Xiao C et al. (2016), Chromatin-remodelling factor Brg1 regulates myo...

XB-ART-52843

Nat Commun 2016 Dec 08;7:13787. doi: 10.1038/ncomms13787.

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Chromatin-remodelling factor Brg1 regulates myocardial proliferation and regeneration in zebrafish.

Xiao C , Gao L , Hou Y , Xu C , Chang N , Wang F , Hu K , He A , Luo Y , Wang J , Peng J , Tang F , Zhu X , Xiong JW .

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The zebrafish possesses a remarkable capacity of adult heart regeneration, but the underlying mechanisms are not well understood. Here we report that chromatin remodelling factor Brg1 is essential for adult heart regeneration. Brg1 mRNA and protein are induced during heart regeneration. Transgenic over-expression of dominant-negative Xenopus Brg1 inhibits the formation of BrdU+/Mef2C+ and Tg(gata4:EGFP) cardiomyocytes, leading to severe cardiac fibrosis and compromised myocardial regeneration. RNA-seq and RNAscope analyses reveal that inhibition of Brg1 increases the expression of cyclin-dependent kinase inhibitors such as cdkn1a and cdkn1c in the myocardium after ventricular resection; and accordingly, myocardial-specific expression of dn-xBrg1 blunts myocardial proliferation and regeneration. Mechanistically, injury-induced Brg1, via its interaction with Dnmt3ab, suppresses the expression of cdkn1c by increasing the methylation level of CpG sites at the cdkn1c promoter. Taken together, our results suggest that Brg1 promotes heart regeneration by repressing cyclin-dependent kinase inhibitors partly through Dnmt3ab-dependent DNA methylation.

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Species referenced: Xenopus
Genes referenced: cdkn1a cdkn1c col1a2 gata4 mef2c myc smarca4 tgfb2

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	Figure 1. brg1 is upregulated during cardiac regeneration in zebrafish.In situ hybridization was performed on paraffin sections of mock-operated zebrafish (a) and those with amputated ventricular apexes (b–h) at the indicated time points using a digoxigenin-labelled anti-sense brg1 RNA probe. Note induced expression of brg1 in the injured heart from 1 to 14 d.p.a. (b–f). Dashed lines mark the resection sites; the right upper corner is high-magnification image of the framed area; similar results were confirmed by performing three independent experiments. Scale bar, 100 μm.
	Figure 2. Brg1 is activated in multiple types of cells during cardiac regeneration in zebrafish.(a–f) Immunofluorescence staining of Brg1 and cardiac sarcomere myosin heavy chain (MF20) was performed on paraffin sections of mock-operated zebrafish (a) and those with amputated ventricular apexes (b–f) at the indicated time points. The right upper corners are high-magnification images of the frame area in a–f, showing Brg1 co-localization in MF20-positive myocytes. (g) Co-staining of Brg1 and 4,6-diamidino-2-phenylindole (DAPI) in paraffin sections of amputated apexes at 7 d.p.a. The right upper corner is high-magnification image of the framed area. (h) Immunofluorescence staining of Brg1 and MF20 of amputated heart at 7 d.p.a., showing the co-localization of Brg1 and MF20. The right upper corner is high-magnification image of the framed area. These data were confirmed by performing three independent experiments. Scale bars, 100 μm.
	Figure 3. Inhibition of brg1 impairs cardiac regeneration.(a–d) Representative sections from wild-type siblings (a,c) and Tg(hsp70:dn-xbrg1) (b,d) hearts at 30 d.p.a., evaluated by AFOG staining (a,b), and immunofluorescence staining with anti-myosin heavy chain (MF20) (c,d). Note massive fibrosis (b) and compromised myocardial regeneration (d) in Tg(hsp70:dn-xBrg1) hearts (tg). Dashed lines mark the resection site. (e–g) Paraffin sections of 14 d.p.a. regenerating heart of wild-type sibling (e) and Tg(hsp70:dn-xBrg1) (f) hearts co-stained for BrdU (green), Mef2C (red) and 4,6-diamidino-2-phenylindole (DAPI; blue). Higher-magnification images of areas in squares are shown in the upper-right corners, and Mef2C+/BrdU+ double-positive cardiomyocytes are indicated by arrowheads. (g) Percentages of Mef2C+/BrdU+ cardiomyocytes in the injured area (*P<0.001; n=6 for siblings and 7 for transgenic hearts; data are mean percentages±s.e.m., paired Student’s t-test). (h–j) Paraffin sections of 14 d.p.a. wild-type Tg(gata4:EGFP) sibling (h) and Tg(hsp70:dn-xbrg1; gata4:EGFP) (i) hearts stained with anti-EGFP and DAPI. The average of fluorescence intensity was calculated using Imaris software (j) (P<0.01; n=6; data are mean percentages±s.e.m.; paired Student’s t-test). Scale bars, 100 μm.
	Figure 4. Transgenic inhibition of Brg1 induces expression of cyclin-dependent kinase inhibitors.(a) Heat map of Z-score values showing genes differentially expressed between Tg(hsp70:dn-xBrg1) (tg1 and tg2) and wild-type sibling (sib1 and sib2) hearts. The FPKM (fragments per kilobase of exon per million fragments mapped) value of each gene was normalized using Z-scores. Genes were ranked by the mean Z-scores in the highest-expression group. (b) Tg(hsp70:dn-xBrg1) and wild-type sibling zebrafish were heat-shocked daily from 5 to 14 d.p.a., and total RNA was isolated from their hearts at 14 d.p.a. Quantitative PCR showed that the cdkn and meis genes, as well as fibrotic markers (col1a1a, col1a2, tgfb1a, tgfb2, tgfb3 and vimentin) were upregulated in transgenic hearts (P<0.05, P<0.001; data are mean fold changes after normalized to GAPDH and expressed as mean±s.e.m.; paired Student’s t-test). (c) Quantitative PCR showed higher expression of brg1 but lower expression of cdkn1c in wild-type hearts at 3 and 5 d.p.a. than mock hearts, suggesting a repressive role of brg1 in regulating cdkn1c. GAPDH was used to normalize the RNA level (P<0.01, ***P<0.001; data are mean±s.e.m.; one-way analysis of variance followed by Dunnett’s multiple comparison test, mock served as control).
	Figure 5. cdkn1a and cdkn1c are induced and enriched in the myocardium of Tg(hsp70:dn-xBrg1) transgenic hearts.(a–h) RNAscope in situ hybridization analysis with cdkn1a (a–d) and cdkn1c (e–h) probes on frozen sections of uninjured wild-type (WT) hearts (a,e), injured WT hearts at 3 d.p.a. (b,f), injured WT sibling hearts at 14 d.p.a. (c,g) and injured Tg(hsp70:dn-xBrg1) transgenic hearts at 14 d.p.a. (d,h). Note the robust induction of cdkn1a (d) and cdkn1c (h) induction in Tg(hsp70:dn-xBrg1) transgenic hearts compared with WT sibling hearts at 14 d.p.a. after heat shock. Black arrowheads indicate the cdkn1a or cdkn1c signals. The panels below a–h are higher-magnification images of areas in squares of a–h. (i–p) Bright-field images of cdkn1a (i–l) and cdkn1c (m–p) expression by RNAscope merged with immunostaining signal images of MF20 on frozen sections of uninjured WT hearts (i,m), injured WT hearts at 3 d.p.a. (j,n), injured WT sibling hearts at 14 d.p.a. (k,o) and injured Tg(hsp70:dn-xbrg1) transgenic hearts at 14 d.p.a. (i,p). Higher-magnification images of squared areas of i–p are shown below their respective panels. Note that both cdkn1a (i) and cdkn1c (m) are normally expressed in cardiomyocytes in uninjured WT hearts, and that they are highly induced in MF20-positive cardiomyocytes of Tg(hsp70:dn-xBrg1) transgenic hearts (l,p) compared with WT sibling hearts (k,o) at 14 d.p.a. White arrowheads show cdkn1a or cdkn1c signals in cardiomyocytes. Tg, Tg(hsp70:dn-xBrg1); (+) HS, heat shock; Scale bars, 100 μm.
	Figure 6. Myocardial-specific inhibition of Brg1 interferes heart regeneration.(a–c) PCNA+/Mef2C+ proliferating cardiomyocytes decreased in Tg(myl7:CreER; ubi:DsRed-dn-xBrg1) transgenic hearts (b) compared with control Tg(ubi:DsRed-dn-xBrg1) transgenic hearts (a) at 7 d.p.a. Statistics of cardiomyocyte proliferation index is shown (*P<0.05; data presented are mean±s.e.m.; paired Student’s t-test) (c). White arrowheads point to PCNA+/Mef2C+ proliferating cardiomyocytes; n, the number of hearts analysed; ubi:DsRed-dn-xBrg1 stands for Tg(ubi:loxP-DsRed-STOP-loxP-dn-xBrg1); tamoxifen (4-HT) was applied at 3 days before injury. (d,e) AFOG staining revealed accumulated fibrin and fibrosis in Tg(myl7:CreER; ubi:DsRed-dn-xBrg1) transgenic hearts (e) compared with control Tg(ubi:DsRed-dn-xBrg1) transgenic hearts (d) at 30 d.p.a. (f,g) MF20 staining showed compromised myocardial regeneration in Tg(myl7:CreER; ubi:DsRed-dn-xBrg1) transgenic hearts (g) compared with control Tg(ubi:DsRed-dn-xBrg1) transgenic hearts (f) at 30 d.p.a. 4/4, all 4 hearts analysed showed the same phenotype. Scale bars, 100 μm.
	Figure 7. Brg1 represses cdkn1c expression by increasing the level of DNA methylation in its promoter region.(a) Methylation patterns of 10 individual CpG sites in the cdkn1c promoter of Tg(hsp70:dn-xBrg1) and wild-type sibling hearts after daily heat shock from 5 to 14 d.p.a. Upper panel, schematic of 10 CpG island sites (set A) of the cdkn1c promoter region and transcription start site (TSS); lower panels, cdkn1c methylation patterns of wild-type sibling (sibling) and dn-xBrg1 transgenic (tg) hearts, with open circles for ‘unmethylated’ and filled circles for ‘methylated’ CpG islands. Methylated DNA sequences were obtained by bisulfite sequencing. Note decreased methylation of ckkn1c promoter in dn-xBrg1 transgenic hearts (b). (c) cdkn1c promoter methylation of 10 individual CpG sites (set A) of mock, 3 d.p.a. and 5 d.p.a. wild-type hearts. The percentages of unmethylated (white) and methylated (black) DNA from a and b are shown in b and d. (e) Left panel, ChIP assays with anti-Brg1 antibody. Right panel, quantitation of Brg1 immunoprecipitated cdkn1c promoter in wild-type mock, 3 d.p.a. and 5 d.p.a. hearts. Data are presented as Brg1 enrichment relative to control IgG. The 335 bp DNA fragment within the cdkn1c promoter region (−1,625 to −1,290 bp) was amplified from immunoprecipitated DNA of mock, 3 d.p.a. and 5 d.p.a. hearts by anti-Brg1 antibody or control IgG. (f) Immunoprecipitation by anti-Myc antibody in 293T cells over-expressing Brg1 and Myc-tagged Dnmt3ab. (g) Upper panel, immunoprecipitation of Brg1 and Myc-Dnmt3ab by Myc antibody or control IgG antibody in H9C2 cells over-expressing Brg1 and Myc-tagged Dnmt3ab. Lower panel, immunoprecipitation of dn-xBrg1-Flag and Myc-Dnmt3ab by Myc antibody or IgG antibody in H9C2 cells over-expressing dn-xBrg1-Flag and Myc-Dnmt3ab. (h) Luciferase reporter assays showed that over-expression of zebrafish brg1 and dnmt3ab synergistically suppressed the transcription of cdkn1c in 293T cells. 293T cells were transfected/infected with the indicated adenoviral constructs and luciferase reporter constructs, and those cells were then collected and measured for luciferase activity at 24 h after transfection/infection. Equal amounts of adenovirus were used for each group. Firefly luciferase activity was normalized by Renilla luciferase activity (P<0.05, **P<0.001; data are mean±s.e.m.; one-way analysis of variance followed by Bonferroni’s multiple comparison test).
	Figure 8. siRNA knockdown of either cdkn1a or cdkn1c partially rescues proliferating cardiomyocytes in the Tg(hsp70: dn-xBrg1) heart.(a,b) Quantitative PCR showed that nanoparticle-encapsulated siRNA efficiently decreased the RNA levels of cdkn1a and cdkn1c in wild-type hearts at 2 d.p.a., into which control and cdkn1a (a) or cdkn1c (b) siRNA were injected at 1 d.p.a. The RNA level was normalized to GAPDH (P<0.05, P<0.001; data presented are mean±s.e.m.; paired Student’s t-test). (c–h) Ventricular apex amputation was performed in wild-type siblings and Tg(hsp70:dn-xBrg1) zebrafish, followed by heat shock treatment for 30 min daily from 5 to 14 d.p.a. The Mef2C+/BrdU+ double-positive cardiomyocytes were comparable in control siRNA-injected (d) and uninjected (c) hearts. Either encapsulated cdkn1a (f) or cdkn1c (g) siRNA partially rescued the ratio of Mef2C+/BrdU+ double-positive cardiomyocytes in Tg(hsp70:dn-xBrg1) hearts compared with those in uninjected control transgenic hearts (e). Scale bar, 100 μm. (h) Statistics of c–g (P<0.01, ***P<0.001; data are mean±s.e.m.; one-way analysis of variance followed by Bonferroni’s multiple comparison test). The number (n) of hearts analysed in each group is indicated in each bar.

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