Arid1a restrains Kras-dependent changes in acinar cell identity
- Memorial Sloan Kettering Cancer Center, United States
- Hannover Medical School, Germany
- University of Michigan School of Medicine, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1 with 1 supplement
A mouse model for inducible and reversible Arid1a depletion in vivo.
(A) Schematic of KC-RIK model. shRNAs against Arid1a and Renilla are targeted into ES cells derived from KC-RIK mice. Experimental cohorts are generated via blastocyst injection of positive clones. …
Figure 1—figure supplement 1
MAPK pathway activation and Arid1a depletion in KC-RIK-shArid1a mice.
(A) Immunohistochemistry staining for phospho-Erk as an indicator of LSL-KrasG12D activation in KC-RIK-shRen and shArid1a.6421 animals after 2 weeks of dox treatment. Scalebar 100 μm. (B) …
Figure 2 with 2 supplements
Acute Arid1a knockdown induces rapid changes in acinar cell fate in the setting of KrasG12D.
(A) Kaplan-Meier curve of KC-RIK-shRen and –shArid1a cohort after dox administration. (B) H and E staining of KC-RIK-shRen and –shArid1a mice after 2 weeks on dox. Top panels, scalebar 600 μm; …
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Figure 2—source data 1
Quantification of pancreatic lesions upon acute Arid1a knockdown.
- https://doi.org/10.7554/eLife.35216.008
Figure 2—figure supplement 1
Acute Arid1a knockdown impacts animal health and pancreas function in the setting of oncogenic Kras.
(A) Plot of survival versus percent chimerism of KC-RIK -shRen and -shArid1a mice placed on dox at five weeks of age. (B) Representative image of fecal samples from KC-RIK shRen and shArid1a mice. (C…
Figure 2—figure supplement 2
Histological changes induced upon acute Arid1a knockdown are maintained after 1 year on dox.
(A) H and E staining of pancreas tissue from KC-RIK-shRen and KC-RIK-shArid1a.6421 animals after one year on dox. (B) Co-immunofluorescence of Arid1a and GFP in shRen and shArid1a animals after one …
Figure 3 with 3 supplements
Arid1a is not required for maintenance of acinar cells in the setting of wild-type Kras.
(A) Representative gross image of pancreas from C-RIK-shRen and C-RIK-shArid1a.6421 animals on dox for 2 weeks. (B) H and E staining of pancreas tissue from C-RIK-shRen and C-RIK-shArid1a.6421 …
Figure 3—figure supplement 1
Arid1a depletion in the setting of wild-type Kras.
(A) Western blot of pancreas lysate from C-RIK -shRen and -shArid1a mice placed on dox at five weeks of age. (B) Immunofluorescence staining of C-RIK-shRen and -shArid1a tissue for Arid1a and …
Figure 3—figure supplement 2
Arid1a is not required for acinar regeneration after caerulein-induced pancreatitis.
(A) Quantification of inflammation (assessing edema and immune infiltration) upon caerulein treatment in C-RIK -shRen and -shArid1a mice (N = 3 mice per genotype). (B) Representative images of …
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Figure 3—figure supplement 2—source data 1
Quantification of inflammation scores in C-RIK-shRen and C-RIK-shArid1a animals upon caerulein-induced pancreatitis.
- https://doi.org/10.7554/eLife.35216.012
Figure 3—figure supplement 3
Regeneration acinar cells in C-RIK-shArid1a mice re-express acinar markers and retain Arid1a silencing.
(A) Immunofluorescence staining of C-RIK-shRen and -shArid1a pancreata at 2 and 9 days post induction of pancreatitis for acinar marker Cpa1 and GFP with DAPI to mark nuclei. Scalebar 50 μm. (B) …
Figure 4 with 1 supplement
Temporal context impacts the outcome of Arid1a depletion in the pancreas.
(A) Schematic of dox administration timepoints for Arid1a silencing cohorts. (B) Gross morphology and mKate2 fluorescence of KC-RIK-shRen and KC-RIK-shArid1a pancreata from indicated dox cohorts at …
Figure 4—figure supplement 1
Mucinous lesions in KC-RIK-shArid1a mice placed on dox during embryonic development.
(A) Quantification of IPMN lesions vs ADM/PanIN lesions per mm2 tissue area in KC-RIK-shArid1a.6421 animals placed on dox at embryonic day 7 (E7) and after post-natal day 5 (P5). Arid1a suppression …
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Figure 4—figure supplement 1—source data 1
Quantification of lesions in KC-RIK-shArid1a and control animals placed on dox during pancreatic development.
- https://doi.org/10.7554/eLife.35216.016
Figure 5
Perturbation of acinar cell fate is not reversed upon Arid1a restoration.
(A) Schematic of dox administration timepoints for Arid1a restoration. (B) Gross morphology and mKate2 fluorescence of KC-RIK-shRen and KC-RIK-shArid1a pancreata on dox and after 4 weeks of dox …
Figure 6 with 1 supplement
Acute Arid1a loss results in rapid transcriptional shifts in cell identity.
(A) Principal component analysis of RNAseq data for KC-RIK-shRen, -shArid1a.6421 and –shArid1a.1803 animals after 5 days on dox. three animals per group. (B) Unsupervised clustering of samples by …
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Figure 6—source data 1
QPCR validation of transcriptional changes observed by RNAseq.
- https://doi.org/10.7554/eLife.35216.020
Figure 6—figure supplement 1
Transcriptional changes induced upon Arid1a loss are reflected by histological expression changes.
(A) Immunofluorescence staining KC-RIK-shRen and -shArid1a tissue for the Arid1a and GFP at RNAseq collection timepoint (5 days dox). Nuclei are labeled with DAPI. (B) Representative …
Figure 7 with 1 supplement
Acute Arid1a silencing decreases chromatin accessibility.
(A) Principal component analysis of ATACseq data for KC-RIK-shRen and shArid1a.6421 animals after acute silencing. (B) Relative comparison of dynamic ATACseq peaks in acute KC-RIK-shArid1a.6421 …
Figure 7—figure supplement 1
Peaks with decreased accessibility upon Arid1a depletion are enriched for intronic/intergenic regions and acinar TF binding sites.
(A) Read count per million mapped reads for gene body, transcription factor start site and putative enhancer regions of genes. (B) Genomic annotation of dynamic peaks upon Arid1a suppression vs all …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (M. musculus) | KC-RIK-shArid1a.6421 | This paper | ||
| Strain, strain background (M. musculus) | KC-RIK-shArid1a.1803 | This paper | ||
| Strain, strain background (M. musculus) | KC-RIK-shRen.713 | This paper | ||
| Antibody | GFP antibody (chicken polyclonal) | Abcam | Abcam Cat# ab13970, RRID:AB_300798 | |
| Antibody | GFP antibody (rabbit monoclonal) | Cell Signaling Technology | Cell Signaling Technology Cat# 2956P, RRID:AB_10828931 | |
| Antibody | mKate2 (rabbit polyclonal) | Evrogen | Evrogen Cat# AB233, RRID:AB_2571743 | |
| Antibody | Arid1a (rabbit polyclonal) | Cell Signaling Technology | Cell Signaling Technology Cat# 12354, RRID:AB_2637010 | |
| Antibody | Arid1a (mouse monoclonal) | Santa Cruz | Santa Cruz Biotechnology Cat# sc-32761, RRID:AB_673396 | |
| Antibody | Ki67 (mouse) | BD Biosciences | BD55609 | |
| Antibody | Cpa1 (goat polyclonal) | R and D | R and D Systems Cat# AF2765, RRID:AB_2085841 | |
| Antibody | Clusterin (goat polyclonal) | Santa Cruz | Santa Cruz Biotechnology Cat# sc-6419, RRID:AB_673567 | |
| Antibody | Sox9 (rabbit polyclonal) | Millipore | Millipore Cat# AB5535, RRID:AB_2239761 | |
| Antibody | CD19 Troma III (rat monoclonal) | Developmental Studies Hybridoma Bank | DSHB Cat# TROMA-III, RRID:AB_2133570 | |
| Antibody | Cleaved casp3 (rabbit monoclonal) | Cell Signaling Technology | Cell Signaling Technology Cat# 9664, RRID:AB_2070042 | |
| Antibody | CK8 (mouse monoclonal) | Biolegend | BioLegend Cat# 904801, RRID:AB_2565043 | |
| Antibody | CD45 (rat monoclonal) | Abcam | Abcam Cat# ab25386, RRID:AB_470499 | |
| Antibody | E cadherin (monoclonal) | BD Biosciences | BD Biosciences Cat# 610181, RRID:AB_397580 | |
| Antibody | donkey- anti-chicken CF488 | Sigma | Sigma-Aldrich Cat# SAB4600031, RRID:AB_2721061 | |
| Antibody | goat-anti-chicken AF488 | Life Technologies | Molecular Probes Cat# A-11039, RRID:AB_142924 | |
| Antibody | goat anti-rabbit AF594 | ThermoFisher | Thermo Fisher Scientific Cat# A-11037, RRID:AB_2534095 | |
| Nextera Tn5 transposase | Illumina | FC-121–1030 | ||
| Commercial assay or kit | Taqman copy number assay for GFP | Life Technologies/Thermo Fisher | 4400291 | |
| NEBNext High-Fidelity 2x PCR Master Mix | New England Biolabs | NEB M0541 | ||
| Software, algorithm | MSigDB database | http://software.broadinstitute. org/gsea/msigdb | ||
| Software, algorithm | BEDTools suite | http://bedtools.readthedocs.io | ||
| Software, algorithm | Homer v4.5 | http://homer.ucsd.edu/ | ||
| Other | ImmPress HRP | Vector | Vector Laboratories Cat# MP-7451, RRID:AB_2631198 | |
| Other | SuperSignal West Femto substrate | ThermoFisher | ||
| Other | Doxycycline diet (625 mg/kg) | Harlan | ||
| Other | M15 + LIF media | PMID: 24395249 | ||
| Other | DNase I | Sigma | DN25-100MG | |
| Other | Soybean Trypsin Inhibitor | Sigma | T9003-250mg | |
| Other | Dispase | Roche |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.35216.023
