pvt1 pas p mice  (Jackson Laboratory)

Journal: Cell reports

Article Title: Long noncoding RNA-dependent control of Myc transcriptional bursting

doi: 10.1016/j.celrep.2025.116439

Figure Lengend Snippet: (A) smRNA-FISH visualization of nascent Pvt1 ( Pvt1i , green) and Myc ( Myci , red) ( left ) and mature ( Myce , green) and nascent ( Myci , red) Myc ( right ) using indicated probes in wild-type (WT) MEFs. DNA stained with DAPI, blue. Scale bar is 5 μM. Enlarged images highlight signal pattern and co-localization at sites of transcription. (B) Quantification of the fraction of cells ( n ≥ 50) with indicated number of Pvt1i and Myci foci per cell from experiment in (A). Foci were counted independent of size in two biological replicates. (C) Schematic of the Pvt1/Myc locus, highlighting the insertion of the polyadenylation signal (PAS) in exon 1a of Pvt1 . (D) qRT-PCR detection of Pvt1a , Pvt1b , and total Pvt1 levels in RNA isolated from littermate WT (+/+) and homozygous PAS mutant ( P/P ) MEFs. (E and F) smRNA-FISH visualization of nascent Pvt1 ( Pvt1i , green) and Myc ( Myci , red) (E) and mature ( Myce , green) and nascent ( Myci , red) Myc (F) using indicated probes in +/+ and P/P MEFs. DNA stained with DAPI, blue. Scale bar is 5 μM. Enlarged images highlight signal pattern and co-localization at sites of transcription. (G) Violin plot showing the distribution of the number of Myci foci per cell in +/+ and P/P MEFs in three biological replicates (Rep). n indicates the number of cells scored in each experiment. Red lines indicate median. (H) Average Myci foci per cell from experiments in (G). (I) Distribution of Myci foci per cell from experiments in (G), showing fraction of cells with indicated number of Myci foci. (J) RT-qPCR detection of nascent and spliced Myc in RNA isolated from +/+ and P/P MEFs. (K) Representative immunoblot and quantification of Myc protein levels in whole-cell extracts (WCEs) from +/+ and P/P MEFs. Hsp90, loading control. Ladder markers (kilodalton [kDa]) indicated. (L) Cumulative frequency distribution plot showing the upregulation of genes from GSEA Hallmark geneset MYC_TARGETS_V1 relative to an expression-matched control set in P/P relative to +/+ MEFs in RNA-seq analysis of 4 biological replicates. KS test used to determine p value. In this figure, bar graphs show individual values and mean ± SEM of indicated number of biological replicates. Paired t test (D and H–K) and unpaired t test (G), * p < 0.05, ** p < 0.01, and *** p < 0.01; ns, not significant. See also , , , and .

Article Snippet: Pvt1 PAS (P) mice were generated using CRISPR/Cas9-mediated engineering in C57BL/6J blastocysts at Jackson Laboratory.

Techniques: Staining, Quantitative RT-PCR, Isolation, Mutagenesis, Western Blot, Control, Expressing, RNA Sequencing

Journal: Cell reports

Article Title: Long noncoding RNA-dependent control of Myc transcriptional bursting

doi: 10.1016/j.celrep.2025.116439

Figure Lengend Snippet: (A) Schematic of the two alternative transcription start sites of Pvt1 , highlighting the p53 response element (p53RE, pink star) and visualizing Pvt1 isoform transcriptional states (black [ Pvt1a ] or orange [ Pvt1b ] solid [high expression] or dashed [low/no expression] lines) in wild-type ( Pvt1 + / + ; p53 + / + ), PAS mutant only ( Pvt1 P/P ; p53 + / + ), and combined Pvt1 mutant and p53 knockout ( Pvt1 P/P ; p53 Δ/Δ ) MEFs. Percentages indicate the observed transcriptional output of total Pvt1 in each cell line normalized to wild-type cells. (B) qRT-PCR detection of Pvt1a , Pvt1b , and total Pvt1 levels in RNA isolated from littermate Pvt1 P/P ; p53 + / + and Pvt1 P/P ; p53 Δ/Δ MEFs. (C) Representative examples of smRNA-FISH visualization of nascent Pvt1 ( Pvt1i , green) and Myc ( Myci , red) ( left ) and mature ( Myce , green) and nascent ( Myci , red) Myc ( right) using indicated probes in Pvt1 P/P ; p53 Δ/Δ MEFs. DNA stained with DAPI, blue. Scale bar is 5 μM. Enlarged images highlight signal pattern. (D) Violin plot showing the distribution of the number of Myci foci per cell in littermate Pvt1 + / + ; p53 + / + and three independent Pvt1 P/P ; p53 Δ/Δ MEFs lines. n indicates the number of cells scored in each experiment. Red lines indicate median. (E) Average Myci foci per cell from experiments in (D). Paired t test compares the three independent Pvt1 P/P ; p53 Δ/Δ MEF lines to the littermate Pvt1 + / + ; p53 + / + control. (F) Distribution of Myci foci per cell from experiments in (D), showing fraction of cells with indicated number of Myci foci. Statistical analysis performed as in (E). (G) qRT-PCR detection of nascent and spliced Myc in RNA isolated from indicated MEFs. In this figure, bar graphs show individual values and mean ± SEM of indicated number of biological replicates. Paired t test (B and G) and unpaired t test (D), * p < 0.05, ** p < 0.01, and *** p < 0.01; ns, not significant.

Article Snippet: Pvt1 PAS (P) mice were generated using CRISPR/Cas9-mediated engineering in C57BL/6J blastocysts at Jackson Laboratory.

Techniques: Expressing, Mutagenesis, Knock-Out, Quantitative RT-PCR, Isolation, Staining, Control

Journal: Cell reports

Article Title: Long noncoding RNA-dependent control of Myc transcriptional bursting

doi: 10.1016/j.celrep.2025.116439

Figure Lengend Snippet: (A) Growth curve analyses showing cumulative cell number over passaging of three independent +/+ and P/P MEF littermate pairs. (B) Fraction of BrdU-positive cells in (A). (C) Representative brightfield images of cells from (A) visualized at passage 14. +/+ MEFs show features of cellular senescence, while P/P MEFs maintain high mitotic index (arrowheads). (D) Representative examples and quantification of colony formation assay in +/+ and P/P cells expressing empty vector (EV) or E1A oncogene. (E) Representative H&E images of lungs from 5-month-old Kras-LA1; Pvt1 + / + ( n = 18) and Kras-LA1; Pvt1 P/P ( n = 18) mice. Enlarged images highlight grade 1 and mixed grade 1–2 tumors in Kras-LA1; Pvt1 + / + and Kras-LA1; Pvt1 P/P lungs, respectively. (F) Quantification of tumor burden in mice from (E). (G) Quantification of the fraction of tumors assigned to AAH-1, 2–3, or 4+ grade categories in mice from (E). Numbers above bars indicate the number of tumors scored in each condition. (H) Quantification of the number of pHH3-positive cells per tumor area in mice from (E). (I) Schematic of experiment and representative H&E images of lungs from KP; Pvt1 + / + ( n = 8) and KP; Pvt1 P/P ( n = 6) mice at 12 weeks post-tumor initiation (pti) by intratracheal infection with adenoviral Cre (AdCre). (J) Quantification of tumor burden in mice from (E). (K) Quantification of the fraction of tumors assigned to AAH-1, 2–3, or 4+ grade categories in mice from (I). Numbers above bars indicate the number of tumors scored in each condition. In this figure, bar graphs show individual values and mean ± SEM of indicated number of biological replicates. Unpaired t test, * p < 0.05, ** p < 0.01, and *** p < 0.01.

Article Snippet: Pvt1 PAS (P) mice were generated using CRISPR/Cas9-mediated engineering in C57BL/6J blastocysts at Jackson Laboratory.

Techniques: Passaging, Colony Assay, Expressing, Plasmid Preparation, Infection

Journal: Cell reports

Article Title: Long noncoding RNA-dependent control of Myc transcriptional bursting

doi: 10.1016/j.celrep.2025.116439

Figure Lengend Snippet: (A) ATAC-seq read coverage of left, Myc , and right Pvt1 gene bodies, showing comparable patterns in +/+ and P/P MEFs in 4 biological replicates, with the exception of one significantly reduced peak in the promoter of Pvt1a (FDR < 0.01), indicated by a purple star. (B) Volcano plot showing log2FC (fold change) of peaks in Pvt1 mutant (PAS) compared to wild-type (WT) MEFs from ATAC-seq experiment in (A). Dot colors represent peaks with significantly (FDR < 0.01) reduced (blue), increased (red), or unaltered (black) chromatin accessibility. Green arrow highlights the peak in the Pvt1a promoter. (C) Top : ATAC-seq and RNA-seq profiles of the Myc/Pvt1 locus in +/+ and P/P samples included for reference. Middle : HiC arc plot visualization of loops in the Myc/Pvt1 locus detected from merged reads of two biological replicates of +/+ and P/P MEFs. Differential enrichment of loops between +/+ and P/P MEFs is not statistically significant (black) and can be attributed to increased sequencing depth in P/P compared to +/+ MEFs . Bottom : arc plot visualization of significantly (FDR < 0.001) depleted (blue) or enriched (red) intrachromosomal contacts (ICs) in the Myc/Pvt1 locus in P/P compared to +/+ MEFs. (D) Volcano plot showing log2FC (fold change) of chromosome 15 ICs in Pvt1 mutant (PAS) compared to WT MEFs from HiC experiment in (C). Dot colors represent ICs that are significantly (FDR < 0.001) depleted (blue), enriched (red), or unaltered (black). Green halos highlight differential contacts in the Myc/Pvt1 locus, illustrated in (C). See also and , , and .

Article Snippet: Pvt1 PAS (P) mice were generated using CRISPR/Cas9-mediated engineering in C57BL/6J blastocysts at Jackson Laboratory.

Techniques: Mutagenesis, RNA Sequencing, Sequencing

Journal: Cell reports

Article Title: Long noncoding RNA-dependent control of Myc transcriptional bursting

doi: 10.1016/j.celrep.2025.116439

Figure Lengend Snippet: (A) Schematic of the Pvt1/Myc locus and Pvt1 promoter eletion strategy. (B) qRT-PCR detection of Pvt1a , Pvt1b , and total Pvt1 levels in RNA isolated from p53-deficient control gRNA (Con) and Pvt1 promoter deletion (ΔPro) MEF lines. (C) qRT-PCR detection of nascent and spliced Myc in RNA isolated from Con and ΔPro MEFs. (D) Representative immunoblot and quantification of Myc protein levels in whole-cell extracts (WCEs) from Con and ΔPro MEFs. Hsp90, loading control. Ladder markers (kilodalton [kDa]) indicated. (E) Growth curve analysis of Con and ΔPro MEFs. (F) smRNA-FISH visualization of nascent Pvt1 ( Pvt1i , green) and Myc ( Myci , red) using indicated probes in Con and ΔPro MEFs. DNA stained with DAPI, blue. Scale bar is 5 μM. Enlarged images highlight signal pattern and co-localization at sites of transcription. Note p53-deficient cells are aneuploid (3N). (G) Violin plot showing the distribution of the number of Myci foci per cell in Con and ΔPro MEFs in three biological replicates (Rep). n indicates the number of cells scored in each experiment. Red lines indicate median. (H) Average Myci foci per cell from experiments in (G). (I) Distribution of Myci foci per cell from experiments in (G). (J) Left : representative loci illustrating inactive, active, and bursting Myc transcriptional states in smRNA-FISH co-localization of indicated probes; Pvt1 is constitutively transcribed. Bursting Myc loci are defined as nuclear regions containing ≥4 Myci independent foci within a 20 μm 2 area. Right : quantification of the fraction of cells with bursting Myc loci in Con and ΔPro MEFs. (K) Combined IF-smRNA-FISH visualization of nascent Pvt1 ( Pvt1i , green) and Myc ( Myci , red) ( left ) and Brd4 (green) and Myc ( Myci , red) ( right ) in Con and ΔPro MEFs. Scale bar is 5 μM. Enlarged images illustrate signal and co-localization patterns. (L) Quantification of the fraction of Myci foci that were found to co-localize (Brd4+) or not co-localize (Brd4−) with Brd4 in experiment in (K) in 4 biological replicates. In this figure, bar graphs show individual values and mean ± SEM of indicated number of biological replicates. Paired t test (B–D, H–J, and L) and unpaired t test (G), * p < 0.05, ** p < 0.01, and *** p < 0.01; ns, not significant. See also .

Article Snippet: Pvt1 PAS (P) mice were generated using CRISPR/Cas9-mediated engineering in C57BL/6J blastocysts at Jackson Laboratory.

Techniques: Quantitative RT-PCR, Isolation, Control, Western Blot, Staining

Journal: Cell reports

Article Title: Long noncoding RNA-dependent control of Myc transcriptional bursting

doi: 10.1016/j.celrep.2025.116439

Figure Lengend Snippet: (A) Representative smRNA-FISH visualization of nascent Pvt1 ( Pvt1i , green) and Myc ( Myci , red) using indicated probes in triploid p53-deficient MEFs with heterozygous Pvt1 promoter deletion (ΔPro/ΔPro/+, Het). DNA stained with DAPI, blue. Scale bar is 5 μM. Enlarged images highlight signal pattern at Pvt1 -proficient wild-type (WT) and Pvt1 -deficient mutant (ΔPro) loci. (B) Quantification of the fraction of WT and ΔPro alleles in Het MEFs with bursting Myc loci, as defined in . (C) Violin plot showing the distribution of the mean intensity (MI) of Myci foci at WT and ΔPro alleles in Het MEFs in two biological replicates (Rep). MI is represented as arbitrary units (a.u.). n indicates the number of alleles scored in each experiment. Red lines indicate median. Unpaired t test (G), * p < 0.05 and *** p < 0.01. (D) Average Myci MI per allele from experiments in (C). (E) Distribution of Myci MI per allele from experiments in (C). In this figure, bar graphs show individual values and mean ± SEM of indicated number of biological replicates. Color of individual data points indicates paired samples.

Article Snippet: Pvt1 PAS (P) mice were generated using CRISPR/Cas9-mediated engineering in C57BL/6J blastocysts at Jackson Laboratory.

Techniques: Staining, Mutagenesis

Journal: Cell reports

Article Title: Long noncoding RNA-dependent control of Myc transcriptional bursting

doi: 10.1016/j.celrep.2025.116439

Figure Lengend Snippet: Top : constitutively transcribed Pvt1 (green) maintains elevated levels of local RNA abundance in the vicinity of Myc promoter. This limits the number of Myc molecules (red) that are transcribed during each burst prior to reaching the local RNA concentration threshold that triggers dissociation from transcriptional condensates. Middle : reduced baseline Pvt1 abundance in loss-of-function studies permits the production of a greater number of Myc molecules in a longer burst before the same threshold is reached. Bottom : increased baseline Pvt1 abundance during the cellular response to stress limits the production of Myc molecules in a shorter burst before the same threshold is reached.

Article Snippet: Pvt1 PAS (P) mice were generated using CRISPR/Cas9-mediated engineering in C57BL/6J blastocysts at Jackson Laboratory.

Techniques: Concentration Assay