Journal: Research Square

Article Title: Malignant A-to-I RNA editing by ADAR1 drives T-cell acute lymphoblastic leukemia relapse via attenuating dsRNA sensing

doi: 10.21203/rs.3.rs-2444524/v2

Figure Lengend Snippet: A-B. Expression of ADAR1 isoforms in patient 076 and patient 081 was measured by RT-qPCR and western blot in Lin − CD34 + LIC− enriched population. C . Expression of MDA5 was determined in Lin − CD34 + LIC-enriched cells of patient 076 and patient 081. D-E. Expression of ADAR1 isoforms in three T-ALL cell lines, CUTTL1, SUP-T1, and Jurkat. F-G . MDA5 and PKR mRNA expression (F) and protein level (G) were determined in T-ALL cell lines. H. Cell counts of shRNA control, shADAR1, shMDA5, and co-knockdown of shADAR1 and shMDA5 were assessed after 3-days post lentiviral transduction. Data from three independent experiments are shown. Error bars represent mean with SEM. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 unpaired Student t-test.

Article Snippet: Lentiviral vectors (pLV-shRNA-EGFP or mCherry:T2A:Puro-U6) was purchased (VectorBuilder) and wild-type and mutant ADAR1 E912A (pCDH-EF1-T2A-copGFP) were produced according to published protocol .

Techniques: Expressing, Quantitative RT-PCR, Western Blot, shRNA, Control, Knockdown, Transduction

Journal: Research Square

Article Title: Malignant A-to-I RNA editing by ADAR1 drives T-cell acute lymphoblastic leukemia relapse via attenuating dsRNA sensing

doi: 10.21203/rs.3.rs-2444524/v2

Figure Lengend Snippet: A. Western blot showing ADAR1 expression in modified Jurkat T-ALL cell lines, including wildtype (wt), ADAR1 KO (KO), and added back wildtype ADAR1 by lentiviral overexpressing vector (add back). b-actin was used as loading control. B . Jurkat cells were stimulated with various concentrations of IFNb and the gene expression of ADAR1 was determined (n = 2 independent experiments). C . Quantification of ADAR1 p150 and p110 isoforms upon IFNb treatment at 10 ng/mL for 24 hours (n = 3 independent experiments). D . Cell viability was quantified in Jurkat cells upon IFNb stimulation (n = 3 independent experiments). E . Western blot showing levels of dsRNA sensor (PRK, MDA5, DDX58) and apoptosis markers (PARP1 and cleaved PARP1). Cells were stimulated with IFNb (1 ng/mL) for 48 hours. b-actin is used as loading control. F . NanoString analysis of gene expression in wildtype (wt), ADAR1 KO, and ADAR1 E912A overexpressing Jurkat cells stimulated with IFNb (1ng/mL, 48 hrs), (n = 2 independent experiments). Insert: western blots showing ADAR1 expression in wildtype, ADAR1 KO, and ADAR1 E912A add back cells. G, Immunofluorescent staining to detect the localization of dsRNA (J2 antibody) in wildtype (wt), ADAR1 KO, and ADAR1 E912A Jurkat cells stimulated with IFNb (1 ng/mL, 24 hrs). H . Quantification of total dsRNA dots and percentage of nuclear dsRNA from Jurkat cells treated with IFNb (1 ng/mL, 24 hrs, 10 cells/condition. I . Model of pathways mediated by ADAR1 in T-ALL LICs. *p<0.05, **p<0.01, and ****p<0.0001, unpaired student t-test. Error bars represent mean with SEM in all graphs. Error bars represent mean with SEM in all graphs.

Article Snippet: Lentiviral vectors (pLV-shRNA-EGFP or mCherry:T2A:Puro-U6) was purchased (VectorBuilder) and wild-type and mutant ADAR1 E912A (pCDH-EF1-T2A-copGFP) were produced according to published protocol .

Techniques: Western Blot, Expressing, Modification, Plasmid Preparation, Control, Gene Expression, Staining

Journal: bioRxiv

Article Title: Engineered SMCHD1 and D4Z4 mutations reveal roles of D4Z4 heterochromatin disruption and feedforward DUX4 network activation in FSHD

doi: 10.1101/2022.10.14.512332

Figure Lengend Snippet: A. Upregulation of DUX4 target genes are comparable in both haplotypes at the myoblast stage. RT-qPCR analyses of SMCHD1 depletion efficiency and expression of two DUX4 target genes (TRIM43 and MBD3L2) in permissive or non-permissive myoblasts treated with control shRNA and shRNA specific to SMCHD1. Data are presented as fold change in expression relative to the respective control shRNA-treated myoblasts. B. Similar experiments as in (A) in myotubes. Following lentiviral shRNA infection, myoblasts were differentiated into myotubes for 3 days and RT-qPCR analyses were performed for SMCHD1, TRIM43 and MBD3L2 as indicated. C. RT-qPCR comparison of shSMCHD1-treated myotubes and FSHD2 patient myotubes on day 3 of differentiation, demonstrating the vast overexpression of TRIM43 and MBD3L2 in patient myotubes.

Article Snippet: To construct the H3.X and LEUTX overexpression lentiviral plasmids pLVX_H3.X and pLVX_LEUTX, human H3.X and LEUTX ORF sequences were amplified from FSHD1 myotube cDNA using primer pairs H3.X_PCR and LEUTX_PCR respectively, and cloned into pLVX Lentiviral vector (Addgene, plasmid #135182).

Techniques: Quantitative RT-PCR, Expressing, Control, shRNA, Infection, Comparison, Over Expression

Journal: bioRxiv

Article Title: Engineered SMCHD1 and D4Z4 mutations reveal roles of D4Z4 heterochromatin disruption and feedforward DUX4 network activation in FSHD

doi: 10.1101/2022.10.14.512332

Figure Lengend Snippet: A. Western blot analysis the SMCHD1 protein expression in the cell lines used in the study. Lysates of immortalized control and FSHD1 and FSHD2 patient myoblasts, SMCHD1 mutants (SM), D4Z4 deletion mutants (DEL) and double mutants (DEL_SM) were subjected to western blot analysis using antibody specific for SMCHD1. β-Tubulin serves as a loading control. B. Determination of the 4q and 10q D4Z4 repeat number. Examples of control and DEL mutant clones are shown. Genomic DNA was digested with EcoRI/HindIII (E/H) or EcoRI/BlnI (E/B) and subjected to PFGE. They were then blot-hybridized with the 4q/10q specific “1-kb” D4Z4 probe. E/H digestion leaves intact two 4q and two 10q D4Z4 arrays, while BlnI in an E/B only cleaves 10qD4Z4 repeat units. Size markers (in kb) are shown on the left. Arrowheads and stars indicate 4q and 10q D4Z4, respectively. The arrow indicates a band around 6.6kb, which should be 2 D4Z4 repeat units caused by incomplete digestion. The two 4q D4Z4 repeat arrays are contracted, while the 10q D4Z4 bands size show no change. C. D4Z4 gRNA targeting resulted in repeat contraction and recombination, leaving the last repeat with the DUX4 gene intact at the 4qA allele in DEL mutant cells. Top: schematic diagram of D4Z4 array in 4qA allele of parental cell with gRNA target sites for D4Z4 deletion (purple bars) as well as crRNA target sites designed for nanopore sequencing were shown at the top panel. D4Z4 cluster in 4qA, 4qB and 10q alleles of DEL3 were shown below. 10q D4Z4 sequences were confirmed by SNP analysis. The large triangle represented a 3.3kb D4Z4 unit and its orientation. The small and partial triangle represented partial D4Z4 units and their orientation. The endonucleases (EcoRI/HindIII) cut sites, which generated the fragments detected in PFGE, are indicated. D. Control, SMCHD1 mutant SM1 and D4Z4 contraction mutant DEL3 myoblasts were differentiated and analyzed for DUX4 target ( TRIM43, LEUTX and MBD3L2 ) RNA expression levels. Data are expressed as relative expression (mean with standard deviation). The gene expression over GAPDH was normalized to the TRIM43 value of DEL, which was set to be 1. * P < 0.05, **P < 0.01 and ***P < 0.001 vs. control. E. Corresponding data from double mutant (DEL_SM) and FSHD2 were added to (D) for comparison. F. DUX4 depletion by lentiviral shRNA abolished activation of DUX4 target genes ( TRIM43, LEUTX and MBD3L2 ) compared to control shRNA (shCTRL). Cells were harvested at 4 days of myotube differentiation. DUX4 target expression levels were determined by RT-qPCR. Y-axis is relative expression (mean with standard deviation) with the expression in shCTRL-transduced samples as one. **P < 0.01 and ***P < 0.001. G. Stochasticity of target gene expression in DEL3. Two aliquots of the same clone at different passage numbers and replicates of a re-cloned cell line were differentiated for 4 days and analyzed for MBD3L2 expression by RT-qPCR. H. Variegation is also observed in multiple DEL mutant clones, including DEL9 as in ( G) . I. Comparison of MBD3L2 expression level in early myotube of Control (n=11), single mutant DEL (n=23), and double mutant DEL_SM (n=10). The dots on each boxplot represent the individual data in each repeat.

Article Snippet: To construct the H3.X and LEUTX overexpression lentiviral plasmids pLVX_H3.X and pLVX_LEUTX, human H3.X and LEUTX ORF sequences were amplified from FSHD1 myotube cDNA using primer pairs H3.X_PCR and LEUTX_PCR respectively, and cloned into pLVX Lentiviral vector (Addgene, plasmid #135182).

Techniques: Western Blot, Expressing, Control, Mutagenesis, Clone Assay, Nanopore Sequencing, Generated, RNA Expression, Standard Deviation, Gene Expression, Comparison, shRNA, Activation Assay, Quantitative RT-PCR, Targeted Gene Expression

Journal: bioRxiv

Article Title: Engineered SMCHD1 and D4Z4 mutations reveal roles of D4Z4 heterochromatin disruption and feedforward DUX4 network activation in FSHD

doi: 10.1101/2022.10.14.512332

Figure Lengend Snippet: A. Double mutant cells were treated with or without 5AzaC as indicated. IF signals of H3.X/Y and LEUTX in myoblasts (left panel) and LEUTX in early myotubes (right panel) were quantified as integrated intensity in each myoblast/myotube using ImageJ software. FSHD2 patient early myotubes are shown for comparison. The DUX4 integrated density values in each myoblast/myotube were measured using ImageJ software. Based on the highest positive myoblasts/myotubes number of all, same number of values in each group were used for graph and data analysis. All the data were normalized to the corresponding mean value of the 5AzaC treated samples (release day1 for the myoblasts). Red dots represent mean values. (N=300 myotubes or 1000 for myoblasts). ***p<0.001, by one-tailed student’s t-test. B. Incorporation of H3.X/Y into DUX4 targets in control and double mutant at Day 4 with or without 5AzaC. Cells were treated with 5AzaC for 48 hours before differentiation. Significant incorporation of H3.X/Y is shown by the asterisks with the indicated comparisons. C. Box plots of representative early and late DUX4 target gene expression in double mutant cells were shown to compare myoblast, early and late myotube stages as indicated. Expression values are in log2 normalized TPM. D. The expression level of DUX4 target gene LEUTX in double mutant DEL4_SM_A transduced with lentivirus carrying shControl, shH3.X/Y or shDUX4. Real-time RT-PCRs were carried out before or 5 days after the induction of differentiation. Three biological replicates for each sample were performed. Data are presented as mean ± SD; **p<0.01, ***p<0.001, by onetailed student’s t-test. Results presented as fold difference compared to shControl differentiated sample. E. Control and DEL4_SM_A myoblasts were transduced with a lentiviral empty vector or a lentiviral vector expressing H3. X. Differentiation was induced at 48 hours after transduction. For myoblasts or early myotubes as indicated, the mRNA expression level of the downstream target genes was assessed by real-time RT-qPCR. Data are presented as mean ± SD; *p<0.05, **p<0.01, ***p<0.001, by one-tailed student’s t-test. Results presented as fold difference compared to empty vector infected double mutant cells. F. Similar experiments as in (E) , but DEL4_SM_A myoblasts were transduced with a lentiviral vector expressing LEUTX. G. Overexpression of H3.X and LEUTX in MB or MT was assessed by western blot. Pan histone H3 antibody was used as control as indicated. Lanes 1 and 2: mock transfection. Lanes 3 and 4: H3.X OE. Lanes 5 and 6: LEUTX OE. The endogenous LEUTX is upregulated in H3.X OE myotubes (lane 4). H. TF binding motifs at the promoter of H3.X/Y. Binding motifs for DUX4 and the putative LEUTX motif (OTX2) within 1 kb upstream and 0.5 kb downstream of the transcription start site for H3.X/Y were identified by using the MoLoTool provided in HOCOMOCO v11, with P -values less than or equal to 0.001. Visualization was done on the UCSC genome browser using GENCODE v36 for the H3.X/Y genes model. I. The effects of control, H3.X/Y or LEUTX overexpression (as in E-G ) on 64 DUX4 target genes in DEL_SM myotubes Day 5 are assessed by RNA-seq and displayed in box plots. P -values are calculated using Wilcoxon t-test indicated at the top. J. Similar analysis was performed with H3.X/Y or LEUTX shRNA depletion compared to the same control as in (I) on DUX4 target gene expression in DEL_SM myotubes Day 7.

Article Snippet: To construct the H3.X and LEUTX overexpression lentiviral plasmids pLVX_H3.X and pLVX_LEUTX, human H3.X and LEUTX ORF sequences were amplified from FSHD1 myotube cDNA using primer pairs H3.X_PCR and LEUTX_PCR respectively, and cloned into pLVX Lentiviral vector (Addgene, plasmid #135182).

Techniques: Mutagenesis, Software, Comparison, One-tailed Test, Control, Targeted Gene Expression, Expressing, Transduction, Plasmid Preparation, Quantitative RT-PCR, Infection, Over Expression, Western Blot, Transfection, Binding Assay, RNA Sequencing, shRNA