Brg1 coordinates multiple processes during retinogenesis and is a tumor suppressor in retinoblastoma

Abstract

Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulate retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.

Keywords: Epigenetics; Mouse; Retina development; Retinoblastoma; SWI/SNF.

Fig. 1.

Brg1-deficient retinae are hypocellular. (A) Isolated mouse retinae with lens. (B) Micrographs of activated caspase 3 immunofluorescence (red) of E14.5 retinal cryosections, with DAPI nuclear counterstaining (blue). (C) Quantitative real-time PCR analysis using TaqMan probes for E14.5 retinae. Each bar is the mean and s.d. of replicate PCR from triplicate samples. (D) Retinal cryosections stained with X-gal showing clones of cells derived from single retinal progenitor cells infected with a replication-incompetent retrovirus expressing nuclear β-galactosidase. (E) The proportion of clones for each clone size for wild-type (NIN) and Brg1-deficient (NIN-Cre) retinae. (F) EdU staining (red) of E14.5 retinal cryosections, with DAPI nuclear counterstaining (blue). (G) Dissociated cells stained with EdU (red) and DAPI (blue). (H) The proportion of EdU⁺ cells from retinal sections and dissociated cell scoring at E14.5. (I) EdU staining (red) with DAPI nuclear countertstain (blue) and detection of [³H]-thymidine in overlaid autoradiographic emulsion (DIC and brightfield images). Arrows indicate EdU⁺ cells and arrowheads indicate [³H]-thymidine⁺ cells. (J) The proportion of EdU⁺ cells among [³H]-thymidine⁺ cells at each time point for wild-type and Brg1-deficient retinae. DIC, differential interference contrast. Scale bars: 25 µm in B,D,F; 10 µm in G,I.

Fig. 2.

Cell fate specification and differentiation in Brg1-deficient retinae. (A,B) Dissociated cells immunostained for recoverin expression (red) with DAPI nuclear counterstain (blue) in P12 wild-type and Brg1-deficient retinae. Arrows indicate representative recoverin-immunopositive cells. (C,D) The proportion of cells immunopositive for cell type-specific markers at P12 and P21. Each bar represents the mean and s.d. of scoring of 250 cells in duplicate across triplicate samples. (E) Histograms of normalized cell type-specific gene expression signature scoring at P12 and P21 for wild-type and Brg1-deficient retinae from gene expression array analysis. Scale bars: 10 µm.

Fig. 3.

Brg1-deficient retinae have defects in retinal lamination. (A) Heat map of gene expression array analysis for P12 and P21 wild-type and Brg1-deficient retinae. Each column is a biological replicate. (B) Quantitative PCR analysis of Wif1 expression using TaqMan probes. Each bar is the mean and s.d. of duplicate PCR reactions from triplicate samples. *P<0.05. (C-E) Immunofluorescent staining of calbindin, Chx10 and Pax6 (red) of P12 wild-type and Brg1-deficient retinae, with green nuclear counterstain. Arrows indicate immunopositive cells. (F) Brightfield micrograph of Toluidine Blue-stained section of wild-type and Brg1-deficient retinae. m, mitotic figure; asterisks, dying cells; arrow, rosette. (G) Wild-type (top) and Brg1-deficient (bottom) E14.5 retinal cryosections stained for pH3 (red) and with nuclear counterstain (blue). Arrows indicate pH3-immunopositive cells. A representative mitotic cell is magnified in the inset. (H) Wild-type and Brg1-deficient retinal sections immunostained for Par3 (red) and with DAPI (blue) at P21. Arrows indicate localization of Par3 immunofluorescence. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; onbl, outer neuroblastic layer; inbl, inner neuroblastic layer; RPE, retinal pigment epithelium. Scale bars: 25 µm.

Fig. 4.

Brg1 target genes are involved in cell polarity and cell adhesion. (A) Heat map of gene expression array analysis of five genes dysregulated at E14.5 and P0 in Brg1-deficient retinae. Each column is a biological replicate. (B) Immunoblot of PP2Ac and β-actin expression in wild-type and Brg1 P0 retinae. Actin, loading control. (C) Quantitation of PP2Ac protein expression from duplicate samples. Each bar is the mean and s.d. of PP2Ac expression normalized to β-actin expression. (D) Heat map of correlation for promoter-proximal Brg1 ChIP peaks across different tissues. Promoter-proximal sites are within 1 kb of the transcriptional start site of a gene. (E) Heat map of correlation for promoter-distal Brg1 ChIP peaks across different tissues. (F) Representative ChIP-Seq traces for an active promoter-proximal site. (G) Representative ChIP-Seq traces for a bivalent promoter-proximal site. (H) Box plot of expression of Brg1 target genes with active or bivalent epigenetic marks at P0, P12 and P21. ESC, embryonic stem cell; RMA, robust multiarray average.

Fig. 5.

Identification and characterization of Brg1 promoter-distal sites in P0 retinae. (A) Each promoter-distal peak for Brg1 is represented by a horizontal line and the intensity represents the number of normalized reads at that position for Brg1, H3K27Ac, H3K27me3 or H3K4me1. All peaks are centered at 0 in the plot and span 10 kb upstream and downstream from the center of the peak. The five classes (active, bivalent, isolated, latent, repressed) of promoter-distal regions are indicated. (B-F) ChIP-Seq traces of genomic regions representative of each type of promoter-distal Brg1 binding site. (G-K) Heat map of correlation for the five types of promoter-distal Brg1 ChIP peaks across different tissues.

Fig. 6.

Brg1-repressed promoters are activated during photoreceptor differentiation. (A) ChIP-Seq traces for a repressed Brg1 promoter-proximal site in the Slc17a7 (VgluT1) promoter of the P0 retina that is activated at later stages of development (P12-14 and P21). (B) ChIP-Seq traces for a repressed Brg1 promoter-proximal site in the Sv2b promoter of the P0 retina that is activated at later stages of development (P12-14 and P21). Both Slc17a7 and Sv2b are downregulated in the P12 and P21 Brg1-deficient retinae. Biological duplicate ChIP-Seq is shown for each histone mark at each stage.

Fig. 7.

Brg1 is a tumor suppressor in retinoblastoma. (A) Quantitative PCR analysis for nestin expression at P12 and P21 using TaqMan probes. Each bar is the mean and s.d. of duplicate PCR reactions in triplicate samples. (B) P6 retinal sections stained for EdU (red) and counterstained with DAPI to label nuclei (blue). Arrows indicate representative immunopositive cells in the central and peripheral retina. (C) P12 Chx10-Cre;Brg1^Lox/Lox;Rosa-YFP retinae. Mosaic regions with Cre-mediated recombination are indicated by green immunofluorescence and EdU⁺ nuclei are indicated by red fluorescence (arrows). Yellow dashed lines indicate the boundaries of the domains where Cre was active and Brg1 was inactivated. (D,E) Survival curves for genetically engineered mouse models of retinoblastoma with Brg1 conditional inactivation. (F) Retinoblastomas from Chx10-Cre;Rb^Lox/Lox;p107^+/−;Brg1^Lox/Lox mice stained with Hematoxylin and Eosin. Scale bars: 10 µm in B,C; 25 µm in F.