pcas9 mcherry empty  (Addgene inc)

Journal: Nature Communications

Article Title: ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress

doi: 10.1038/s41467-021-24206-5

Figure Lengend Snippet: a Genome browser representations of ATRX, ATRX-GFP, H3.3, and H3K9me3 ChIP-seq at predicted G4 regions in ESCs. Data represented as read density in reads per kilobase per million mapped reads (RPKM) normalized to an external standard for each data set. Gray boxes indicate predicted G4 regions. b ChIP-seq analysis of ATRX enrichment in ESCs. Pie chart represents the percentage of ATRX-enriched regions containing G4 consensus motifs (169/435, 39%). c ATRX ChIP-seq average profiles in ESCs at ATRX-enriched regions containing G4 consensus motifs (G4) compared with ATRX-enriched regions that do not contain a G4 consensus motif (non-G4). d Schematic of ESCs synchronization protocol. Cells are incubated with thymidine for 14 h, washed, and treated in medium with nocodazole for 7 h. After washing, mitotic cells are released in medium and incubated with EdU in prior cell fixation for downstream experiments. Cells in G1, early S, and late S phase were analyzed 1, 2, and 8 h after release, respectively. e Representative images demonstrating ATRX and G4 co-localization by proximity ligation assay (PLA) in synchronized ESCs. Green—PLA (ATRX-G4). Red—EdU-labeling, indicative of newly synthesized DNA. Blue—DAPI nuclear stain. Scale bar equals 10 μm ( n = 1 independent experiment). f Quantification of signal intensity from ATRX-G4 PLA foci in G1 ( n = 47), early S ( n = 53), and late S ( n = 34) phases of ESCs. Quantification of PLA data are presented as box-and-whisker plots marking a horizontal median line. The bottom and the top of the boxes correspond to the 25 and 75th percentiles, and the internal band is the 50th percentile (median). The plot whiskers show down to the minimum and up to the maximum value. One-way ANOVA, p = 0.2554 (G1 vs. early S), p = 0.2760 (G1 vs. late S), and p = 0.9905 (early S vs. late S).

Article Snippet: To tag the endogenous ATRX locus with GFP, pCAS9-mCherry empty, pCAS9-mCherry-Frame +1, and pCRISPaint-TagGFP2-PuroR plasmids were purchased from Addgene.

Techniques: ChIP-sequencing, Incubation, Proximity Ligation Assay, Labeling, Synthesized, Staining, Whisker Assay

Journal: Nature Communications

Article Title: ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress

doi: 10.1038/s41467-021-24206-5

Figure Lengend Snippet: a Representative images demonstrating EdU and G4 co-localization by proximity ligation assay (PLA) in synchronized ESCs at early S phase. Green—PLA (EdU-G4). Blue—DAPI nuclear stain. Scale bar equals 10 μm ( n = 2 independent experiments). b Quantification of signal intensity from EdU-G4 PLA foci in wild-type ( n = 48), ATRX KO ( n = 35) ( p = 2 × 10 −10 ) and DAXX KO ( n = 52) ( p = 1 × 10 −10 ) ESCs. The bottom and the top of the boxes correspond to the 25th and 75th percentiles, and the internal band is the 50th percentile (median). The plot whiskers show down to the minimum and up to the maximum value. Statistical significance determined by a One-way ANOVA test. *** p < 0.001. c Genome browser representations of ATRX, ATRX-GFP ChIP-seq, and BG4 CUT&Tag at observed G4 regions in ESCs. d – f CUT&Tag analysis of G4 enrichment in ESCs. d Venn diagram showing the unique and overlaps between wild-type and ATRX KO ESCs. e , f Pie chart of genomic region annotation in wild-type ( e ) and ATRX KO ESCs ( f ). g Box plots representing EdU-seq read counts of early S phase in wild-type, ATRX KO ( p = 8.9 × 10 −11 ) and DAXX KO ( p = 1.6 × 10 −6 ) ESCs at observed G4 regions that are only identified in ATRX KO ( n = 6301). Data are representative of two independent experiments. The bottom and the top of the boxes correspond to the 25 and 75th percentiles, and the internal band is the 50th percentile (median). The plot whiskers correspond to 1.5 interquartile range. Statistical significance determined by Wilcoxon–Mann–Whitney test. *** p < 0.001.

Article Snippet: To tag the endogenous ATRX locus with GFP, pCAS9-mCherry empty, pCAS9-mCherry-Frame +1, and pCRISPaint-TagGFP2-PuroR plasmids were purchased from Addgene.

Techniques: Proximity Ligation Assay, Staining, ChIP-sequencing, MANN-WHITNEY

Journal: Nature Communications

Article Title: ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress

doi: 10.1038/s41467-021-24206-5

Figure Lengend Snippet: a Genome browser representations of ATRX, ATRX-GFP, H3.3 ChIP-seq and ATAC-seq at predicted G4 regions in ESCs. Box plots representing ATAC-seq read counts at ATRX-enriched G4 and non-G4 regions in wild-type ESCs compared to ( b ) ATRX KO ( p = 5.6 × 10 −4 ), ( c ) DAXX KO ( p = 1.7 × 10 −4 ), and ( d ) H3.3 KO ( p = 4.4 × 10 −5 ) ESCs. e Box plots representing ATAC-seq read counts at ATRX-enriched G4 and non-G4 regions in ATRX KO ESCs exogenously expressing either wild-type ( p = 0.003) or mutant ATRX (L1238A p = 0.47; K1562R p = 0.57) constructs. f Box plots representing ATAC-seq read counts at ATRX-enriched G4 and non-G4 regions in H3.3 KO ESCs exogenously expressing either wild-type H3.2 ( p = 0.68), H3.3 ( p = 0.047), or mutant ( p = 0.87) H3.3 constructs. Data are representative of two independent experiments. The bottom and the top of the boxes correspond to the 25th and 75th percentiles, and the internal band is the 50th percentile (median). The plot whiskers correspond to 1.5 interquartile range. Statistical significance determined by Wilcoxon–Mann–Whitney test compared to ATRX KO ( e ) and H3.3 KO in ( f ). * p < 0.05; ** p < 0.01; *** p < 0.001. ns not significant.

Article Snippet: To tag the endogenous ATRX locus with GFP, pCAS9-mCherry empty, pCAS9-mCherry-Frame +1, and pCRISPaint-TagGFP2-PuroR plasmids were purchased from Addgene.

Techniques: ChIP-sequencing, Expressing, Mutagenesis, Construct, MANN-WHITNEY

Journal: Nature Communications

Article Title: ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress

doi: 10.1038/s41467-021-24206-5

Figure Lengend Snippet: a Genome browser representations of ATRX, ATRX-GFP, and H3K9me3 ChIP-seq at predicted G4 regions in ESCs. Box plots representing ChIP-seq read counts for H3K9me3 at ATRX-enriched G4 and non-G4 regions in wild-type ESCs compared to ( b ) ATRX KO ( p = 4.8 × 10 −4 ), ( c ) DAXX KO ( p = 3.1 × 10 − 7 ) and ( d ) H3.3 KO ( p = 2.1 × 10 −8 ) cells. Data are representative of two independent experiments. The bottom and the top of the boxes correspond to the 25 and 75th percentiles, and the internal band is the 50th percentile (median). The plot whiskers correspond to 1.5 interquartile range. Statistical significance determined by Wilcoxon–Mann–Whitney test. *** p < 0.001. e ChIP-qPCR of H3K9me3 at ATRX-enriched G4 regions ( n = 7) in ATRX KO ESCs exogenously expressing either wild-type or mutant ATRX constructs. f ChIP-qPCR of H3K9me3 at ATRX-enriched G4 regions ( n = 7) in H3.3 KO ESCs exogenously expressing either wild-type H3.2, H3.3, or mutant H3.3 constructs. Data represent mean ± SD. Statistical significance determined by one-way ANOVA compared to ATRX KO + ATRX in ( e ) and H3.3 KO + H3.3 in ( f ). ** p < 0.01; *** p < 0.001. p = 2.9 × 10 −4 (vector vs. ATRX addback in e ), p = 1.8 × 10 −3 (ATRX addback vs. L1238A in e ), and p = 3.8 × 10 −4 (ATRX addback vs. K1562R in e ). p = 4.9 × 10 −4 (H3.3 KO vs. H3.3 addback in f ), p = 6.6 × 10 –3 (H3.2 addback vs. H3.3 addback in f ), and p = 5.6 × 10 −4 (H3.3 LIAA vs. H3.3 addback in f ).

Article Snippet: To tag the endogenous ATRX locus with GFP, pCAS9-mCherry empty, pCAS9-mCherry-Frame +1, and pCRISPaint-TagGFP2-PuroR plasmids were purchased from Addgene.

Techniques: ChIP-sequencing, MANN-WHITNEY, ChIP-qPCR, Expressing, Mutagenesis, Construct, Plasmid Preparation

Journal: Nature Communications

Article Title: ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress

doi: 10.1038/s41467-021-24206-5

Figure Lengend Snippet: a Genome browser representations of ATRX, ATRX-GFP, H3.3-H3K9me3 reChIP-seq, H3.3 ChIP-seq, and ATAC-seq at predicted G4 regions in ESCs. Box plots representing ( b ) H3.3-H3K9me3 reChIP-seq ( p = 5.0 × 10 −6 ), ( c ) H3.3 ChIP-seq ( p = 0.47), and ( d ) ATAC-seq ( p = 0.12) at ATRX-enriched G4 and non-G4 regions in wild-type ESCs compared to ESET KO ESCs. The bottom and the top of the boxes correspond to the 25 and 75th percentiles, and the internal band is the 50th percentile (median). The plot whiskers correspond to 1.5 interquartile range. Statistical significance determined by Wilcoxon–Mann–Whitney test. *** p < 0.001; ns: not significant. e Cell viability of wild-type and ESET KO ESCs treated with 1 μM of PDS for 3 days. Mock-treated cells at day 0 were taken as 100% survival. Data represented as mean ± SD ( n = 9). Statistical significance determined by Mann–Whitney U test. *** p < 0.001. p = 9 × 10 −10 (WT vs. ESET KO in e ).

Article Snippet: To tag the endogenous ATRX locus with GFP, pCAS9-mCherry empty, pCAS9-mCherry-Frame +1, and pCRISPaint-TagGFP2-PuroR plasmids were purchased from Addgene.

Techniques: ChIP-sequencing, MANN-WHITNEY