Journal: Life Science Alliance

Article Title: DUSP4 protects BRAF- and NRAS-mutant melanoma from oncogene overdose through modulation of MITF

doi: 10.26508/lsa.202101235

Figure Lengend Snippet: (A) SKMEL28 cells were transfected with siRNA against DUSP4 or nontargeting control with or without trametinib (Tram) at 0.25 nM. After 48 h, cells were collected, and RNA-seq experiments were performed. Volcano plots display differentially expressed genes from the RNA-seq analysis between siDUSP4 and siNT or between siDUSP4+ Tram and siDUSP4 (right panel). The vertical axis (y-axis) corresponds to the mean expression value of log 10 (q-value), and the horizontal axis (x-axis) displays the log 2 fold change value. Positive x-values represent up-regulated genes, and negative x-values represent down-regulated genes. MITF target genes are labeled in red . (B) Cells were treated as in (A), and 48 h later, lysates were analyzed by immunoblot. (C) Transcription factor activities were inferred based on the RNA-seq data using the msviper (Virtual Inference of Protein-Activity by Enriched Regulon analysis) algorithm. The activity of transcription factors was analyzed comparing y-axis: NES for siDUSP4 versus siDUSP4+ Tram or x-axis: NES for siNT versus siDUSP4. Positive values in both axes represent increased activity, whereas negative values represent decreased activity. (D) Cells were transfected with siRNA against DUSP4, MITF, and nontargeting as the control. The cell growth was analyzed by measuring cell confluence over time. Cell growth values were normalized against time 0. Data are mean ± SEM, n = 3. Statistical significance was calculated between siNT versus siDUSP4 and siMITF conditions at 108 h. (E) Lysates from transfected cells were also analyzed by immunoblotting. (F) BRAF V600E cells from melanoma (SKMEL28 and COLO829), colorectal cancer (LS411N), and glioblastoma (DBTRG-05MG) were transfected with siRNA against BRAF and DUSP4. The cell growth was analyzed by measuring cell confluence over time. Cell growth values were normalized against time 0. Data are mean ± SEM, n = 3. Statistical significance was calculated between siNT versus siDUSP4 and siBRAF conditions at 120 h.

Article Snippet: Reverse transfection using Lipofectamine RNAiMAX Transfection Reagent (#13778150; Thermo Fisher Scientific) and Opti-MEM (#51985026; Thermo Fisher Scientific) was performed according to the siTOOLS Biotech protocol using 3 nM final concentration of siPOOL nontargeting control and siPOOL against DUSP4, DUSP6, DUSP10, MITF, BRAF (siTOOLs Biotech).

Techniques: Transfection, Control, RNA Sequencing, Expressing, Labeling, Western Blot, Activity Assay

Journal: Life Science Alliance

Article Title: DUSP4 protects BRAF- and NRAS-mutant melanoma from oncogene overdose through modulation of MITF

doi: 10.26508/lsa.202101235

Figure Lengend Snippet: (A) mRNA levels of MITF and its related genes were analyzed by RT–qPCR 48-h post-transfection and were referred to the expression level of siNT cells. Data are normalized to GAPDH and represent mean of three independent experiments. Statistical significance was calculated between siNT versus NT+T and siDUSP4 and siDUSP4 versus siDUSP4+ Tram. (B, C) Cells were transfected with siRNA against nontargeting control and DUSP4, DUSP6, DUSP10. (B, C) Forty-eight hours later, cell lysates were analyzed by Western blot (B), and DUSP10 mRNA levels were measured by RT–qPCR (C). Data are mean ± SEM, n = 2. (D) BRAF-mutant cell lines from different tissues were transfected with siRNA against DUSP4 and BRAF. After 48 h, cell lysates were analyzed by immunoblot. (E) MITF protein levels were assessed in the indicated BRAF-mutant cell lines by immunoblot.

Article Snippet: Reverse transfection using Lipofectamine RNAiMAX Transfection Reagent (#13778150; Thermo Fisher Scientific) and Opti-MEM (#51985026; Thermo Fisher Scientific) was performed according to the siTOOLS Biotech protocol using 3 nM final concentration of siPOOL nontargeting control and siPOOL against DUSP4, DUSP6, DUSP10, MITF, BRAF (siTOOLs Biotech).

Techniques: Quantitative RT-PCR, Transfection, Expressing, Control, Western Blot, Mutagenesis

Journal: Life Science Alliance

Article Title: DUSP4 protects BRAF- and NRAS-mutant melanoma from oncogene overdose through modulation of MITF

doi: 10.26508/lsa.202101235

Figure Lengend Snippet: (A) BRAF V600E cells labeled in purple (SKMEL28, A375) and NRAS Q61K/R cells labeled in green (SKMEL30, SKMEL2, respectively) were transfected with siRNA against DUSP4, MITF, and nontargeting control. After 48 h, cell lysates were analyzed by Western blot. The upper cartoons classify melanoma cells according on their MITF expression levels. High levels of MITF are shown in brown, whereas low levels of MITF are labeled in pink. Band intensities were analyzed by ImageJ software, and the P-ERK/GAPDH and MITF/GAPDH ratios are indicated in the histogram. Data represent mean ± SEM of three independent experiments. Statistical significance was calculated against siNT for each different cell line. (B) Cells were treated as in (A), and cell growth was analyzed by measuring cell confluence over time. Graphs show cell growth curves normalized against time 0. Data are mean ± SEM, n = 3. Statistical significance was calculated between siNT versus siDUSP4 condition at 102 h. (C) Patient-derived cell lines containing NRAS Q61L/K mutation (WM1366 and WM3623, respectively) were transfected as in (A). After 48 h, cell lysates were analyzed by Western blot. (D) WM1366 and WM3623 cells were treated as in (A), and cell growth was analyzed by measuring cell confluence over time. Graphs show cell growth curves normalized against time 0. Data are mean ± SEM, n = 3. Statistical significance was calculated between siNT versus siDUSP4 condition at 120 h. (E) Co-expression analysis of DUSP4 and MITF in human melanoma. Bivariate and rank correlation analysis of combined gene expression data (n = 124) from four separate studies available through cBioportal . Source data are available for this figure.

Article Snippet: Reverse transfection using Lipofectamine RNAiMAX Transfection Reagent (#13778150; Thermo Fisher Scientific) and Opti-MEM (#51985026; Thermo Fisher Scientific) was performed according to the siTOOLS Biotech protocol using 3 nM final concentration of siPOOL nontargeting control and siPOOL against DUSP4, DUSP6, DUSP10, MITF, BRAF (siTOOLs Biotech).

Techniques: Labeling, Transfection, Control, Western Blot, Expressing, Software, Derivative Assay, Mutagenesis, Gene Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cis -element within the ARF locus mediates repression of p16 INK4A expression via long-range chromatin interactions

doi: 10.1073/pnas.1909720116

Figure Lengend Snippet: The p16INK4A-P2A-mCherry reporter allele recapitulates endogenous transcription of p16INK4A. (A) Schematic diagram of the transcriptional repression of p16INK4A by the CRISPR interference system. The dCas9-KRAB fusion protein was guided to the p16INK4A promoter with 3 individual sgRNAs. (B) qRT-PCR was performed on the p16mCherry/+;dCas9-KRAB cells with 3 different p16INK4A-sgRNAs using primers targeting coding sequences of mCherry and p16INK4A. (C) Flow cytometry analysis was performed on the p16mCherry/+;dCas9-KRAB cells with 2 p16INK4A-sgRNAs. The mean value of the mCherry density in each group was calculated and obtained by 3 replicates. The P value was calculated by a 2-tailed t test. (D) The correlation of transcription reduction in mCherry and p16INK4A in response to dCas9-KRAB–mediated transcription repression (from B) was calculated by Pearson’s correlation test (n = 6). (E) qRT-PCR was performed on the p16mCherry/+ cells with or without the treatment of doxorubicin for 24 h by using specific primers targeting the mRNA sequence of p16INK4A. Three biological replicates were performed. The P value was calculated by a 2-tailed t test. (F) qRT-PCR was performed on the p16mCherry/+ cells with or without the treatment of doxorubicin for 24 h by using specific primers targeting the mRNA sequence of mCherry. Three biological replicates were performed. The P value was calculated by a 2-tailed t test.

Article Snippet: A set of 2,029 sgRNA oligos that target H3K27ac and ATAC-seq positive peaks defined in the TAD containing INK4 / ARF in IMR90, HCT116, and SEM cells as well as an additional 20 nontargeting control sgRNAs with no detectable match to the human genome were designed for array-based oligonucleotide synthesis (CustomArray).

Techniques: CRISPR, Quantitative RT-PCR, Flow Cytometry, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cis -element within the ARF locus mediates repression of p16 INK4A expression via long-range chromatin interactions

doi: 10.1073/pnas.1909720116

Figure Lengend Snippet: Noncoding pooled CRISPR screening identified a distal repressive element of p16INK4A residing within the ARF locus. (A) Schematic diagram of a working model of dCas9-KRAB– and Cas9-mediated noncoding screening. (B) The global correlation of sgRNA distribution in dCas9-KRAB and Cas9 screens in the p16mCherry/+ reporter cell line. (C) The global distribution of all sgRNAs in a selected region of the INK4/ARF locus from 3 screens in the p16mCherry/+ reporter cell line using dCas9-KRAB, Cas9, and no effector control. The red arrow indicates the most enriched sgRNAs in a 42-bp region of the ARF exon1β and intron 1. (D) Two candidate sgRNAs binding to the plus and minus strands of the most enriched 42-bp core DNA sequence near the ARF promoter were designed for validation (ARF-sgRNA-3 and -4). The red arrows indicate the orientation of the sgRNA binding sequences. (E) Western blotting analysis of ARF and p16INK4A in dCas9-KRAB– and Cas9-expressing SEM cells infected with ARF-sgRNA-3, -4, and the nontargeting sgRNA control.

Article Snippet: A set of 2,029 sgRNA oligos that target H3K27ac and ATAC-seq positive peaks defined in the TAD containing INK4 / ARF in IMR90, HCT116, and SEM cells as well as an additional 20 nontargeting control sgRNAs with no detectable match to the human genome were designed for array-based oligonucleotide synthesis (CustomArray).

Techniques: CRISPR, Control, Binding Assay, Sequencing, Biomarker Discovery, Western Blot, Expressing, Infection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cis -element within the ARF locus mediates repression of p16 INK4A expression via long-range chromatin interactions

doi: 10.1073/pnas.1909720116

Figure Lengend Snippet: Physical interactions between ARF, p16INK4A, and p15INK4B were detected by 3C. (A) Next-generation Capture-C was performed on parental human SEM cells for 2 replicates. Two specific anchor probes (Bait 1 and Bait 2) were designed to hybridize to the H3K27ac peaks that overlap each of the promoters of p16INK4A, ARF, and p15INK4B to capture chromatin interactions with the respective promoters within the INK4/ARF locus. (B) CUT&RUN was performed on p16mCherry/+;dCas9-KRAB cells infected individually with 2 sgRNAs targeting the p16INK4A repressive element adjacent to the ARF promoter and 2 sgRNAs targeting the p16INK4A promoter. A Cas9 antibody that recognizes dCas9 was used for a pull-down assay. ChIP-seq tracks of CTCF and H3K27ac were included to indicate the open chromatin status of the locus.

Article Snippet: A set of 2,029 sgRNA oligos that target H3K27ac and ATAC-seq positive peaks defined in the TAD containing INK4 / ARF in IMR90, HCT116, and SEM cells as well as an additional 20 nontargeting control sgRNAs with no detectable match to the human genome were designed for array-based oligonucleotide synthesis (CustomArray).

Techniques: Capture-C, Infection, Pull Down Assay, ChIP-sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cis -element within the ARF locus mediates repression of p16 INK4A expression via long-range chromatin interactions

doi: 10.1073/pnas.1909720116

Figure Lengend Snippet: The ARF/p16INK4A chromatin interaction and transcriptional regulation is functionally detected in NBL cells. (A) qRT-PCR analysis of p16INK4A in the human NBL cell line SK-N-SH infected with lentiviral dCas9-KRAB and ARF-sgRNAs-3 and -4. NT-sgRNA is a nontargeting sgRNA control. Four biological replicates were performed. The P value was calculated by a 2-tailed t test. (B) qRT-PCR analysis of ARF in the human NBL cell SK-N-SH infected with lentiviral dCas9-KRAB and ARF-sgRNAs-3 and -4. Four biological replicates were performed. The P value was calculated by a 2-tailed t test. (C) Western blotting analysis of ARF and p16INK4A in the human NBL cell line SK-N-SH infected with lentiviral dCas9-KRAB and ARF-sgRNA-3 and -4 compared to NT-infected controls. (D) Representative image of SK-N-SH cells stably expressing dCas9-KRAB and ARF-sgRNAs-3, -4, and NT-sgRNA at day 3. (E) Quantification of cell proliferation by cell number count. Two biological replicates were performed. The P value was calculated by a 2-tailed t test. (F) Next-generation Capture-C was performed on SK-N-SH cells stably expressing dCas9-KRAB and ARF-sgRNAs-3, -4, and NT-sgRNA. Anchor probes that hybridized to the p16INK4A promoter were utilized to capture chromatin interactions. Boxes 2 and 3 highlight ARF and p15INK4B promoters which were interacting with the indicated p16INK4A anchor regions. Box 1 indicates a negative control noncoding region. (G) Quantification of interaction frequency between the anchor regions to Boxes 1, 2, and 3. Signal value from the .bw file of Capture-C was normalized by probe signal for each experiment.

Article Snippet: A set of 2,029 sgRNA oligos that target H3K27ac and ATAC-seq positive peaks defined in the TAD containing INK4 / ARF in IMR90, HCT116, and SEM cells as well as an additional 20 nontargeting control sgRNAs with no detectable match to the human genome were designed for array-based oligonucleotide synthesis (CustomArray).

Techniques: Quantitative RT-PCR, Infection, Control, Western Blot, Stable Transfection, Expressing, Capture-C, Negative Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cis -element within the ARF locus mediates repression of p16 INK4A expression via long-range chromatin interactions

doi: 10.1073/pnas.1909720116

Figure Lengend Snippet: Loss-of-function CRISPR screening of human transcriptional factors in the p16INK4A reporter cell line. (A) Schematic diagram of a working model of Cas9-mediated CRISPR screening targeting 1,639 human TFs. (B) Gene ranking of looping factors (YY1, first; CTCF, 1,001st) enriched from screening. The enrichment score of 7 sgRNAs against each TF was combined by MAGeCK algorithm. (C, Top) The overall distribution of all sgRNAs from the screening. (C, Bottom) The Log2[Fold Change (top10/bottom10)] ratio for all sgRNAs targeting CTCF, YY1, and the top 10 negative regulators from D and NT sgRNAs were overlaid on a gray gradient depicting the overall distribution. NT: 100 gRNAs; TF: 7 sgRNAs/each. (D) Gene ranking of top 10 negative candidate regulators enriched from screening analysis by MAGeCK algorithm. (E) Immunoblotting of YY1 in p16mCherry/+;Cas9 cells targeted individually with 2 sgRNAs against the coding sequence of human YY1. GAPDH was probed as a loading control. (F) The p16mCherry/+;Cas9 cells were targeted with Cas9 and 2 sgRNAs identified from TF screening targeting the coding region of human YY1. Flow cytometry analysis of mCherry reporter activity was subsequently conducted in YY1-targeted cells compared with NT control. (G) YY1-binding motif prediction by JASPAR algorithm. (H) CUT&RUN of YY1 was conducted in p16mCherry/+;dCas9-KRAB cells infected with ARF-sgRNA-4 and NT-sgRNA for 2 replicates. Tracks were shown at the viewpoint of the INK4/ARF locus. (I) CUT&RUN of ZNF217 was conducted in p16mCherry/+;dCas9-KRAB cells infected with ARF-sgRNA-4 and NT-sgRNA. Tracks were shown at the viewpoint of the INK4/ARF locus. (J) CUT&RUN of ZNF217 was conducted in p16mCherry/+;dCas9-KRAB cells infected with ARF-sgRNA-4 and NT-sgRNA. Tracks of ZNF217 were shown for viewpoint at a randomly chosen locus.

Article Snippet: A set of 2,029 sgRNA oligos that target H3K27ac and ATAC-seq positive peaks defined in the TAD containing INK4 / ARF in IMR90, HCT116, and SEM cells as well as an additional 20 nontargeting control sgRNAs with no detectable match to the human genome were designed for array-based oligonucleotide synthesis (CustomArray).

Techniques: CRISPR, Western Blot, Sequencing, Control, Flow Cytometry, Activity Assay, Binding Assay, Infection