Journal: Cancer Immunology Research

Article Title: HDAC3 inhibition promotes anti-tumor immunity by enhancing CXCL10 mediated chemotaxis and recruiting of immune cells

doi: 10.1158/2326-6066.cir-22-0317

Figure Lengend Snippet: Figure 2. Hdac3 deficiency suppresses tumor growth and is dependent on host immune responses. A, WT, Hdac1/, Hdac2/, and Hdac3/ MC38 cells were inoculated in C57BL/6 mice subcutaneously, and tumor growth was monitored (n ¼ 5/group). WT, Hdac3þ/, and Hdac3/ MC38 and MCA205 cells were inoculated in C57BL/6 (B and C) and nude mice (D and E) subcutaneously, and tumor growth was monitored (n ¼ 5/group). Hdac3 knockdown and knockout was confirmed via Western blot analysis (top, B and C). F, WT and Hdac3/ MCA205 cells were injected into C57BL/6 mice intravenously, and the OS of mice was monitored (n ¼ 8/group). G–I, The OS of patients with gastric cancer was compared between individuals bearing tumors with high or low Hdac1, 2, 3 mRNA transcription. HR, 95% confidence interval, and P value from the log-rank test are shown. Western blot images (B and C) are presentative results from three independent experiments. Experiments (A– F) were repeated two times, and the dot plots (A–E) are shown as mean SD. Significance was calculated with nonparametric Mann–Whitney U test. ns, not significant; , P < 0.01; , P < 0.001.

Article Snippet: To generate wild-type (WT) and H134/135Q-mutant HDAC3overexpressing cells, WT and H134/135Q-mutant HDAC3 coding sequences were cloned and inserted into VP64-GFP (Addgene_70228) vector by standard molecular procedure.

Techniques: Knockdown, Knock-Out, Western Blot, Injection, MANN-WHITNEY

Journal: Cancer Immunology Research

Article Title: HDAC3 inhibition promotes anti-tumor immunity by enhancing CXCL10 mediated chemotaxis and recruiting of immune cells

doi: 10.1158/2326-6066.cir-22-0317

Figure Lengend Snippet: Figure 4. Hdac3 deficiency upregulates chemokine signaling and TCR signaling pathways. A–F, Total RNA from WT or Hdac3þ/ MCA205 tumors (n ¼ 3/group) was extracted and sequenced by RNA-seq. Expression changes in Hdac3þ/ versus WT MCA205 whole tumors were compared by KEGG analysis (A and B). Significantly enriched KEGG annotated classification bar chart (A) and distribution point map (B) are shown. Expression changes in Hdac3þ/ versus WT MCA205 tumors were compared by GSEA (C–E). Chemokine signaling (C), cytokine–cytokine receptor interaction (D), and TCR signaling pathways (E) are shown. Heat map shows differentially expressed genes associated with the chemokine signaling pathway and T cells in WT and Hdac3þ/ MCA205 tumors (F). The RNA-seq was performed once.

Article Snippet: To generate wild-type (WT) and H134/135Q-mutant HDAC3overexpressing cells, WT and H134/135Q-mutant HDAC3 coding sequences were cloned and inserted into VP64-GFP (Addgene_70228) vector by standard molecular procedure.

Techniques: Protein-Protein interactions, RNA Sequencing, Expressing

Journal: Cancer Immunology Research

Article Title: HDAC3 inhibition promotes anti-tumor immunity by enhancing CXCL10 mediated chemotaxis and recruiting of immune cells

doi: 10.1158/2326-6066.cir-22-0317

Figure Lengend Snippet: Figure 6. HDAC3 directly binds and deacetylates Cxcl10 gene promotor. A and B, The Cxcl10 gene promotor loci are displayed. Tracks depict HDAC3 ChIP (A) signal, as well as ChIP for H3K27ac (B) in WT, Hdac3þ/, and Hdac3/ MCA205 cells treated with IFNb (10 ng/mL) or vehicle control treatment for 3 hours. Cxcl10 promotor occupancy of HDAC3 (C)and H3K27ac (D) proteins in WT, Hdac3þ/, andHdac3/MCA205 cells analyzed by ChIP q-PCR. Eand F, TheCxcl10gene promotorloci are displayed. Tracks depict HDAC3 ChIP (E) signal, as well as ChIP for H3K27ac (F) in Hdac3/ MCA205 cells overexpressing WT or H134/135Q mutant HDAC3, or vector control. Cxcl10 promotor occupancy of HDAC3 (G) and H3K27ac (H) proteins in Hdac3/ MCA205 cells overexpressing WT or H134/135Q-mutant HDAC3, or vector control as analyzed by ChIP q-PCR. I, The mRNA expression of Cxcl10 in Hdac3/ MCA205 cells overexpressing WT, H134/135Q mutant HDAC3 or vector control as analyzed by qRT-PCR. MCA205 cells were treated with 100 U/mL IFNa, 10 ng/mL IFNb, or 100 U/mL IFNg for 6 hours before being lysed for RNA extraction. J–K, WT MCA205 and vector control, WT and H134/135Q-mutant HDAC3 overexpressing Hdac3/ MCA205 cells were inoculated in C57BL/6 mice or nude mice subcutaneously (n ¼ 5/group), and tumor growth was monitored. ChIP q-PCR (C, D, G, H) and qRT-PCR (I) were repeated two times, and data are shown as mean SD. Experiments (J and K) were repeated two times, and the dot plots are shown as mean SD. Significance was calculated with nonparametric Mann– Whitney U test. ns, not significant; , P < 0.05; , P < 0.001; and , P < 0.0001. NC; negative control.

Article Snippet: To generate wild-type (WT) and H134/135Q-mutant HDAC3overexpressing cells, WT and H134/135Q-mutant HDAC3 coding sequences were cloned and inserted into VP64-GFP (Addgene_70228) vector by standard molecular procedure.

Techniques: Control, Mutagenesis, Plasmid Preparation, Expressing, Quantitative RT-PCR, RNA Extraction, MANN-WHITNEY, Negative Control

Journal: Cell Genomics

Article Title: Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing

doi: 10.1016/j.xgen.2025.100770

Figure Lengend Snippet:

Article Snippet: The lentiviral VP64 dCas9 VP64 plasmid (Addgene #232179) was generated by modifying Addgene #59791 to replace GFP with BSD blasticidin resistance.

Techniques: DNA Methylation Assay, Sequencing, Recombinant, Plasmid Preparation, Expressing, Software

Journal: Nature biotechnology

Article Title: Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers

doi: 10.1038/nbt.3199

Figure Lengend Snippet: The dCas9 p300 Core fusion protein activates transcription of endogenous genes from proximal promoter regions. ( a ) Schematic of dCas9 fusion proteins dCas9 VP64 , dCas9 FL p300 , and dCas9 p300 Core . Streptococcus pyogenes dCas9 contains nuclease inactivating mutations D10A and H840A. The D1399 catalytic residue in the p300 HAT domain is indicated. ( b ) Western blot showing expression levels of dCas9 fusion proteins and GAPDH in co-transfected cells (full blot shown in ). ( c ) Relative mRNA expression of IL1RN , MYOD , and OCT4 , determined by qRT-PCR, by the indicated dCas9 fusion protein co-transfected with four gRNAs targeted to each promoter region (Tukey-test, * P -value < 0.05, n = 3 independent experiments each, error bars: s.e.m.). Numbers above bars indicate mean expression. FLAG, epitope tag; NLS, nuclear localization signal; HA, hemagglutinin epitope tag; CH, cysteine-histidine-rich region; Bd, bromodomain; HAT, histone acetyltransferase domain.

Article Snippet: An HA epitope tag was added to dCas9 (no effector) by removing the VP64 effector domain from dCas9 VP64 via AscI/PacI restriction sites and using isothermal assembly to include an annealed set of oligos containing the appropriate sequence as per manufacturers instruction (NEB cat. #2611). pcDNA-dCas9 FLp300 (dCas9 FLp300 ) was created by amplifying full-length p300 from pcDNA3.1-p300 (Addgene, plasmid #23252) in two separate fragments and cloning these fragments into the dCas9 VP64 backbone via isothermal assembly.

Techniques: Residue, Western Blot, Expressing, Transfection, Quantitative RT-PCR, FLAG-tag

Journal: Nature biotechnology

Article Title: Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers

doi: 10.1038/nbt.3199

Figure Lengend Snippet: The dCas9 p300 Core fusion protein activates transcription of endogenous genes from distal enhancer regions. ( a ) Relative MYOD mRNA production in cells co-transfected with a pool of gRNA s targeted to either the proximal or distal regulatory regions and dCas9 VP64 or dCas9 p300 Core ; promoter data from (Tukey-test, * P -value <0.05 compared to mock-transfected cells, Tukey test † P -value <0.05 between dCas9 p300 Core and dCas9 VP64 , n = 3 independent experiments, error bars: s.e.m.). The human MYOD locus is schematically depicted with corresponding gRNA locations in red. CE, MyoD core enhancer; DRR, MyoD distal regulatory region. ( b ) Relative OCT4 mRNA production in cells co-transfected with a pool of gRNAs targeted to the proximal and distal regulatory regions and dCas9 VP64 or dCas9 p300 Core ; promoter data from (Tukey-test, * P -value <0.05 compared to mock-transfected cells, Tukey test † P -value <0.05 between dCas9 p300 Core and dCas9 VP64 , n = 3 independent experiments, error bars: s.e.m.). The human OCT4 locus is schematically depicted with corresponding gRNA locations in red. DE, Oct4 distal enhancer; PE, Oct4 proximal enhancer. ( c ) The human β-globin locus is schematically depicted with approximate locations of the hypersensitive site 2 (HS2) enhancer region and downstream genes ( HBE , HBG , HBD , and HBB ). Corresponding HS2 gRNA locations are shown in red. Relative mRNA production from distal genes in cells co-transfected with four gRNAs targeted to the HS2 enhancer and the indicated dCas9 proteins. Note logarithmic y-axis and dashed red line indicating background expression (Tukey test among conditions for each β-globin gene, † P -value <0.05, n = 3 independent experiments, error bars: s.e.m.). n.s., not significant.

Article Snippet: An HA epitope tag was added to dCas9 (no effector) by removing the VP64 effector domain from dCas9 VP64 via AscI/PacI restriction sites and using isothermal assembly to include an annealed set of oligos containing the appropriate sequence as per manufacturers instruction (NEB cat. #2611). pcDNA-dCas9 FLp300 (dCas9 FLp300 ) was created by amplifying full-length p300 from pcDNA3.1-p300 (Addgene, plasmid #23252) in two separate fragments and cloning these fragments into the dCas9 VP64 backbone via isothermal assembly.

Techniques: Transfection, Expressing

Journal: Nature biotechnology

Article Title: Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers

doi: 10.1038/nbt.3199

Figure Lengend Snippet: dCas9 p300 Core targeted transcriptional activation is specific and robust. ( a–c ) MA plots generated from DEseq2 analysis of genome-wide RNA-seq data from HEK293T cells transiently co-transfected with dCas9 VP64 ( a ) dCas9 p300 Core ( b ) or dCas9 p300 Core (D1399Y) ( c ) and four IL1RN promoter-targeting gRNAs compared to HEK293T cells transiently co-transfected with dCas9 and four IL1RN promoter-targeting gRNAs. mRNAs corresponding to IL1RN isoforms are shown in blue and circled in each panel. Red labeled points in panels b–c correspond to off-target transcripts significantly enriched after multiple hypothesis testing ( KDR, (FDR = 1.4 × 10 −3 ); FAM49A, (FDR = 0.04); p300, (FDR = 1.7 × 10 −4 ) in panel b ; and p300, (FDR = 4.4 × 10 −10 ) in panel c .

Article Snippet: An HA epitope tag was added to dCas9 (no effector) by removing the VP64 effector domain from dCas9 VP64 via AscI/PacI restriction sites and using isothermal assembly to include an annealed set of oligos containing the appropriate sequence as per manufacturers instruction (NEB cat. #2611). pcDNA-dCas9 FLp300 (dCas9 FLp300 ) was created by amplifying full-length p300 from pcDNA3.1-p300 (Addgene, plasmid #23252) in two separate fragments and cloning these fragments into the dCas9 VP64 backbone via isothermal assembly.

Techniques: Activation Assay, Generated, Genome Wide, RNA Sequencing, Transfection, Labeling

Journal: Nature biotechnology

Article Title: Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers

doi: 10.1038/nbt.3199

Figure Lengend Snippet: The dCas9 p300 Core fusion protein acetylates chromatin at a targeted enhancer and corresponding downstream genes. ( a ) The region encompassing the human β-globin locus on chromosome 11 (5,304,000 – 5,268,000; GRCh37/hg19 assembly) is shown. HS2 gRNA target locations are indicated in red and ChIP-qPCR amplicon regions are depicted in black with corresponding green numbers. ENCODE/Broad Institute H3K27ac enrichment signal in K562 cells is shown for comparison. Magnified insets for the HS2 enhancer, HBE , and HBG1/2 promoter regions are displayed below. ( b–d ) H3K27ac ChIP-qPCR enrichment (relative to dCas9; red dotted line) at the HS2 enhancer, HBE promoter, and HBG1/2 promoters in cells co-transfected with four gRNAs targeted to the HS2 enhancer and the indicated dCas9 fusion protein. HBG ChIP amplicons 1 and 2 amplify redundant sequences at the HBG1 and HBG2 promoters (denoted by ‡). Tukey test among conditions for each ChIP-qPCR region, * P -value <0.05 (n = 3 independent experiments, error bars: s.e.m.).

Article Snippet: An HA epitope tag was added to dCas9 (no effector) by removing the VP64 effector domain from dCas9 VP64 via AscI/PacI restriction sites and using isothermal assembly to include an annealed set of oligos containing the appropriate sequence as per manufacturers instruction (NEB cat. #2611). pcDNA-dCas9 FLp300 (dCas9 FLp300 ) was created by amplifying full-length p300 from pcDNA3.1-p300 (Addgene, plasmid #23252) in two separate fragments and cloning these fragments into the dCas9 VP64 backbone via isothermal assembly.

Techniques: ChIP-qPCR, Amplification, Comparison, Transfection

Journal: Nature biotechnology

Article Title: Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers

doi: 10.1038/nbt.3199

Figure Lengend Snippet: The dCas9 p300 Core fusion protein activates transcription of endogenous genes from regulatory regions with a single gRNA. ( a–c ) Relative ( a ) IL1RN, ( b ) MYOD or ( c ) OCT4 mRNA produced from cells co-transfected with dCas9 p300 Core or dCas9 VP64 and gRNAs targeting respective promtoters (n = 3 independent experiments, error bars: s.e.m.). Relative MYOD ( d ) or OCT4 ( e ) mRNA produced from cells co-transfected with dCas9 p300 Core and indicated gRNAs targeting the indicated MYOD or OCT4 enhancers (n = 3 independent experiments, error bars: s.e.m.). DRR, MYOD distal regulatory region; CE, MYOD core enhancer; PE, OCT4 proximal enhancer; DE, OCT4 distal enhancer. (Tukey test between dCas9 p300 Core and single OCT4 DE gRNAs compared to mock-transfected cells, * P -value <0.05, Tukey test among dCas9 p300 Core and OCT4 DE gRNAs compared to All, † P -value <0.05,). Relative HBE ( f ) or HBG ( g ) mRNA production in cells co-transfected with dCas9 p300 Core and the indicated gRNAs targeted to the HS2 enhancer (Tukey test between dCas9 p300 Core and single HS2 gRNAs compared to mock-transfected cells, * P -value <0.05, Tukey test among dCas9 p300 Core and HS2 single gRNAs compared to All, † P <0.05, n = 3 independent experiments, error bars: s.e.m.). HS2, β-globin locus control region hypersensitive site 2; n.s., not significant using Tukey test.

Article Snippet: An HA epitope tag was added to dCas9 (no effector) by removing the VP64 effector domain from dCas9 VP64 via AscI/PacI restriction sites and using isothermal assembly to include an annealed set of oligos containing the appropriate sequence as per manufacturers instruction (NEB cat. #2611). pcDNA-dCas9 FLp300 (dCas9 FLp300 ) was created by amplifying full-length p300 from pcDNA3.1-p300 (Addgene, plasmid #23252) in two separate fragments and cloning these fragments into the dCas9 VP64 backbone via isothermal assembly.

Techniques: Produced, Transfection, Control

Journal: Nature biotechnology

Article Title: Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers

doi: 10.1038/nbt.3199

Figure Lengend Snippet: The p300 Core can be targeted to genomic loci by diverse programmable DNA-binding proteins. ( a ) Schematic of the Neisseria meningitidis (Nm) dCas9 fusion proteins Nm -dCas9 VP64 and Nm -dCas9 p300 Core . Neisseria meningitidis dCas9 contains nuclease-inactivating mutations D16A, D587A, H588A, and N611A. ( b–c ) Relative ( b ) HBE or ( c ) HBG mRNA in cells co-transfected with five individual or pooled (A–E) Nm gRNAs targeted to the HBE or HBG promoter and Nm -dCas9 VP64 or Nm -dCas9 p300 Core . ( d–e ) Relative ( d ) HBE or ( e ) HBG mRNA in cells co-transfected with five individual or pooled (A–E) Nm gRNAs targeted to the HS2 enhancer and Nm -dCas9 VP64 or Nm -dCas9 p300 Core . ( f ) Schematic of TALEs with domains containing IL1RN -targeted repeat variable diresidues (Repeat Domain). ( g ) Relative IL1RN mRNA in cells transfected with individual or pooled (A–D) IL1RN TALE VP64 or IL1RN TALE p300 Core encoding plasmids. ( h ) Schematic of ZF fusion proteins with zinc finger helices 1–6 (F1–F6) targeting the ICAM1 promoter. ( i ) Relative ICAM1 mRNA in cells transfected with ICAM1 ZF VP64 or ICAM1 ZF p300 Core . Tukey-test, * P -value <0.05 compared to mock-transfected control, n = 3 independent experiments each, error bars: s.e.m. NLS, nuclear localization signal; HA, hemagglutinin tag; Bd, bromodomain; CH, cysteine-histidine-rich region; HAT, histone acetyltransferase domain.

Article Snippet: An HA epitope tag was added to dCas9 (no effector) by removing the VP64 effector domain from dCas9 VP64 via AscI/PacI restriction sites and using isothermal assembly to include an annealed set of oligos containing the appropriate sequence as per manufacturers instruction (NEB cat. #2611). pcDNA-dCas9 FLp300 (dCas9 FLp300 ) was created by amplifying full-length p300 from pcDNA3.1-p300 (Addgene, plasmid #23252) in two separate fragments and cloning these fragments into the dCas9 VP64 backbone via isothermal assembly.

Techniques: DNA Binding Assay, Transfection, Control

Journal: bioRxiv

Article Title: Pathogen-driven CRISPR screens identify TREX1 as a regulator of DNA self-sensing during influenza virus infection

doi: 10.1101/2023.02.07.527556

Figure Lengend Snippet: A , Cartoon of re-engineered TRPPC NS genome segment and TRPPCa-mediated gene expression. The sgRNA directs VP64-dCas9 to specific genome targets while two MS2 hairpins inserted in the sgRNA recruit MCP-p65-HSF1. B , TRPPCa of a luciferase reporter in 293T cells. Cell were transfected with vectors expressing viral genomic RNA for NS, Split NS that lacks an sgRNA, or TRPCC NS targeting the reporter promoter. Activation was measured in the presence (+RNP, right) or absence (left) of the viral replication machinery. C , Multicycle replication of IAV harboring a TRPPC- NS segment in A549 cells. D , Virally delivered sgRNA activates reporter gene expression in a multicycle infection. A549-CRISPRa cells were inoculated with virus encoding the indicated NS segment (MOI = 0.05), or mock treated, and luciferase reporter was measured over the course of infection. E , Virally delivered sgRNAs activate expression of host genes from the endogenous locus. A549-CRISPRa cells were inoculated with TRPPC viruses (MOI = 5) targeting the indicated gene, a non-targeting control (C) or mock. Host gene expression was measured at 8 hpi via RT-qPCR. F , A pool of 34 TRPPC viruses targeting a collection of 10 potential pro- or antiviral host genes were subject to 4 rounds of selection in A549-CRISPRa cells cells. Viruses present at each stage of selection were quantified by deep-sequencing and normalized sgRNA composition is depicted. Viruses activating proviral genes enriched at least 3-fold are colored green, while viruses activating antiviral genes that are depleted at least 3-fold are colored red. Graph is representative of mean values for 2 replicate screens. G , TRPPCa screens are highly reproducible. Comparison of two biological replications shows nearly identical relative enrichment of TRPPC viruses targeting the indicated host genes after 4 rounds of selection. H , TRPPC results reflect changes in viral replication. Multicycle replication in A549-CRISPRa cells of individual TRPPC viruses targeting specific host genes (MOI = 0.01). Data are shown as grand mean of 3 replicates ± SEM (B, D) or mean ± s.d. (C, E, F, H). T tests (B), two-way ANOVA with Dunnett’s multiple comparisons test against WT (C, D, H), and one-way ANOVA with Dunnett’s multiple comparisons tests (E) were performed (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: Lentivirus packaging plasmids included psPAX2 (Addgene 12260), pCMV-VSV-G (Addgene 8454), pLenti dCAS-VP64_Blast (Addgene 61524), pLenti MS2-P65-HSF1_Hygro (Addgene 61426), and pLX304 (dCas9-VP64, MS2-p65-HSF1).

Techniques: Expressing, Luciferase, Transfection, Activation Assay, Infection, Quantitative RT-PCR, Selection, Sequencing