Journal: PLOS Biology

Article Title: PRMT5 regulates alternative splicing of TCF3 under hypoxia to promote EMT and invasion in breast cancer

doi: 10.1371/journal.pbio.3003444

Figure Lengend Snippet: A) Representation of PRMT5 promoter luciferase construct cloned upstream of F-Luc gene in pGL3 basic vector. B) Luciferase assay showing an increase in luciferase activity under hypoxia in MDA-MB-231 cells. C) Immunoblot showing decrease in PRMT5 expression upon CTCF KD under hypoxia in MCF7 cells. D) MCF7 CTCF ChIP-seq track showing CTCF enrichment at PRMT5 promoter under hypoxia. E) Schematic representation of CTCF binding site at PRMT5 promoters along with the primer positions. F) MeDIP-qPCR showing decrease in DNA methylation at PRMT5 promoter in MCF7 cells normoxia vs. hypoxia. G) CTCF Chip qPCR showing enrichment in CTCF binding at PRMT5 promoter in MCF7 cells normoxia vs. hypoxia. H) Schematic representation of the dCAS9-DNMT3A epigenetic system targeting CTCF binding site of PRMT5 promoter. I) MeDIP qPCR showing enrichment of DNA methylation at PRMT5 promoter under hypoxia post dCAS9-DNMT3A sgCTCFbs vs. sgControl transfections. J) CTCF ChIP qPCR showing reduction in CTCF binding at PRMT5 promoter under hypoxia post dCAS9-DNMT3A sgCTCFbs vs. sgControl transfection. K) Immunoblot showing decrease in PRMT5 expression upon transfection with dCAS9-DNMT3A sgCTCFbs vs. sgControl. L) Luciferase assay showing decrease in luciferase activity in promoter construct harboring a mutated CTCF binding site. Error bars, mean ± SEM; two-tailed t test, one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 3 biological replicates. Numerical data of (B), (F–G), (I–J), (L) available in S1 Data, sheet “Figure 2.” Fig 2H, Created in BioRender. Shukla, S. (2025) https://BioRender.com/74kzhl6 .

Article Snippet: For creating the dCAS9-PRMT5 construct, firstly, the DNMT3A CD was digested out of the dCAS9-DNMT3A-puroV2 plasmid (Addgene, 74407) using BamHI and FseI .

Techniques: Luciferase, Construct, Clone Assay, Plasmid Preparation, Activity Assay, Western Blot, Expressing, ChIP-sequencing, Binding Assay, Methylated DNA Immunoprecipitation, DNA Methylation Assay, ChIP-qPCR, Transfection, Two Tailed Test

Journal: PLOS Biology

Article Title: PRMT5 regulates alternative splicing of TCF3 under hypoxia to promote EMT and invasion in breast cancer

doi: 10.1371/journal.pbio.3003444

Figure Lengend Snippet: A) Genome track from UCSC genome browser showing TCF3 Intronic Conserved Region (ICR). B) H3R8me2s and H4R3me2s ChIP qPCR depicting increased histone symmetric arginine dimethylation at TCF3-ICR region in MCF7 cells, normoxia vs. hypoxia. C) Schematic representation of the dCAS9-PRMT5 epigenome editing vector targeted at the TCF3-ICR region. D) H3R8me2s and H4R3me2s ChIP qPCR depicting increased histone symmetric arginine dimethylation at TCF3-ICR region after transfection with dCAS9-PRMT5-EV vs. dCAS9-PRMT5-sgICR in MCF7 shPRMT5 cells. E) qRT-PCR showing decrease in exon 18A/18B ratio after transfection with dCAS9-PRMT5-EV vs. dCAS9-PRMT5-sgICR in MCF7 shPRMT5 cells. F) MeDIP qPCR showing increase in DNA methylation at TCF3-ICR region in MDA-MB-231 cells, normoxia vs. hypoxia. G) DNMT3A ChIP qPCR showing change in DNMT3A binding at TCF3-ICR region in MDA-MB-231 cells treated with DMSO (Nx vs. Hx) or 5 µM GSK591(Hx). H) MeDIP qPCR showing decrease in DNA methylation at TCF3-ICR region in MDA-MB-231 cells after treatment with DMSO vs. 5 µM GSK591under hypoxia. I) DNMT3A ChIP qPCR showing change in DNMT3A binding at TCF3-ICR region after transfection with dCAS9-PRMT5 construct in PRMT5-depleted MCF7 cells under hypoxia. J) MeDIP qPCR showing increase in DNA methylation at TCF3-ICR region after transfection with dCAS9-PRMT5 construct in PRMT5-depleted MCF7 cells under hypoxia. K) MeCP2 ChIP qPCR depicting change in MeCP2 binding at TCF3-ICR region in MDA-MB-231 cells treated with DMSO (Nx vs. Hx) or 5 µM GSK591(Hx). L) MeCP2 ChIP qPCR depicting change in MeCP2 binding at TCF3-ICR region after transfection with dCAS9-PRMT5 construct in PRMT5-depleted MCF7 cells under hypoxia. Error bars, mean ± SEM; two-tailed t test, one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, *** p < 0.0001, n = 3 biological replicates. Numerical data of (B), (D–L) available in S1 Data, sheet “Figure 5.” 5C, Created in BioRender. Shukla, S. (2025) https://BioRender.com/jcru48k .

Article Snippet: For creating the dCAS9-PRMT5 construct, firstly, the DNMT3A CD was digested out of the dCAS9-DNMT3A-puroV2 plasmid (Addgene, 74407) using BamHI and FseI .

Techniques: ChIP-qPCR, Plasmid Preparation, Transfection, Quantitative RT-PCR, Methylated DNA Immunoprecipitation, DNA Methylation Assay, Binding Assay, Construct, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Locus-specific DNA methylation of Mecp2 promoter leads to autism-like phenotypes in mice

doi: 10.1038/s41419-020-2290-x

Figure Lengend Snippet: a Graphical representation of the Mecp2 locus showing sgRNAs target location. Transcription Start Site (TSS) is indicated by black arrow. The location of targets (1–5) for sgRNAs (Mecp2_1–5) are indicated by blue arrows. The Mecp2 exon is indicated by blue rectangle. b Representative methylation pattern as analyzed by bisulfite sequencing. c Targeted DNA methylation of Mecp2 promoter led to downregulation of Mecp2 expression in N2a cells. Expression of Mecp2 was calculated by quantitative PCR at 4 days after transfection. DDdCas9, DNMT3L-DNMT3A-dCas9; DDmutdCas9, DNMT3L-DNMT3Amut-dCas9; sc sgRNA, scrambled sgRNA. Data were statistically analyzed by Student’s t test (** p < 0.01; NS, no significance), and shown as the mean ± s.e.m. ( n = 3 from three independent experiments).

Article Snippet: Plasmids were deposited in Addgene with following catalog numbers: pGL3-U6-sgRNA (Addgene, 51133), pSt1374-N-NLS-DNMT3L-L-DNMT3A-Ldcas9-NLS (Addgene, 112209), pSt1374-N-NLS-DNMT3L-L-DNMT3Amut-L-dcas9-NLS (Addgene, 112210), pAAV-CAG-C-intein-C-spC9-H840AN863A-2xNLS-hGH (Addgene, 112211), pAAV-CAG-NLS-DNMT3A-L-N-spC9-Nintein-hG (Addgene, 112212), pAAV-5U6- sgRNAs-hsyn-EGFP (Addgene, 112213), pSt1374m-EF1alpha-N-NLS-DNMT3L-LDNMT3A-L-dcas9 -NLS (Addgene, 112214) and pSt1374m-EF1alpha-N-NLS-DNMT3L-L-DNMT3Amut-L-dcas9-NLS (Addgene, 112215).

Techniques: Methylation, Methylation Sequencing, DNA Methylation Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection

Journal: Cell Death & Disease

Article Title: Locus-specific DNA methylation of Mecp2 promoter leads to autism-like phenotypes in mice

doi: 10.1038/s41419-020-2290-x

Figure Lengend Snippet: a Schematic illustration of experimental procedure for targeted DNA methylation in vivo. Constructs expressing DNMT3L-DNMT3A-dCas9 or DNMT3L-DNMT3Amut-dCas9 along with sgRNAs targeting the Mecp2 TSS region were pooled together and injected into mouse zygotes as indicated. b DNA methylation of targeted regions in the tail of male mice was analyzed by bisulfite sequencing. Each dot indicates one individual mouse. n = 8 mice/group; Data were statistically analyzed by Student’s t test (*** p < 0.001), and shown as the mean ± s.e.m. c Representative methylation pattern in the tails of male mice as determined by bisulfite sequencing. d DNA methylation of target regions in the hippocampus (left) and parietal cortex (right) of male mice were analyzed by bisulfite sequencing. Each dot indicates one individual mouse. n = 8 mice/group; Data were statistically analyzed by Student’s t test (*** p < 0.001), and shown as the mean ± s.e.m. e Methylation in the tail of male mice. Black portion of the circles indicates the methylation ratio in each CpG site. The results include all the sequencing data from mice described in ( b ). The Mecp2 sgRNAs (2–4) are indicated by blue rectangles.

Article Snippet: Plasmids were deposited in Addgene with following catalog numbers: pGL3-U6-sgRNA (Addgene, 51133), pSt1374-N-NLS-DNMT3L-L-DNMT3A-Ldcas9-NLS (Addgene, 112209), pSt1374-N-NLS-DNMT3L-L-DNMT3Amut-L-dcas9-NLS (Addgene, 112210), pAAV-CAG-C-intein-C-spC9-H840AN863A-2xNLS-hGH (Addgene, 112211), pAAV-CAG-NLS-DNMT3A-L-N-spC9-Nintein-hG (Addgene, 112212), pAAV-5U6- sgRNAs-hsyn-EGFP (Addgene, 112213), pSt1374m-EF1alpha-N-NLS-DNMT3L-LDNMT3A-L-dcas9 -NLS (Addgene, 112214) and pSt1374m-EF1alpha-N-NLS-DNMT3L-L-DNMT3Amut-L-dcas9-NLS (Addgene, 112215).

Techniques: DNA Methylation Assay, In Vivo, Construct, Expressing, Injection, Methylation Sequencing, Methylation, Sequencing

Journal: Cell Death & Disease

Article Title: Locus-specific DNA methylation of Mecp2 promoter leads to autism-like phenotypes in mice

doi: 10.1038/s41419-020-2290-x

Figure Lengend Snippet: a Schematic illustration of structure and assembly of AAV-DNMT3A-split-dCas9 system. b Representative methylation pattern as determined by bisulfite sequencing in cultured neurons infected with AAV-DNMT3A-N-dCas9, AAV-C-dCas9 and AAV-5U6-sgRNAs (methylation) or AAV-5U6-sgRNAs only (control). c Schematic illustration of viral injection point. The red arrow indicates the injection direction; the blue dots indicate the injected points. d , e Social behavior as examined by the three-chamber test. d Left: Time of mice interacting with either the stranger mouse (S) or the empty cage (E) in the second phase. Right: The ratio of interaction time with a stranger mouse to interaction time with an empty cage. (E) Left: Time of mice interacting with either the unfamiliar mice (S2) or the familiar mice (S1) in the third phase. Right: The ratio of interaction time with an unfamiliar mouse (S2) to a familiar mouse (S1). Methylation n = 10, control n = 12. f Anxiety-related behavior as measured by the time in the open arms in the elevated plus-maze assay (EPM). Methylation n = 11, control n = 9. g Memory-related behavior as measured by the total time spent on interacting with novel-object recognition (right) and discrimination ratio (left) over a 10 min period in object recognition assay. Methylation n = 10, control n = 8. h Social memory-related behavior measured by the total consumption (left) and consumption ratio (right) of cocaine and cinnamon eaten by the male observer mice in the social transmission of food preference assay. Coc, cocaine; Cin, cinnamon. Methylation male mice were indicated in black and control male mice were indicated in gray. Methylation n = 11, control n = 16. Data are shown as the mean ± s.e.m, the number of mouse were indicated in respective bars. Statistical analysis was performed by Student’s t test (NS, p > 0.05; * p < 0.05; ** p < 0.01; and *** p < 0.001).

Article Snippet: Plasmids were deposited in Addgene with following catalog numbers: pGL3-U6-sgRNA (Addgene, 51133), pSt1374-N-NLS-DNMT3L-L-DNMT3A-Ldcas9-NLS (Addgene, 112209), pSt1374-N-NLS-DNMT3L-L-DNMT3Amut-L-dcas9-NLS (Addgene, 112210), pAAV-CAG-C-intein-C-spC9-H840AN863A-2xNLS-hGH (Addgene, 112211), pAAV-CAG-NLS-DNMT3A-L-N-spC9-Nintein-hG (Addgene, 112212), pAAV-5U6- sgRNAs-hsyn-EGFP (Addgene, 112213), pSt1374m-EF1alpha-N-NLS-DNMT3L-LDNMT3A-L-dcas9 -NLS (Addgene, 112214) and pSt1374m-EF1alpha-N-NLS-DNMT3L-L-DNMT3Amut-L-dcas9-NLS (Addgene, 112215).

Techniques: Methylation, Methylation Sequencing, Cell Culture, Infection, Control, Injection, EPM Assay, Transmission Assay