-1 and GATA-2 expression in CD34+ HPCs of E culture (Bottom panel). ( I ) Q-PCR (left panel) and immunoblot (right panel) analysis of GATA-1 and GATA-2 expression in CD34+ HPCs transduced with lentivirus-expressing siRNAs against GATA-1 or control on day 11 of E culture. Error bars represent standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( J ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in CD34+ HPCs treated as described in (I). Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Standard Deviation, Western Blot, Functional Assay, Activity Assay, Mutagenesis, Luciferase, Transfection, Construct, Control, Transduction

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: A regulatory circuit involving GATA-1, GATA-2 and miR-27a/24 in erythropoiesis. ( A ) A schematic representation of the regulatory circuit comprised GATA-1, GATA-2, miR-27a and miR-24 in erythroid differentiation. ( B , C ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with scramble or miRNA mimics and inhibitors (B, r miR-27a and C, miR-24). ( D ) Q-PCR analysis of Pri-27a∼24 abundance in K562s treated as described in (B) and (C). Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Lenti-miR-27a/24 or Lenti-GFP control for 11 days. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( F ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Zip-miRNA or Zip-GFP for 11 days. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Transfection, Standard Deviation, Transduction, Control

Journal: bioRxiv

Article Title: Defective BRCA1-mediated DNA end resection drives tandem duplication formation and FANCM synthetic lethality

doi: 10.64898/2026.02.20.706968

Figure Lengend Snippet: A. Schematic of RPA accumulation on ssDNA in response to DNA end resection of a Cas9 induced DSB, targeted to the I-SceI site within the HR reporter at Rosa26 . Intensity of RPA ChIP-seq signal reflects the efficiency of DNA end resection. B. Cas9 DSB-induced RPA ChIP-seq signals in two independent experiments (REP#1 and REP#2) using Brca1 genotypes shown. Brca1 allele abbreviations as in . See STAR Methods for details. C . Quantitation of RPA enrichment averaged over 30 kb window flanking I-SceI site. Signals were normalized to WT for each of the replicates. Statistical analysis: unpaired t -test with Welch’s correction. ns: not significant, p *<0.05.

Article Snippet: The ChIP libraries were prepared according to the NEB Ultra II DNA Library Prep Kit (NEB, #E7645).

Techniques: ChIP-sequencing, Quantitation Assay

Journal: bioRxiv

Article Title: Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing

doi: 10.1101/2023.03.27.534417

Figure Lengend Snippet: a, Schema for analysis of BM primitive SLAM LSK HSPCs. Murine femurs and tibias were harvested, flushed, and crushed, to collect maximal yield of bone and marrow cells. BM labeled cells were flow sorted for the SLAM LSK markers: Live\Ter-119 neg \Lineage neg \Sca-1 + \c-Kit + \CD150 + \CD48 neg . Next, combined multiome single-nuclei RNA/ATAC (snRNA/ATAC) sequencing analysis was performed. b, Weighted nearest neighbor (WNN) UMAP with hematopoietic stem and progenitor cell type annotation for hematopoietic stem cell (HSC), multi-potent progenitor (MPP), megakaryocyte progenitor (MkP), and erythrocyte progenitor (EryP) sub-cluster representation. c, Engrafting LTR-HSC transcriptional signature (from Rodriguez-Fraiticelli et al . 2020) assigned on a WNN UMAP space. d, Heatmap representation of differential transcriptional nuclei output from distinct HSPC sub-clusters by averaged Z-score, with selected genes presented. e, Heatmap representation of differential ChromVAR motif activity in distinct HSPC sub-clusters by averaged Z-score, with selected TF motifs presented.

Article Snippet: Human hematopoietic CD34 + HSPCs from either cord blood (CB) or mobilized peripheral blood (mPB) donors, were isolated using human CD34 microbead kit (Miltenyi Biotec) and by passing twice through LS columns (Miltenyi Biotec) attached to a magnetic stand, achieving a purity >95%.

Techniques: Labeling, Sequencing, Activity Assay

Journal: bioRxiv

Article Title: Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing

doi: 10.1101/2023.03.27.534417

Figure Lengend Snippet: WT, Fli-1 ROSAΔ , and Fli-1 ROSAΔ with conditionally inducible Notch1 internal component overexpression transgene (Fli-1 ROSAΔ N1-IC iOE ) BM LSK HSPCs were isolated and expanded as described in Fig. S5A. Harvested cells were analyzed by flow cytometry and/or flow sorted for LSK HSPCs which were competitively co-transplanted with congenic SJL BM cells into lethally irradiated congenic SJL recipient mice. a, Representative images of co-cultures at the end point before harvest. Yellow arrows indicate megakaryocytes. Note expansion of round hematopoietic cells in Fli-1 ROSAΔ N1-IC iOE co-cultures without any appearance of megakaryocytes. Bar = 100 µM. b, Frequency of LSK HSPCs was determined by flow cytometry and fold expansion was calculated. One-way ANOVA multiple comparisons was used; n=4 BM donor mice per genotype, with 2 technical replicates per donor. c, Frequency of chimerism indicating engraftment levels as determined by flow cytometry. One-way ANOVA multiple comparisons was used; n=8 recipient mice per genotype. d-g, WT, Fli-1 ROSAΔ , and Fli-1 ROSAΔ with conditionally inducible Notch1 internal component overexpression transgene (Fli-1 ROSAΔ N1-IC iOE ) BM LSK HSPCs were isolated and expanded. After 48 hours of expansion in co-culture, cells were harvested and hematopoietic LSK HSPCs were sorted and applied for single cell RNA-seq analysis; n=4 per genotype “pooled” together. d, Dimensionality reduction by UMAP of single cell transcriptomes from co-cultured and sorted LSK HSPCs. e. Dot plot for the E-SLAM HSC markers EPCR, CD34, and CD150 per cluster identity. Dot plot size indicates percent expression in cluster and color intensity indicates average expression score. f. Distribution of clusters 7 and 15 in UMAP for HSC identified populations among LSK HSPC clusters based on Garnette and the Dot plot from panel (e). g, Dot plot for cell cycle scores (S phase and G2/M phases) per cluster identity. Dot plot size indicates percent expression in cluster and color intensity indicates average expression score. h, Cell cycle status classification in UMAP for clusters 7 and 15: WT (left UMAP), Fli-1 ROSAΔ (middle UMAP) and Fli1 ROSAΔ N1-IC iOE (right UMAP). i-l, BM cells were harvested, 16 weeks post engraftment of WT and “rescued” Fli-1 ROSAΔ N1-IC iOE long term repopulating HSCs and pooled from n=4 recipient mice (from 4 different donors) per genotype. CD45.2 + /Lin − cells were flow sorted and applied for single cell RNA-seq analysis. (HSC: hematopoietic stem cells, MPP: multi-potent progenitors, LPP: lymphoid-primed progenitors, MEP: megakaryocyte/erythroid progenitors). i, Dimensionality reduction by UMAP of single cell transcriptomes from donor-derived lineage-negative BM samples (CD45.2 + /Lin − ) following transplantation of WT or Fli1 ROSAΔ N1-IC iOE LSK HSPC. j, Cell type classification of hematopoietic sub-populations using Garnette: WT (left panel) and Fli1 ROSAΔ N1-IC iOE (right panel) samples in UMAP, and distribution of cell types between samples (bar plot). k, Distribution of WT and Fli1 ROSAΔ N1-IC iOE sample cells in UMAP among populations classified as multi-lineage stem/progenitor cell types (HSC, MPP, or LPP) using Garnette, and distribution of cell types between samples (bar plot). See also Table S5. l , Heatmaps of gene-set scores for the subset of multi-lineage stem and progenitor cell types (HSC, MPP, LPP) in UMAP (left panels), and violin plots of gene-set scores between samples (WT vs Fli1 ROSAΔ N1-IC iOE ) (right panels), for HSC molecular overlap signature genes (from Wilson et al . 2015) and engrafting long term HSC signature genes (from Rodriguez-Fraiticelli et al . 2020). Gene-set scores in violin plots are shown only for the subset of cells classified as HSC cell type. Wilcoxon Rank Sum Test was used to determine p-Values.

Article Snippet: Human hematopoietic CD34 + HSPCs from either cord blood (CB) or mobilized peripheral blood (mPB) donors, were isolated using human CD34 microbead kit (Miltenyi Biotec) and by passing twice through LS columns (Miltenyi Biotec) attached to a magnetic stand, achieving a purity >95%.

Techniques: Over Expression, Isolation, Flow Cytometry, Irradiation, Co-Culture Assay, RNA Sequencing, Cell Culture, Expressing, Derivative Assay, Transplantation Assay

Journal: bioRxiv

Article Title: Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing

doi: 10.1101/2023.03.27.534417

Figure Lengend Snippet: a-d, Sorted CD45 + /CD34 + HSPC from cord blood (CB) and adult mobilized peripheral blood (mPB) sources, were co-cultured on top of a vascular niche for 48h to encourage HSPC activation. Next, cells were harvested and CD45 + /CD34 + HSPC were sorted and applied for single cell RNA-seq analysis. a-c, Dimensionality reduction by UMAP of single cell transcriptomes from co-cultured and sorted CD45 + /CD34 + HSPC, displaying cell distribution by CB and mPB source (a), cluster identity (b), and single cell cycle phase (c). d, Dot plot for cell cycle scores (S phase and G2/M phases) per cellular source (CB or mPB), for total cells in analysis (upper panel) and for HSC\MPP cluster (lower panel). Dot plot size indicates percent expression in cluster and color intensity indicates average expression score. e, To highly enrich for HSCs, the mononuclear fraction from CB (n=3) and mPB (n=4) sources was isolated, labeled, and sorted for CD45 + /CD34 + /CD38 − /CD45RA − /CD90 + /CD49f + . Sorted cells were lysed and processed for RNAseq analysis. GSEA analysis plots for HSC activation signatures (left panels) and for HSC quiescence signatures (right panels) acquired from Venezia et al., 2004 (upper panels) and from Roy et al., 2021 (lower panels), showing a positive enrichment for the activation signatures in CB HSCs and positive enrichment for quiescence signatures in mPB HSCs. f, g, UMAP projections of the single cell ATAC-seq enrichment analysis for sorted HSPCs of all 10 signatures identified in Takayama et al., 2021. Colors indicate the degree of enrichment in each cell (blue, depleted; red, enriched). Scale bar indicates enrichment Z-score. f, Enrichment for activated HSPCs (upper panel) and quiescent HSPCs (lower panel) signatures defined in in Takayama et al., 2021., as calculated by chromVAR. g, Enrichment for called peaks from the FLI-1 HSPC ChIP-seq (Fli-1 signature) data set (Beck et al., 2013) overlayed on single cell ATAC-seq HSPC data set (Takayama et al., 2021), as analyzed by chromVAR.

Article Snippet: Human hematopoietic CD34 + HSPCs from either cord blood (CB) or mobilized peripheral blood (mPB) donors, were isolated using human CD34 microbead kit (Miltenyi Biotec) and by passing twice through LS columns (Miltenyi Biotec) attached to a magnetic stand, achieving a purity >95%.

Techniques: Cell Culture, Activation Assay, RNA Sequencing, Expressing, Isolation, Labeling, ChIP-sequencing

Journal: bioRxiv

Article Title: Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing

doi: 10.1101/2023.03.27.534417

Figure Lengend Snippet: a, Schema of experimental design. Sorted human CD34 + HSPCs from cord blood (CB) or mobilized peripheral blood (mPB) sources were transduced by electroporation with FLI-1 modified-RNA molecules (2 µg FLI-1 modRNA per 10 5 cells) and expanded for 1 week on top of E4orf1 vascular niche cells in a sub-optimal ratio of 1:3 (HSPCs:ECs). Next, expansion co-cultures were analyzed and transplanted into immunodeficient NSG KitW41 mice without myeloablative preconditioning. b , Fold expansion of CB derived hematopoietic subtypes after 1 week in co-culture following transduction with FLI-1 (red) or control (blue) modified-RNA. Unpaired two tailed t-test was used; n = 4 CB donors. Each mark represents the averaged triplicate (n = 3 technical repeats) per donor. c , Fold expansion of mPB derived hematopoietic subtypes after 1 week in co-culture following transduction with FLI-1 (red) or control (blue) modified-RNA. Unpaired two tailed t-test was used; n = 4 mPB donors. Each mark represents the averaged triplicate (n = 3 technical repeats) per donor. d, Representative flow dot plots of mPB HSPC analysis post 1 week of expansion in co-culture following transduction with FLI-1 or control modified-RNA. The population of CD34 + \CD38 neg HSPCs is colored in red. e, Human CD45 chimerism analysis in peripheral blood (PB), spleen, and bone marrow (BM) of NSG KitW41 mice. Tissues were harvested and analyzed by flow cytometry 16 weeks post transplantation. Unpaired two tailed t-test was used; n = 6 mPB donors. f, Frequency of BM engrafted human HSPCs as determined by flow cytometry 16 weeks post transplantation. Unpaired two tailed t-test was used; n = 6 mPB donors. g, Representative flow dot plots for BM engrafted human CD34 + and CD34 + \CD38 neg HSPCs acquired 16 weeks post transplantation. Unpaired two tailed t-test was used; n = 6 mPB donors. h, Total BM from primary NSG KitW41 recipient mice was transplanted into secondary NSG KitW41 recipient mice without myeloablative preconditioning. Human CD45 chimerism in the BM of recipient mice was determined by flow cytometry 16 weeks post secondary transplantation. Unpaired two tailed t-test was used; n = 6 mPB donors.

Article Snippet: Human hematopoietic CD34 + HSPCs from either cord blood (CB) or mobilized peripheral blood (mPB) donors, were isolated using human CD34 microbead kit (Miltenyi Biotec) and by passing twice through LS columns (Miltenyi Biotec) attached to a magnetic stand, achieving a purity >95%.

Techniques: Electroporation, Modification, Derivative Assay, Co-Culture Assay, Transduction, Control, Two Tailed Test, Flow Cytometry, Transplantation Assay

Journal: PLoS Genetics

Article Title: Tet1 regulates epigenetic remodeling of the pericentromeric heterochromatin and chromocenter organization in DNA hypomethylated cells

doi: 10.1371/journal.pgen.1009646

Figure Lengend Snippet: (A) : Representative immunostaining images of the surface-spread nuclei of the wild-type, Dnmt1 -KO and Dnmt1/Tet1 -DKO ESCs. The yellow squares indicate the enlarged areas shown on the bottom. The white dashed circles indicate the PCH. (B) : Quantification of immunostaining of H2AK119ub, related to (A). The enrichment score is calculated by the relative signal intensity of PCH to the entire chromosome. An average of 3 chromosomes was calculated for each cell. n = 20 per cell line. (C) : ChIP-qPCR for H2AK119ub at the major satellite repeats in the wild-type, Dnmt1 -KO and Dnmt1/Tet1 -DKO ESCs. Each bar represents relative enrichment to wild-type after normalization against H2A ChIP. n = 3 per cell line. (D) : Representative immunostaining images of surface-spread nuclei of the wild-type, Dnmt1 -KO and Dnmt1/Ring1A/1B -TKO ESCs. Cells were analyzed 4 d after the transfection of the CRISPR cassette. (E) : Boxplot showing the number of distinct chromocenters in the nuclei of ESCs, related to (D). Wild-type, n = 97; Dnmt1 -KO, n = 75; Dnmt1/Ring1A/1B -TKO, n = 64. P values were calculated using the Mann-Whitney U-test. *** P < 0.001; ** P < 0.01. Scale bar, 5 μm.

Article Snippet: DNA was purified with QIAquick PCR purification kit (QIAGEN) and quantified by Thunderbird qPCR Mix (TOYOBO, QPK-201) with the major satellite specific primers (5’-GACGACTTGAAAAATGACGAAATC-3’ and 5’-CATATTCCAGGTCCTTCAGTGTGC-3’).

Techniques: Immunostaining, ChIP-qPCR, Transfection, CRISPR, MANN-WHITNEY