Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: cAMP signaling induces CRTC2 nuclear translocation and condensate formation. a) Live‐cell imaging of ectopically expressed CRTC2‐eGFP, CREB‐eGFP, CREM‐eGFP, and ATF1‐eGFP in 293T cells without (DMSO) and with forskolin (FSK) treatment (upper). Quantification of condensates number per nucleus (lower). b) In vitro droplet formation assay with recombinant CRTC2‐IDR‐eGFP at different protein concentrations (left). Quantification of the size of droplets (right). c) Representative images of the in vitro FRAP experiment with recombinant CRTC2‐IDR‐eGFP (upper). Quantification of FRAP data for CRTC2‐IDR‐eGFP puncta (lower). d) In vitro droplet formation assay of recombinant eGFP fusion proteins fused with wild‐type (WT) CRTC2‐IDR or CRTC2‐IDR mutants (left). Quantification of the size of droplets (right). e) Live‐cell imaging of ectopically expressed CRTC2‐eGFP in 293T cells. Arrows indicate representative CRTC2 puncta that fused over time. The dotted line area indicates the nucleus. f) Representative images of the FRAP experiment with ectopically expressed CRTC2‐eGFP in 293T cells (upper). The dotted line area indicates the nucleus. Quantification of FRAP data for CRTC2‐eGFP puncta (lower). g) Live‐cell images of ectopically expressed WT CRTC2‐eGFP or CRTC2‐IDR‐R>A mutant (R>A‐eGFP) in 293T cells (left). Quantification of cells with eGFP foci and western blot analysis of CRTC2‐eGFP or CRTC2‐IDR‐R>A expression (right). h) Live‐cell snapshots of ectopically expressed mCherry‐CRY2 fusion proteins fused with WT CRTC2‐IDR (upper) or CRTC2‐IDR‐R>A mutant (lower) in 293T cells before and after blue light stimulation (left). Quantification of cells with mCherry foci before and after blue light stimulation (right). Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001. n.s., not significant. Scale bar, 5 µm (a), 10 µm (b,d). All results are from more than three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Translocation Assay, Live Cell Imaging, In Vitro, Tube Formation Assay, Recombinant, Mutagenesis, Western Blot, Expressing

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: P‐TEFb interacts with CRTC2 and enhances CRTC2‐dependent transcription. a) Immunofluorescence analysis of the co‐localization of CRTC2 and CycT1 in HeLa cells treated with DMSO or FSK (left). Line scans along the dotted lines in the images (right). b) Co‐IP assay examining the interaction between endogenous CRTC2 and P‐TEFb in 293T cells treated with DMSO or FSK for 1 h. c) Co‐IP assay examining the interactions between FLAG‐CDK9 and HA‐CRTC2 in 293T cells treated with the indicated concentration of FSK or H89. d) Co‐IP assay examining the interactions between FLAG‐CDK9 and HA‐CRTC2 in 293T cells ectopically expressing Myc‐SIK1. e) Co‐IP assay examining the interactions between FLAG‐CDK9 and HA‐CRTC2 (WT or S171/274A mutants) in 293T cells. f) Quantification of CRE‐luc luciferase activity in 293T cells transfected with the indicated amounts of FLAG‐CDK9 plasmids and treated with DMSO or FSK (10 µ m ) for 6 h. g) Quantification of CRE‐luc luciferase activity in 293T cells treated with flavopiridol (FP, 300 n m ) and FSK (10 µ m ) for 6 h. h) Quantification of CRE‐luc luciferase activity in HA‐CRTC2‐expressing 293T cells infected with lentivirus carrying sgRNA against CDK9 (sgCDK9) or GFP (sgControl) and treated with DMSO or FSK (10 µ m ) for 6 h. i) Quantification of CRE‐luc luciferase activity in FLAG‐CDK9‐expressing 293T cells infected with lentivirus carrying sgRNA against CRTC2 (sgCRTC2) or GFP (sgControl) and treated with DMSO or FSK (10 µ m ) for 6 h. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001; n.s., not significant. All results are from more than three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Immunofluorescence, Co-Immunoprecipitation Assay, Concentration Assay, Expressing, Luciferase, Activity Assay, Transfection, Infection

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 extracts P‐TEFb from HEXIM1‐containing inhibitory complex. a) Live‐cell imaging of 293T cells co‐expressing CDK9‐mCherry and CRTC2‐eGFP or R>A‐eGFP and treated with DMSO or FSK (10 µ m ) for 1 h (left). The dotted line area indicates the nucleus. Quantification of cells with CDK9‐mCherry foci upon treatment with DMSO or FSK (right). b) Immunofluorescence analysis of co‐condensates of CRTC2‐IDR‐mCherry‐CRY2 with CDK9 or CycT1 in 293T cells before or after blue light stimulation (left). Quantification of cells with CRTC2‐IDR co‐condensates with CDK9 or CycT1 (right). c) Live‐cell imaging of 293T cells co‐expressing CycT1‐CFP, HEXIM1‐RFP, CRTC2‐eGFP, and R>A‐eGFP as indicated. d) Quantification of cells containing CycT1‐CFP foci. e) RNA‐IP assay examining the association between FLAG‐CDK9 and 7SK snRNA in 293T cells co‐expressing HA‐CRTC2 or HA‐R>A and treated with DMSO or FSK. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001. All results are from more than three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Live Cell Imaging, Expressing, Immunofluorescence

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 forms nuclear droplets in cystic epithelial cells. a) Western blot analysis of CRTC2 levels in kidneys from WT and Pkd1 −/− mice ( n = 3). b) Representative immunohistochemistry (IHC) images of CRTC2 in kidneys of WT and Pkd1 −/‐ mice (left). Quantification of the signal intensity of nuclear CRTC2 (right) ( n = 5). Cy, large cyst. c) Representative IF images of CRTC2 in the kidney of WT and Pkd1 −/‐ mice. Cy, large cyst. d) Representative IHC images of CRTC2 in kidneys from healthy people ( n = 5) and ADPKD patients ( n = 19). e) Quantification of the signal density of nuclear CRTC2 of (d). f) Correlation between the signal intensity of nuclear CRTC2 and eGFR in patients with ADPKD. Pearson's correlation coefficients are displayed for each graph. p values for (f) were determined using linear regression analysis. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. *** p < 0.001, * p < 0.05. Scale bar, 20 µm (b–d).

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Western Blot, Immunohistochemistry

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: Loss of CRTC2 delays cyst formation in an ADPKD mouse model. a) Experimental design of the ADPKD mouse model. b) Representative kidneys from mice at P29 with indicated genotype. ( n = 8 or 9 biologically independent mice per group). c) KW/BW ratios of the indicated groups of mice in the ADPKD model. The number below each bar refers to the number of samples analyzed. d) Hematoxylin and eosin (H&E) staining of kidney sections from mice at P29 with indicated genotype. ( n = 8 or 9 biologically independent mice per group). e) Cystic index of H&E‐stained kidneys from mice at P29 with indicated genotype. The number below each bar refers to the number of samples analyzed. f) Plasma BUN levels of mice from the indicated groups. The number below each bar refers to the number of samples analyzed. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001. Scale bar, 2 mm (b,d).

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Staining, Clinical Proteomics

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: Genome‐wide CRTC2 localization in human ADPKD cells. a) Representative immunofluorescence images of endogenous CRTC2 in WT 9–12 cells. The result is from more than three independent experiments. b) Western blot analysis of whole‐cell lysates from WT 9–12 cells with indicated treatment. The result is from more than three independent experiments. c) Genomic distribution of CRTC2 in WT 9–12 cells. d) Heatmaps of normalized ChIP‐seq signals for CRTC2, H3K4me1, and H3K4me3, H3K27ac. The rows show 3 kb flanking the CRTC2 peak center. e) Boxplots of the normalized counts of H3K4me1, H3K4me3, and H3K27ac signals at CRTC2 binding + or − peaks. f) Boxplots of CRTC2, H3K4me1, H3K4me3, and H3K27ac reads in the indicated groups. Low: CRTC2 reads < 39; medium: CRTC2 reads 40–70; high: CRTC2 reads > 71. g) ChIP‐seq tracks of CRTC2, H3K4me3, H3K27ac, and H3K4me1 on representative genes. h) Motif analysis of CRTC2 peaks. i) GO and KEGG pathway enrichment analyses of CRTC2‐binding genes. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001, ** p < 0.01. Scale bar, 20 µm (a).

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Genome Wide, Immunofluorescence, Western Blot, ChIP-sequencing, Binding Assay

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 regulates the expression of cystogenesis‐associated genes in human ADPKD cells. a) Heatmap of gene expression in WT 9–12 cells transfected with sgControl or sgCRTC2. Rows show the Z scores calculated for each group. b) Volcano plots showing differentially expressed genes. c) Gene set enrichment analysis displaying CRTC2‐binding gene set enriched for genes downregulated in sgCRTC2 cells. NES, normalized enrichment score. d) Venn diagram showing the overlap of sgCRTC2 downregulated genes and CRTC2‐binding genes, referred to as CRTC2‐target genes. e) GO and KEGG pathway enrichment analyses of CRTC2‐target genes. f) Heatmap of expression values of genes enriched in the indicated processes. Rows show the Z scores calculated for each group. g) RT‐qPCR analysis of mRNA levels of representative CRTC2‐target genes in kidneys from the indicated mouse groups. Data are presented as means ± SEM. The unpaired Student's two‐sided t ‐test was used for statistical analysis. **** p < 0.0001. These results are from three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Expressing, Gene Expression, Transfection, Binding Assay, Quantitative RT-PCR

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 activates cystogenesis‐associated genes by promoting paused Pol II release. a) Immunofluorescence analysis of CRTC2 and CycT1 co‐localization in the kidneys of WT and Pkd1 −/‐ mice ( n = 3). b) Immunofluorescence analysis of CRTC2 and CycT1 co‐localization in WT 9–12 cells (left). Line scan of the dashed line in the merged inset image (right). c) Western blot analysis of CRTC2 expression in WT 9–12 cells transfected with the indicated sgRNAs and constructs. d) Immunofluorescence analysis of ectopically expressed TY1‐CRTC2 and TY1‐CRTC2‐R>A in WT 9–12 cells with endogenous CRTC2 knocked out. e) RT‐qPCR analysis of representative CRTC2‐target genes in WT 9–12 cells transfected with the indicated sgRNAs and constructs. f) Cumulative curve of Pol II pausing index (PI) for CRTC2 target genes in normal and ADPKD kidney tissues. g) ChIP‐qPCR analysis of CycT1, Pol II Ser2P, and Pol II occupancy on LDHA and PLAU genes in WT 9–12 cells with endogenous CRTC2 replaced by TY1‐CRTC2 or TY1‐CRTC2‐R>A mutant. The inset bar graphs indicate the Pol II pausing index for LDHA and PLAU genes, respectively. h) Schematic illustration of how CRTC2 triggers the productive elongation of cystogenesis‐associated genes, ultimately leading to ADPKD progression. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar, 20 µm (a,b). All results are from more than three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Immunofluorescence, Western Blot, Expressing, Transfection, Construct, Quantitative RT-PCR, ChIP-qPCR, Mutagenesis