Journal: Frontiers in Microbiology

Article Title: Targeting the Histone Methyltransferase Disruptor of Telomeric Silencing 1-Like Restricts Avian Leukosis Virus Subgroup J Replication by Restoring the Innate Immune Response in Chicken Macrophages

doi: 10.3389/fmicb.2020.603131

Figure Lengend Snippet: Primers used in qRT-PCR.

Article Snippet: Three guide RNAs specific for the chicken DOT1L gene (GeneID: 84444) were designed using CHOPCHOP web tool, were then respectively cloned into U6-sgRNA plasmid (Addgene plasmid #65626).

Techniques: Sequencing

Journal: Frontiers in Microbiology

Article Title: Targeting the Histone Methyltransferase Disruptor of Telomeric Silencing 1-Like Restricts Avian Leukosis Virus Subgroup J Replication by Restoring the Innate Immune Response in Chicken Macrophages

doi: 10.3389/fmicb.2020.603131

Figure Lengend Snippet: Disruptor of telomeric silencing 1-like (DOT1L) expression and H3K79me2 levels are upregulated during avian leukosis virus subgroup J (ALV-J) infection in chicken macrophage cell line HD11. (A,B) Western blot (A) and intensity (B) for DOT1L protein and DOT1L-mediated H3K79me2 levels in HD11 cells at the indicated time points post infection with ALV-J (MOI 2). α -tubulin and total H3 were used as a loading control. (C) The relative DOT1L mRNA levels in HD11 cells at the indicated time points post infection with ALV-J, measured by quantitative real-time reverse transcription PCR (qRT-PCR). GAPDH was used as a normalizing control. (D) The relative ALV-J RNA level in HD11 cells at the indicated time points post infection, normalized to chicken GAPDH mRNA, was measured by qRT-PCR. All the data were shown as mean ± SD (error bars) from three independent experiments. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; unpaired, two-tailed Student’s t -test.

Article Snippet: Three guide RNAs specific for the chicken DOT1L gene (GeneID: 84444) were designed using CHOPCHOP web tool, were then respectively cloned into U6-sgRNA plasmid (Addgene plasmid #65626).

Techniques: Expressing, Virus, Infection, Western Blot, Control, Reverse Transcription, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Targeting the Histone Methyltransferase Disruptor of Telomeric Silencing 1-Like Restricts Avian Leukosis Virus Subgroup J Replication by Restoring the Innate Immune Response in Chicken Macrophages

doi: 10.3389/fmicb.2020.603131

Figure Lengend Snippet: Chemical blockade of DOT1L promotes ALV-J clearance in HD11 cells. (A) Chemical structure of DOT1L inhibitor EPZ004777 (EPZ). (B) The blocking efficiency of EPZ was evaluated by western blotting. HD11 cells were incubated with various concentrations of EPZ or the vehicle control DMSO for 48 h, and the levels of H3K79me2 was determined by western blot (upper panel). Total histone H3 were used as internal control. Relative protein levels were quantified with ImageJ software and normalized to the amount of total H3 (lower panel). (C) Western blot analysis of ALV-J Env protein. HD11 cells were infected with ALV-J (MOI 2) in the presence of indicated concentrations of EPZ or vehicle control. ALV-J Env protein and the levels of H3K79me2 was determined at 48 hpi. β -actin and histone H3 were used as internal control (upper panel). Relative protein levels of ALV-J Env were quantified with ImageJ software and normalized to the amount of β-Actin (lower panel). HD11 cells were incubated with 5 μM of EPZ004777 for 24 h and then infected with ALV-J (MOI 2) for 48 h. Viral RNA levels were measured by qRT-PCR. GAPDH mRNA level was measured as an internal control (D) .Viral RNA was Viral yield in the supernatants was analyzed by (TCID 50 ) assay at 48 h after infection (E) . All the data were shown as mean ± SD (error bars) from three independent experiments. * p < 0.05; ** p < 0.01; unpaired, two-tailed Student’s t -test.

Article Snippet: Three guide RNAs specific for the chicken DOT1L gene (GeneID: 84444) were designed using CHOPCHOP web tool, were then respectively cloned into U6-sgRNA plasmid (Addgene plasmid #65626).

Techniques: Blocking Assay, Western Blot, Incubation, Control, Software, Infection, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Targeting the Histone Methyltransferase Disruptor of Telomeric Silencing 1-Like Restricts Avian Leukosis Virus Subgroup J Replication by Restoring the Innate Immune Response in Chicken Macrophages

doi: 10.3389/fmicb.2020.603131

Figure Lengend Snippet: Generation and identification of DOT1L-knockout HD11 cell line. (A) The genomic architecture of chicken DOT1L gene and the CRISPR/cas9 strategy for DOT1L knockout. (B) DNA sequencing results of the chicken DOT1L allele between the wild-type (WT) and the DOT1L knockout cells clone (C) HD11 morphology after DOT1L depletion. Scale bar, 500 μm. (D) Western blot detected the levels of DOT1L mediated-H3K79me2 in DOT1L-KO cells and WT cells.

Article Snippet: Three guide RNAs specific for the chicken DOT1L gene (GeneID: 84444) were designed using CHOPCHOP web tool, were then respectively cloned into U6-sgRNA plasmid (Addgene plasmid #65626).

Techniques: Knock-Out, CRISPR, DNA Sequencing, Western Blot

Journal: Frontiers in Microbiology

Article Title: Targeting the Histone Methyltransferase Disruptor of Telomeric Silencing 1-Like Restricts Avian Leukosis Virus Subgroup J Replication by Restoring the Innate Immune Response in Chicken Macrophages

doi: 10.3389/fmicb.2020.603131

Figure Lengend Snippet: Knockout of DOT1L inhibits ALV-J replication. (A) HD11 and DOT1L-KO cells were infected with ALV-J (MOI 2). After 48 h, the viral RNA levels were measured by qRT-PCR. GAPDH mRNA level was measured as an internal control (left panel). The supernatants were collected to measure the viral titers by a standard TCID 50 method (right panel). (B) Immunofluorescence analyses of ALV-J Env protein. HD11 cells and DOT1L-KO cells were infected with ALV-J at indicated MOI for 48 h, the cells were then visualized with the inverted fluorescence microscope and a specific antibody to the ALV-J Env protein (green), and nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI; blue). Images show a representative image. All the data were shown as mean ± SD (error bars) from three independent experiments. ** p < 0.01; unpaired, two-tailed Student’s t -test.

Article Snippet: Three guide RNAs specific for the chicken DOT1L gene (GeneID: 84444) were designed using CHOPCHOP web tool, were then respectively cloned into U6-sgRNA plasmid (Addgene plasmid #65626).

Techniques: Knock-Out, Infection, Quantitative RT-PCR, Control, Immunofluorescence, Fluorescence, Microscopy, Staining, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Targeting the Histone Methyltransferase Disruptor of Telomeric Silencing 1-Like Restricts Avian Leukosis Virus Subgroup J Replication by Restoring the Innate Immune Response in Chicken Macrophages

doi: 10.3389/fmicb.2020.603131

Figure Lengend Snippet: Gene-expression profiling of HD11 cells and DOT1L-KO cells upon ALV-J stimulation. (A) Mean average (MA) plot of genes differentially expressed in DOT1L-KO cells relative to their expression in HD11 cells. The log2 FPKM and log2 fold change between DOT1L-KO cells and WT cells are represented in the x and y axes, respectively. The red and green dots, respectively, indicate the upregulated and downregulated genes, and the black dots indicate no change in expression. FPKM, fragments per kilobase of transcript per million mapped reads. (B) KEGG pathway analysis showing the top 10 enriched pathways.

Article Snippet: Three guide RNAs specific for the chicken DOT1L gene (GeneID: 84444) were designed using CHOPCHOP web tool, were then respectively cloned into U6-sgRNA plasmid (Addgene plasmid #65626).

Techniques: Gene Expression, Expressing

Journal: Frontiers in Microbiology

Article Title: Targeting the Histone Methyltransferase Disruptor of Telomeric Silencing 1-Like Restricts Avian Leukosis Virus Subgroup J Replication by Restoring the Innate Immune Response in Chicken Macrophages

doi: 10.3389/fmicb.2020.603131

Figure Lengend Snippet: Knockout of DOT1L promotes defense response to ALV-J infection. (A) Heatmap of representative genes involved in type I IFN‐ related genes from DOT1L-KO vs. control cells infected with ALV-J at 24 h. (B) qRT-PCR validations. (C) HD11 cells were infected with ALV-J (MOI 2) in the presence of 5 μM EPZ or vehicle control (DMSO). At 48 hpi, the relative expression of IFIH1 , IFNβ , STAT1 , and MX1 were determined by qRT-PCR. GAPDH were used as internal control. All the data were shown as mean ± SD (error bars) from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; unpaired, two-tailed Student’s t -test.

Article Snippet: Three guide RNAs specific for the chicken DOT1L gene (GeneID: 84444) were designed using CHOPCHOP web tool, were then respectively cloned into U6-sgRNA plasmid (Addgene plasmid #65626).

Techniques: Knock-Out, Infection, Control, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Targeting the Histone Methyltransferase Disruptor of Telomeric Silencing 1-Like Restricts Avian Leukosis Virus Subgroup J Replication by Restoring the Innate Immune Response in Chicken Macrophages

doi: 10.3389/fmicb.2020.603131

Figure Lengend Snippet: Disruptor of telomeric silencing 1-like does not directly regulate the melanoma differentiation-associated protein 5 (MDA5)-mediated IFNβ signaling. HD11 and DOT1L-KO cells were transfected with nothing (Ctrl) or 2 μM poly(I:C) for 8 h. The relative IFIH1 and IFNβ mRNA levels were measured by qRT-PCR and normalized to GAPDH. All the data were shown as mean ± SD (error bars) from three independent experiments. ns, not significant; unpaired, two-tailed Student’s t -test.

Article Snippet: Three guide RNAs specific for the chicken DOT1L gene (GeneID: 84444) were designed using CHOPCHOP web tool, were then respectively cloned into U6-sgRNA plasmid (Addgene plasmid #65626).

Techniques: Transfection, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Targeting the Histone Methyltransferase Disruptor of Telomeric Silencing 1-Like Restricts Avian Leukosis Virus Subgroup J Replication by Restoring the Innate Immune Response in Chicken Macrophages

doi: 10.3389/fmicb.2020.603131

Figure Lengend Snippet: Disruptor of telomeric silencing 1-like deficiency restores the innate immune response to ALV-J infection in a time-dependent manner. (A) HD11 cells and DOT1L-KO cells were infected with ALV-J (MOI 2). Cell lysates were prepared at 0, 24, and 48 hpi for western blot to detect the protein expression levels of MDA5, total‐ and phospho-TBK1, STAT1, and β-actin. (B) Relative protein levels were quantified by densitometry using ImageJ software. All the data were shown as mean ± SD (error bars) from three independent experiments. ** p < 0.01; *** p < 0.001; unpaired, two-tailed Student’s t -test.

Article Snippet: Three guide RNAs specific for the chicken DOT1L gene (GeneID: 84444) were designed using CHOPCHOP web tool, were then respectively cloned into U6-sgRNA plasmid (Addgene plasmid #65626).

Techniques: Infection, Western Blot, Expressing, Software, Two Tailed Test