Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Schematic representation of generation of Tet2 -knockout clones with CRISPR/Cas9. ( B ) Proliferative outgrowth of Cebpa p30/p30 cells with Tet2 indels. ( C ) Volcano plot depicting differentially expressed genes dependent on the Tet2 mutational status in Cebpa p30/p30 cells (n=5–7 per group). ( D ) Experimental setup for evaluating the effect of Tet2 -deficiency ( Tet2 −/− ) in Cebpa DM AML initiation in vivo . ( E ) Myeloid (Mac1 + ) contribution of donor-derived blood and bone marrow (BM) cells evaluated at 12, 24, and 36 weeks after BM transplantation and Cre-LoxP recombination to generate a Cebpa −/p30 and Tet2 −/− hematopoietic compartment (n=3–6 per genotype and timepoint). ( F ) Survival of lethally irradiated recipient mice after BM transplantation and Cre-LoxP recombination (n=12–14/group). ( G ) Volcano plot depicting differentially expressed genes dependent on Tet2 deficiency status in Cebpa −/p30 leukemic blasts (n=3 per group). ( H ) Frequency of proliferating (Ki67 + ) cells in BM of moribund recipient mice (n=3 per group). **=P<0.01, ***=P<0.001, ****=P<0.0001

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Knock-Out, Clone Assay, CRISPR, In Vivo, Derivative Assay, Transplantation Assay, Irradiation

Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Experimental setup for evaluating the effect of Gata2 knockdown, via short hairpin RNA (shRNA) mediated silencing, on Cebpa p30/p30 leukemic cells in a competitive in vivo assay. ( B ) Gata2 mRNA in Cebpa p30/p30 leukemic cells prior to transplantation. ( C ) Representative flow cytometry profiles of input and output of shControl (no knockdown), sh Gata2 A (low knockdown), and sh Gata2 D (high knockdown). ( D ) Competitive advantage of targeting shRNA (GFP + ) vs. non-targeting shRNA (YFP + ) cells in vivo assessed as by flow cytometry (n=3–4 per group). ( E ) Experimental setup for Gata2 CRISPR/Cas9 mutagenesis in Cebpa p30/p30 cells, and outgrowth of heterozygous mutated clones. Percentages of Gata2 mutated clones are indicated. ( F ) Growth curve of Cebpa p30/p30 clones with Gata2 mutation ( Cebpa p30/p30 Gata2 +/MUT ) or wild type Gata2 ( Cebpa p30/p30 Gata2 +/+ ). Red lines mark individual clones. (G) Presence or absence of GATA2 mutations ( GATA2 MUT ) in CEBPA double mutated ( CEBPA DM ) AML cases (n=460) with or without TET2 mutations ( TET2 MUT ) in aggregated data from published cohorts – , , , . *=P<0.05, **=P<0.01

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Knockdown, shRNA, In Vivo, Transplantation Assay, Flow Cytometry, CRISPR, Mutagenesis, Clone Assay

Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Gata2 mRNA expression in mouse Cebpa p30/p30 leukemic granulocyte/monocyte progenitors (GMPs) vs normal GMPs and, ( B ) CEBPA binding to the Gata2 distal hematopoietic enhancer ( G2 DHE; −77kb) region, data from Jakobsen et al. (n=2–4 per group). ( C ) Schematic genomic view of the Gata2 distal hemeatopoietic enhancer ( G2 DHE), including tracks from CEBPA and H3K27Ac chromatin immunoprecipitation sequencing (ChIP-seq) in Cebpa p /p30 cells (data from Heyes et al. ), TET2 ChIP-seq in AML-ETO expressing cells (data from Rasmussen et al. ),Targeting of the G2 DHE by dual-( D ) or single-( E ) guided CRISPR-Cas9 in Cebpa p30/p30 cells using indicated sgRNAs (n=3/condition). ( F ) Experimental setup for evaluating the effects of Cebpa knockout on Gata2 V2 mRNA expression and DNA methylation of the CpG island at the promoter of Gata2 V2 in MLL-fusion driven AML ( iMLL-AF9 ). ( G ) Cebpa and ( H ) Gata2 V2 mRNA expression upon induction of Cre-LoxP recombination and, ( I ) DNA methylation of the Gata2 V2 promoter CpG-island (2 biological replicates per genotype). ( J ) Frequency of GATA2 and/or TET2 mutations ( GATA2 MUT and TET2 MUT , respectively) in CEBPA high expressing ( CEBPA HIGH n=45) vs. CEBPA low expressing (CEBPA LOW n=61) AML cases, data from Beat AML cohort . *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Expressing, Binding Assay, ChIP-sequencing, CRISPR, Knock-Out, DNA Methylation Assay

Journal: bioRxiv

Article Title: Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B cell fate decisions and yields T cell-depleted lymphomas

doi: 10.1101/2023.02.13.528351

Figure Lengend Snippet: A , Odds ratio (OR) analysis of CREBBP and KMT2D mutations among EZB/Cluster3 DLBCL (n=319) and FL_all (merged FL datasets, n=478) cohorts of patients. The p values were calculated by Fisher’s exact test. B , Representative H&E and B220 IHC images of formalin-fixed paraffin-embedded kidney and liver sections prepared from mice euthanized at day 116 post-BMT. The scale bars represent 380 pixels. C , Representative H&E, B220 and Ki67 IHC images of formalin-fixed paraffin-embedded spleen sections from mice euthanized at day 235 post-BMT. The scale bars represent 200 pixels. D , Representative FACS plots show the gating strategy and frequency of B220 + CD38 - FAS + splenic GC B cells in mice at day 235 post-BMT. E , FACS analysis showing the relative abundance of splenic total B cells (B220 + ) normalized to total single cells at day 116 and 235 post-BMT (mean ± SD). Each dot represents a mouse (n=4 mice per genotype).

Article Snippet: HiFi Cas9 Nuclease (IDT; 1081061), Alt-R CRISPR-Cas9 tracrRNA (IDT; 1072534), and Alt-R CRISPR-Cas9 crRNA targeting CREBBP exon 26 or KMT2D exon 4, were assembled in vitro following the manufacturer’s protocol and delivered into OCI-Ly7 cells by electroporation using SF Cell Line 96-well Nucleofector Kit (Lonza; V4SC-2096). ssODN for CREBBP R1446C mutagenesis was added along with RNP complex during electroporation as needed.

Techniques: Formalin-fixed Paraffin-Embedded

Journal: bioRxiv

Article Title: Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B cell fate decisions and yields T cell-depleted lymphomas

doi: 10.1101/2023.02.13.528351

Figure Lengend Snippet: A , Experimental design for generating CREBBP -KO and CREBBP -R1446C, an enzymatically dead mutant, OCI-Ly7 cell lines. ssODN: single-stranded oligodeoxynucleotide. B , Sanger sequencing chromatogram confirming the homozygous knock out or R1446C point mutation in CREBBP . C , Experimental design for generating KMT2D -KO OCI-Ly7 cell line. D , Sanger sequencing chromatogram confirming the homozygous knock out of KMT2D . E , Immunoblotting for endogenous CREBBP and KMT2D in the indicated isogenic OCI-Ly7 cell lines using MED1 as internal loading control. F , GSEA plot using CK vs WT upregulated genes in OCI-Ly7 as the gene set against a ranked gene list based on CK vs epigenetic WT RNA-seq datasets of human BCCA cohort GCB-DLBCL patients. NES, normalized enrichment score. The p value was calculated by an empirical phenotype-based permutation test. The FDR is adjusted for gene set size and multiple hypotheses testing. G-H , GSEA plots using CK vs epigenetic WT ( G ) downregulated or ( H ) upregulated genes in human BCCA cohort GCB-DLBCL patients as the gene set against a ranked gene list based on CK vs WT OCI-Ly7 RNA-seq datasets. NES, normalized enrichment score. The p value was calculated by an empirical phenotype-based permutation test. The FDR is adjusted for gene set size and multiple hypotheses testing. I , Immunoblot for H3K4me1, H3K27ac and H3 in isogenic OCI-Ly7 cells. J-K , Relative densitometry of ( J ) H3K4me1 and ( K ) H3K27ac for panel I . L , Co-IP for assessing interaction between endogenous CREBBP and KMT2D in human SUDHL4 GCB-DLBCL cell line. M , RT-qPCR of indicated genes in different isogenic OCI-Ly7 cells. qPCR signal for each gene was normalized to those of HPRT and then mean WT and presented as log2 fold-change ± SEM. Statistical significance was determined using ordinary one-way ANOVA followed by Tukey-Kramer’s multiple comparisons test (**p < 0.01, ***p < 0.001, ****p < 0.0001). N , Stacked flow cytometry histograms showing the progressive signal decrease for the indicated surface markers in C, K, and CK-deficient OCI-Ly7 cells compared to WT. O , FACS measuring cell surface levels of the indicated markers in different isogenic OCI-Ly7 cells. Mean fluorescence intensity (MFI) of each surface marker was normalized to mean WT and presented as log2 fold-change ± SEM. Each dot represents a biological replicate. Statistical significance was determined using ordinary one-way ANOVA followed by Tukey-Kramer’s multiple comparisons test (****p < 0.0001).

Article Snippet: HiFi Cas9 Nuclease (IDT; 1081061), Alt-R CRISPR-Cas9 tracrRNA (IDT; 1072534), and Alt-R CRISPR-Cas9 crRNA targeting CREBBP exon 26 or KMT2D exon 4, were assembled in vitro following the manufacturer’s protocol and delivered into OCI-Ly7 cells by electroporation using SF Cell Line 96-well Nucleofector Kit (Lonza; V4SC-2096). ssODN for CREBBP R1446C mutagenesis was added along with RNP complex during electroporation as needed.

Techniques: Mutagenesis, Sequencing, Knock-Out, Western Blot, Control, RNA Sequencing, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Flow Cytometry, Fluorescence, Marker

Journal: bioRxiv

Article Title: Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B cell fate decisions and yields T cell-depleted lymphomas

doi: 10.1101/2023.02.13.528351

Figure Lengend Snippet: A , PCA analysis of RNA-seq datasets done in isogenic human OCI-Ly7 GCB-DLBCL cell lines (n=2 per genotype) using all genes normalized by VST. B , GSEA plot using CK vs WT downregulated genes in OCI-Ly7 as gene set against ranked gene list based on CK vs epigenetic WT (epiWT, no mutations in CREBBP , KMT2D , and EZH2 ) RNA-seq datasets of human BCCA cohort GCB-DLBCL patients. NES, normalized enrichment score. The p value was calculated by an empirical phenotype-based permutation test. The FDR is adjusted for gene set size and multiple hypotheses testing. C , t-SNE dimensionality reduction and K-means clustering of union peaks, generated by taking the union of H3K4me3, H3K4me1, and K3K27ac CUT&RUN signals from all isogenic OCI-Ly7 cell lines (WT, C, K, and CK), produced eight distinct clusters (named as C1-C8). D , t-SNE plots showing the basal level and distribution pattern of the indicated histone marks or RNA in WT OCI-Ly7 cells. The projected values are VST normalized counts. E , Read density changes for the indicated histone marks and RNA were projected onto t-SNE plots. The projected values are log2FC signal between the indicated genotypes. F , Heatmaps showing median VST normalized read density (left) or read density change (median log2FC) in C/K/CK relative to WT (middle and right) of the indicated histone marks or RNA-seq for each cluster defined in panel C . Distance to TSS plot shows the distance of union peaks to their closest TSSs. G-H , Average signal profiles (top) and heatmaps (bottom) displaying ( G ) H3K4me1 and ( H ) H3K27ac CUT&RUN signals around peak summit (+/-15kb) or peak center (+/-5kb), respectively at C1 peak regions. I , Venn diagram displaying overlap between CREBBP and KMT2D ChIP-seq peaks in OCI-Ly7. J , Genomic feature annotation of CREBBP-unique, KMT2D-unique, and CREBBP/KMT2D-co-bound ChIP-seq peaks. The definitions for different genomic features are depicted below the bar plot. K , Co-IP for assessing interaction between endogenous CREBBP and KMT2D in OCI-Ly7. CREBBP, KMT2D and IgG control were immunoprecipitated from OCI-Ly7 nuclear extracts. Western blots were performed using anti-CREBBP and KMT2D antibodies. L-M , ChIP-qPCR quantifying binding of endogenous ( L ) KMT2D and ( M ) CREBBP at the indicated gene loci in the indicated isogenic OCI-Ly7 cell lines. ChIP signals from three independent experiments were normalized to input and then to WT and presented as mean ± SD. The p values were calculated by unpaired t test and BH adjusted for multiple comparisons and denoted as follows: ns p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. N , Functional annotation of C1 genes (n=401) by Enrichr and Toppgene ( , ).

Article Snippet: HiFi Cas9 Nuclease (IDT; 1081061), Alt-R CRISPR-Cas9 tracrRNA (IDT; 1072534), and Alt-R CRISPR-Cas9 crRNA targeting CREBBP exon 26 or KMT2D exon 4, were assembled in vitro following the manufacturer’s protocol and delivered into OCI-Ly7 cells by electroporation using SF Cell Line 96-well Nucleofector Kit (Lonza; V4SC-2096). ssODN for CREBBP R1446C mutagenesis was added along with RNP complex during electroporation as needed.

Techniques: RNA Sequencing, Generated, Produced, ChIP-sequencing, Co-Immunoprecipitation Assay, Control, Immunoprecipitation, Western Blot, ChIP-qPCR, Binding Assay, Functional Assay

Journal: Human Molecular Genetics

Article Title: PD-linked CHCHD2 mutations impair CHCHD10 and MICOS complex leading to mitochondria dysfunction

doi: 10.1093/hmg/ddy413

Figure Lengend Snippet: Generation of human isogenic lines harboring PD-related CHCHD2 mutants. ( A ) PD-associated CHCHD2 mutants T61I, Q126X and R145Q on functional domains of human CHCHD2 protein. CHCH domain. ( B ) Schematic overview depicting human CHCHD2 locus and Cas9 targeting strategy with designed guide RNA recognized exon 3 sequences in human CHCHD2 locus. ( C ) Sanger sequencing chromatogram showing the heterozygous point mutation leading to Q126X+/−. Representative results from Q10 were shown. ( D ) Sanger sequencing chromatogram on the reverse DNA strand showing the heterozygous point mutation leading to R145Q+/− (R4), the homozygous point mutant leading to R145Q−/− (R17) at the targeting locus of H9 (+/+) hESCs. * the blocking nonsense mutation to enhance the specificity of Cas9 system.

Article Snippet: Capped and polyadenylated Cas9 mRNA was obtained from pT7-Cas9 vector (Origene) by mMESSAGE Mmachine T7 ULTRA kit (Thermo Fisher Scientific).

Techniques: Functional Assay, Sequencing, Mutagenesis, Blocking Assay

Journal: International journal of oncology

Article Title: Inhibition of basal cell carcinoma cells by cold atmospheric plasma‑activated solution and differential gene expression analysis.

doi: 10.3892/ijo.2020.5009

Figure Lengend Snippet: Figure 9. After 3, 6 and 12 h treatment with PAS, the expression of cas3/cle‑cas3 and cas9/cle‑cas9 in TE354T cells is analyzed by western blotting. Changes in cas3/cle‑cas3 and cas9/cle‑cas9 protein expression in TE354T cells after PAS treatment by (A) western blotting and (B) subsequent densitometry. **P<0.01 vs. respective con. (C) Changes in the ratio of cle‑cas3/cas3 and cle‑cas9/cas9 after PAS treatment. Data are presented as the mean ± SD of three independent experiments. **P<0.01 vs. respective 0 h. PAS, plasma‑activated solution; cas3, caspase 3; cle, cleaved; cas9, caspase 9; con, control; NS, not significant.

Article Snippet: CAP, cold atmospheric plasma; NS, not significant. antibodies used for Cas3, cleaved Cas3 and cleaved Cas9 were goat rabbit anti‐lgG (cat. no. ZB2301; OriGene Technologies, Inc.); the secondary antibodies used for Cas9 were goat mouse anti‐IgG (cat. no. ZB2305; OriGene Technologies, Inc.).

Techniques: Expressing, Western Blot, Control