Journal: EMBO Reports

Article Title: A CRISPR-Cas9 screen reveals genetic determinants of the cellular response to decitabine

doi: 10.1038/s44319-025-00385-w

Figure Lengend Snippet: ( A ) Dose–response curves of HAP1 cells transduced with sgCTRL or two different sgRNAs targeting KDM1A (sg KDM1A #1 and #2) upon DAC treatment at indicated concentrations. Cell viability was measured by CellTiter-Glo after 3 days of DAC treatment. Data are presented with ± SEM. Experiments performed in duplicates. The p values were determined using nonlinear regression followed by the extra sum-of-squares F test. ( B ) Dose–response curves of wild-type HAP1 cell and isogenic KDM1A KO clones upon DAC treatment at indicated concentrations. Cell viability was measured by CellTiter-Glo after 3 days of DAC treatment. Data are presented with ± SEM. Experiments performed in duplicates. The p values were determined using nonlinear regression followed by the extra sum-of-squares F test. ( C ) Survival analysis of WT, KDM1A KO clones, and KDM1A KO clones complemented with either empty vector, wild-type KDM1A (KDM1A-WT), or catalytically inactive KDM1A-AE/KA and KDM1A-3DA mutants after treatment with DAC at indicated concentrations for 3 days. Cell viability was measured by CellTiter-Glo. Data represent the means ± SEM of 2 biological independent clones (2 technique replicates each, n = 4). The p values were determined using two-way ANOVA followed by Dunnett’s multiple comparisons test. ( D ) Immunoblot analysis showing the KDM1A protein level in two HAP1 clones expressing FKBP-KDM1A fusion protein treated with or without dTAG-13 for 2 days with β-ACTIN as a loading control. ( E ) Dose–response curves of two HAP1 clones expressing FKBP-KDM1A fusion protein upon DAC treatment at indicated concentration. Cells were treated with or without dTAG-13 at the same time with DAC. Data are presented with ± SEM. Experiments performed in duplicates. The p values were determined using nonlinear regression followed by the extra sum-of-squares F test. ( F ) Heatmap and bar plot (left) depicting sgRNA abundance changes of KDM1A domain scanning at indicated time points versus Day 0, normalized against non-targeting control sgRNAs. The positions of domain scanning sgRNAs in the coding region of KDM1A are indicated as vertical lines in the above schematic diagram. Heatmap (right) for averaged differential β scores of sgRNAs in KDM1A domains with clustering. ( G ) Survival analysis of WT, and KDM1A KO clones, and KDM1A KO clones complemented with KDM1A-ΔNFR, KDM1A-ΔSWIRM, KDM1A-ΔNFR/ΔSWIRM, and KDM1A-ΔTOWER after treatment with DAC at indicated concentrations for 3 days. Cell viability was measured by CellTiter-Glo. Data represent the means ± SEM of 2 biological independent clones (2 technical replicates each, n = 4). The p values were determined using two-way ANOVA followed by Dunnett’s multiple comparisons test. ( H ) KDM1A ChIP-seq metagene profiles of KDM1A binding peaks in KDM1A KO clone complemented with KDM1A-WT, KDM1A-ΔSWIRM, and KDM1A-ΔTOWER (upper). Heatmap representation of KDM1A occupancies at KDM1A binding peaks in these cells ranked by the intensity of KDM1A ChIP-seq signal (lower). ( I ) Volcano plot showing differential interacted proteins between KDM1A-WT and KDM1A-ΔSWIRM obtained from label-free quantification MS with triplicates. The p values were determined by two-sample t -test using a permutation-based FDR approach. ( J ) Immunoblot analysis for immunoprecipitations of 3×FLAG-tagged KDM1A-WT and KDM1A-ΔSWIRM. ( K ) Dose–response curves of wild-type HAP1 and isogenic ZMYM2 (left) or ZMYM3 (right) KO clones upon DAC treatment at indicated concentrations. Cell viability was measured by CellTiter-Glo after 3 days of DAC treatment. Data are presented with ± SEM. Experiments performed in duplicates. The p values were determined using nonlinear regression followed by the extra sum-of-squares F test. .

Article Snippet: FKBP12 PROTAC dTAG-13 , MCE , HY-114421.

Techniques: Transduction, Clone Assay, Plasmid Preparation, Western Blot, Expressing, Control, Concentration Assay, ChIP-sequencing, Binding Assay, Quantitative Proteomics

Journal: EMBO Reports

Article Title: A CRISPR-Cas9 screen reveals genetic determinants of the cellular response to decitabine

doi: 10.1038/s44319-025-00385-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: FKBP12 PROTAC dTAG-13 , MCE , HY-114421.

Techniques: Recombinant, Sequencing, Software, Cloning, Proliferation Assay, In Situ

Journal: Nature cell biology

Article Title: The transcription factor GABPA is a master regulator of naive pluripotency.

doi: 10.1038/s41556-024-01554-0

Figure Lengend Snippet: Fig. 2 | GABPA activates a group of major ZGA genes by binding to their promoters. a, A diagram showing dTAG13-triggered GABPA degradation at different time windows of mouse pre-implantation development. dTAG13 #1: dTAG13 treatment from zygote (6 hpf) to late blastocyst (112 hpf) stage; dTAG13 #2: dTAG13 treatment from zygote to four-cell (42 hpf) stage (covers ZGA stage). dTAG13 #3: dTAG13 treatment from four-cell to late blastocyst stage (after ZGA). b, The embryo development rate (left) and representative images of blastocyst stage embryos (right) after GABPA degradation (DMSO, n = 43; #1, n = 50; #2, n = 57; #3, n = 54; n represents the total embryos from three independent experiments). The data are presented as mean values ± standard deviation. The black arrows indicate the four-cell or eight-cell arrested embryos. Scale bar, 100 μm. c, An RNA-seq comparison of late two-cell embryos treated with dTAG13 or DMSO from zygote (6 hpf) to late two-cell stage (30 hpf). The x and y axis of the dot plots are log2-normalized counts from RNA-seq. d, The genomic distribution

Article Snippet: The confocal microscope (Zeiss, LSM800) was used for fluorescence detection. dTAG13 treatment dTAG13 (Tocris, 6605) was reconstituted in DMSO to a 5 mM stock.

Techniques: Binding Assay, Standard Deviation, RNA Sequencing, Comparison

Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: dTAG13 , Tocris Bioscience , Fisher Scientific Cat#: 66-055.

Techniques: Recombinant, Protease Inhibitor, Magnetic Beads, Cloning, Isolation, CRISPR, Software, Imaging

Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: (A) Diagram of DHX15’s canonical role in disassembling of intron-lariat spliceosome (ILS) at the end of the splicing cycle and its proposed role in disassembling aberrant splicing intermediates during splicing QC. (B) Schematic of HEK.dDHX15 cell line construction and total and nascent chromatin-associated RNA-seq experiments following rapid dTAG13-induced proteolysis of endogenously FKBPF36V degron-tagged DHX15. (C) Immunoblots of total cell lysates from parental HEK293T.A2 cells and two monoclonal HEK.dDHX15 cell lines were treated with DMSO versus dTAG13 at 100 nM for 2 h. (D) Immunoblots of cytoplasm, nucleoplasm, and chromatin fractions, collected upon the preparation of chromatin-associated nascent RNA. β-actin, cytoplasmic marker; SC35, nucleoplasm and nuclear speckle marker; Histon H3, chromatin-associated protein marker. (E) Illustration of how SI is computed by taking the ratio between spliced exon-exon junction reads and normalized total counts of spliced plus unspliced junction-spanning (exon-intron and intron-exon junction) reads. (F) Example chRNA and totalRNA-seq read coverage of the indicated RBM5 intron in control (DMSO) versus dTAG13-treated HEK.dDHX15 cells. ΔSI is shown. (G) Volcano plots of introns with altered splicing efficiency across six biological replicates upon dTAG13-induced DHX15 depletion. Red/blue, introns exhibiting significant (false discovery rate [FDR] ≤ 0.05) increases/decreases of SI ≥ 0.05; gray, introns exhibiting insignificant or unaltered changes in SI. (H) Scatterplot between chromatin-associated splicing efficiency index (SI.ch) and post-transcriptional splicing efficiency index (SI.postTx) across six biological replicates. (I) Scatterplot (left) and empirical cumulative distribution function (eCDF) plot (right) of DHX15-altered introns and their splicing efficiency changes between steady-state and chromatin-associated nascent state (SI.postTx), across six biological replicates. Magenta/navy (up/down), introns exhibiting significant (FDR ≤ 0.05) increases/decreases of SI ≥ 0.05 upon dTAG13-induced DHX15 depletion in HEK.dDHX15 cells; gray (Unch), introns exhibiting insignificant or unaltered changes in SI. Statistical significance is calculated by Welch’s t test, indicated by asterisks ****p < 0.0001), unless otherwise indicated. See also Figure S1.

Article Snippet: dTAG13 , Tocris Bioscience , Fisher Scientific Cat#: 66-055.

Techniques: RNA Sequencing, Western Blot, Marker, Control

Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: (A) Diagram of splicing QC via the rejection route: DHX15 dissembles splicing intermediates rejected by exon-joining helicase DHX38 to facilitate spliceosomal components recycling and intron-lariat degradation. (B) Immunoblots of total cell lysates from parental HEK293T.A2 cells and two monoclonal HEK.dDHX38 cell lines were treated with DMSO versus dTAG13 at 100 nM for 2 h. (C) Volcano plots of introns with altered splicing efficiency averaged across six biological replicates upon dTAG13-induced DHX38 depletion. Red/blue, introns exhibiting significant (FDR ≤ 0.05) increases/decreases of SI ≥ 0.05; gray, introns exhibiting insignificant or unaltered changes in SI. Left, nascent RNA-seq; right, total RNA-seq. (D) Nascent RNA-seq read coverage of the indicated PRPF31 intron in control (DMSO) versus dTAG13-treated HEK.dDHX38 and HEK.dDHX15 cells. ΔSI is shown by taking the SI difference between dTAG13-treated and control cells. (E) Nascent RNA-seq read coverage of SUPT16H’s two introns (as indicated) with different sensitivity to dTAG13-mediated DHX38 versus DHX15 depletions. (F and G) (F) Venn diagram of shared introns (left) with decreased splicing efficiency, and shared genes (right) with introns that exhibit decreased splicing efficiency upon DHX15 and DHX38 depletion, across six biological replicates each. The size of the intersection and odds ratios (ORs) are shown. Statistical significance of the intersection is calculated by Hypergeometric test in R (****p < 0.0001) (G) Gene Ontology (GO) enrichment of the shared and unique substrate genes between DHX38 and DHX15. Enriched Biological Process (BP) GO terms are shown. Adjusted p value was calculAdjusted p value was calculated by Benjamini-Hochberg method. Seeated by Benjamini-Hochberg method. See also Figure S1.

Article Snippet: dTAG13 , Tocris Bioscience , Fisher Scientific Cat#: 66-055.

Techniques: Western Blot, RNA Sequencing, Control

Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: (A) eCDF of splice sites strength MaxEntScan scores. Magenta/navy (up/down), introns exhibiting significant (FDR ≤ 0.05) increases/decreases of SI ≥ 0.05 upon dTAG13-induced DHX15 depletion in HEK.dDHX15 cells; gray (Unch), introns exhibiting insignificant or unaltered changes in SI. 5ss, 5′ splice site; 3ss, 3′ splice site. (B) Similar to (A), the distribution of counts of branchpoint site (BPS) per intron. (C) Similar to (B), the distribution of distances between BPS and 3ss for each BPS-3ss pair. Statistical significance in (A)–(C) is calculated by Welch’s t test, indicated by asterisks ****p < 0.0001; ns, not significant) unless otherwise indicated. (D and E) (D) Fraction of proximal versus distal alternative 3ss and 5ss usage and (E) fraction of cryptic splicing status in the down, up, or unchanged intron groups upon dTAG13-induced DHX15 depletion. SJ, splicing junction. Statistical significance in (D)–(E) is calculated by chi-squared test, indicated by asterisks (****p < 0.0001, ***p< 0.001, **p < 0.01) unless otherwise indicated. See also Figure S2.

Article Snippet: dTAG13 , Tocris Bioscience , Fisher Scientific Cat#: 66-055.

Techniques:

Journal: Cell reports

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

doi: 10.1016/j.celrep.2023.113223

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: dTAG13 , Tocris Bioscience , Fisher Scientific Cat#: 66-055.

Techniques: Recombinant, Protease Inhibitor, Magnetic Beads, Cloning, Isolation, CRISPR, Software, Imaging

Journal: bioRxiv

Article Title: The pluripotency factor Tex10 finetunes Wnt signaling for PGC and male germline development

doi: 10.1101/2023.02.23.529824

Figure Lengend Snippet: (A) Relative expression levels of Tex10 in mouse tissue/cell line samples (n=30). The male and female PGCs are indicated in orange and green, respectively. Data were obtained from the publicly available dataset . (B) Relative expression levels of Tex10 on day 1, day 3, day 7 testes, and spermatocytes from adult testes. The data were curated from GSE83264. (C) Schematic representation of the Tex10 degron system. Briefly, the Tex10 C terminus was edited using the CRISPR/Cas9 scissor to integrate degron system elements, and the Cre-loxP system was further used to delete the dsRed element. (D) Western blot validation on two cell clones (C1 and C2) of the Tex10 degron system. The top arrow indicates Tex10-HA-tagged FKBP12 F36V protein with a size of around 118 kDa, and the second arrow indicates the wildtype Tex10 protein of 106 kDa. (E) Cellular morphology of embryoid bodies (EBs) treated with DMSO or dTAG13 during six days of in vitro PGCLC specification from EpiLCs. (F) Flow cytometry analysis of PGCLC specification efficiency using cell surface markers SSEA1 and CD61. Percentages of double-positive (SSEA1 + and CD61 + ) cells are indicated at day 2 and day 6 of PGCLC induction for clone C1. (G-H) Quantification of double-positive (SSEA1 + and CD61 + ) percentage in live cells (G) and cell numbers per 20,000 analyzed cells (H) shown with bar plots. Two cell clones C1 and C2 were used as biological replicates, and an ANOVA test was used to detect significance. (I-K) Flow cytometry analysis of PGCLC specification efficiency using cell surface markers SSEA1 and CD61. Percentages of double-positive (SSEA1 + and CD61 + ) cells are indicated at day 6 of PGCLC induction (I). Quantification of double-positive percentage in live cells (J) and cell numbers per 20,000 analyzed cells (K) are shown with bar plots. Two cell clones C1 and C2 were used as biological replicates, and a paired t-test was used to detect significance. Withdrawal for depleting Tex10 only at day one of PGCLC induction and then transferring EBs into medium without dTAG13 to restore Tex10 expression; No Withdrawal for depleting Tex10 for six days of PGCLC induction.

Article Snippet: Expanded clones were treated with or without dTAG13 (500 nM, Toronto Research Chemicals, D710020) and compared with wildtype J1 mESCs for Tex10 expression.

Techniques: Expressing, CRISPR, Western Blot, Biomarker Discovery, Clone Assay, In Vitro, Flow Cytometry, Transferring

Journal: bioRxiv

Article Title: The pluripotency factor Tex10 finetunes Wnt signaling for PGC and male germline development

doi: 10.1101/2023.02.23.529824

Figure Lengend Snippet: (A) Volcano plots show the comparison of transcriptomic profiles of day 2 PGCLC (D2EB) vs. EpiLC stage (bottom volcano), and dTAG13-treated day 2 PGCLC (dD2EB) vs. control (D2EB; top volcano). Venn diagrams show the intersections between 1831 genes upregulated in D2EB vs. EpiLC (log 2 FC > 1 & p-value < 0.05) and 1392 genes downregulated in dD2EB vs. D2EB (log 2 FC < −1 & p-value < 0.05), and between 1278 genes downregulated in D2EB vs. EpiLC (log 2 FC < −1 & p-value < 0.05) and 2454 genes upregulated in dD2EB vs. D2EB (log 2 FC > 1 & p-value < 0.05). Enriched gene ontology biological processes are shown for the two overlapping gene sets. (B) Heatmaps for gene expression profiles of pluripotency, PGC, ectoderm, mesoderm, and endoderm regulation markers in D2EBs vs. dD2EBs. (C) Western blots of key PGC markers in EpiLCs, D2EBs, and dD2EBs. Data from two Tex10-degron clones (C1 & C2) are shown with dTAG13 added at the EpiLC stage and Tex10 depletion happened in dD2EBs. Quantification values for Otx2 relative to Vinculin are shown on top of bands for D2 and dD2 EB samples. (D) Gene set enrichment analysis showing that Wnt signaling but not Bmp signaling is activated by Tex10 depletion on day 2 and day 4 of PGCLC induction. (E) Flow cytometry analysis of PGCLC specification efficiency using cell surface markers SSEA1 and CD61. Percentages of double positive (SSEA1 + and CD61 + ) cells are indicated at day 2 and day 6 of PGCLC induction for clone C1. Quantification of double-positive percentages in live cells and numbers per 20,000 analyzed cells are shown with bar plots. Two cell clones C1 and C2 were used as biological replicates, and an ANOVA test was used to detect significance.

Article Snippet: Expanded clones were treated with or without dTAG13 (500 nM, Toronto Research Chemicals, D710020) and compared with wildtype J1 mESCs for Tex10 expression.

Techniques: Comparison, Control, Gene Expression, Western Blot, Clone Assay, Flow Cytometry

Journal: bioRxiv

Article Title: The pluripotency factor Tex10 finetunes Wnt signaling for PGC and male germline development

doi: 10.1101/2023.02.23.529824

Figure Lengend Snippet: (A) Venn diagram showing the overlap between Tex10 and H3K4me3 ChIP-seq peaks at the D2PGCLC stage. (B) Venn diagram showing the intersection between Tex10-bound/H3K4me3-marked genes and genes downregulated upon Tex10 depletion. (C) Gene ontology biological processes enriched in the 322 genes shown in B. (D) Genome browser tracks of Tex10 and H3K4me3 ChIP-seq signals near Trim27, Tyro3, Psmd3 , and Psmd7 gene loci in D2EBs. Green shaded regions indicate the Tex10 peaks. (E) ChIP-qPCR validation of Tex10 binding and H3K4me3 mark at the promoter regions of Psmd3 and Psmd7 . Tex10 ChIP experiment was performed with an anti-HA tag antibody, and IgG served as a negative control.” (F) Flow cytometry analysis of PGCLC specification efficiency using cell surface markers SSEA1 and CD61. Percentages of double-positive (SSEA1 + and CD61 + ) cells are indicated at day 6 of PGCLC induction for control shRNA (shCtrl) and Psmd7 shRNA (shKD). Quantification of double-positive percentages in live cells and numbers per 20,000 analyzed cells are shown with bar plots. N = 2 biological replicates per condition (two control shRNAs vs. two Psmd7 shRNAs), and an unpaired t-test was used to detect significance. (G) Flow cytometry analysis of PGC specification efficiency using the cell surface markers SSEA1 and CD61. Percentages of double-positive (SSEA1 + and CD61 + ) cells are indicated at day 6 of PGCLC induction for DMSO, dTAG13, and dTAG13 plus Psmd7 ectopic expression (dTAG13+Psmd7) treatment. Quantification of double-positive percentages in live cells and numbers per 20,000 analyzed cells are shown with bar plots. Two cell clones C1 and C2 were used as biological replicates, and a paired t-test was used to detect significance.

Article Snippet: Expanded clones were treated with or without dTAG13 (500 nM, Toronto Research Chemicals, D710020) and compared with wildtype J1 mESCs for Tex10 expression.

Techniques: ChIP-sequencing, ChIP-qPCR, Biomarker Discovery, Binding Assay, Negative Control, Flow Cytometry, Control, shRNA, Expressing, Clone Assay