Gem interacts with Brg1. (A) Co-IP of Brg1 and Gem from HEK293 cell extract. (B) Scanning electron micrograph of adult male eyes from wild-type (panel i), GMR-GAL4/+; UAS-Brm^K804R/+ (panel ii), and GMR-GAL4/Gem^l(2)k03202; UAS-Brm^K804R/+ (panel iii). (Panels i′–iii′) Higher magnification of the ventral half of the eye. (Panels i″–iii″) Transverse eye sections stained for rhabdomeres. Arrowheads mark losses of photoreceptor cells. (C) Overexpression of both Brm^K804R and wild-type Gem in the wing produces synergistic defects. (Panels i,i′) C96-GAL4, UAS-Gem. (Panels ii,ii′) C96-GAL4/+; UAS-Brm^K804R/+. (Panels iii,iii′) C96-GAL4/UAS-Gem; UAS-Brm^K804R/+. Panels i′, ii′, and iii′ are a higher magnification of panels i, ii, and iii, respectively. Note the loss of cells and bristles in panels iii and iii′ (arrowheads).

Geminin interacts with Domain II of Brg1 through its C terminus. (A) Schematics for Brg1 deletion constructs used to map Gem-interacting domain and summary of the result. Numbers denote amino acid positions. (B) Co-IP assays with Myc-tagged Brg1 deletion mutants and Flag-Gem. (C) Schematics for Gem deletion constructs used to map Brg1-interacting domain and summary of the result. Numbers denote amino acid positions. Asterisks depict the substitution of five acidic amino acids to neutral ones with similar side-chains in the Gem C terminus. (D) Co-IP assays with Myc-Gem deletion mutants and Flag-Brg1 Dom II. (E) GST-pull down assay reveals that Gem directly interacts with Brg1. GST and GST-Gem proteins were purified from E. coli, and Myc-Brg1 was generated by in vitro translation. After GST pull-down, bound proteins were analyzed by anti-Myc Western blotting, revealing specific and direct interaction of Gem and Brg1. (F) Amino acid sequences (amino acids 142–177) of wild-type and mutant Gem variants used in G. Acidic amino acids were color-coded in red. In mutant Gem variants, glutamic acid (E) and aspartic acid (D) were replaced with glutamine (Q) and aspargine (N), respectively. Other amino acids were identical and are not shown. Deleted amino acids in GemΔ(BD) were depicted as a dash (—). Δ(BD) indicates deletion of Brg1-binding domain. (G) Co-IP assays using mutated Gem variants.

Brg1 and Gem have overlapping expression patterns but Gem diminishes prior to neuronal differentiation. In situ hybridization for X-Brg1 (A–C) and X-Gem (D–H) in Xenopus embryos. (A,D,G) Dorso-lateral views. (C,F) Dorsal views at tailbud stages; anterior toward left. (B,E,H) Transverse sections at stages shown in A, D, and G, respectively. (I) Transverse section of N-tubulin-stained embryo. In H and I, a dashed line marks the neural plate–somite boundary. (J) Expression profile of Gem and other markers during RA-induced neuronal differentiation of P19 cells.

Misexpression of Gem prevents neurogenesis. (A–O), Two-cell stage Xenopus embryos were injected in one cell as indicated (yellow) with β-galactosidase mRNA coinjection for lineage tracing and analyzed for NCAM (A–C) or N-tubulin (D–O) expression. (CT) C-terminal region. 3EA is an alanine substitution mutant of acidic amino acids 188–190 in Xenopus Gem (which correspond to mouse Gem amino acids 174–176). Like GemE174Q (Fig. 2F,G), Xenopus Gem 3EA has severely reduced Brg1 binding (data not shown). Numbers indicate the number of affected embryos over the total number of embryos. In situ for markers is stained purple, and blue X-gal staining marks the injected side. Injected sides were oriented rightward. (P) Expression levels of Gem variants injected into Xenopus embryos. Embryos were injected as indicated at the two-cell stage and harvested at stage 12 to prepare lysates. Ten embryos were used for each sample, and one embryo-equivalent of lysate was loaded per lane for Western blotting with anti-Myc antibody.

Gem misexpression inhibits neuronal differentiation of mouse P19 cells driven by NeuroD2. P19 cells were transfected as indicated together with GFP expression vectors to identify transfected cells and subjected to immunofluorescence with TuJ1 to detect neurons.

Loss of Gem activity results in premature neurogenesis. (A–H) Xenopus embryos were injected with 2.5 ng (A–D) or 1.5 ng (E,F) of GemMO or 20 ng of control MO (G,H) and probed as indicated (white). Red arrowheads in E and F mark the posterior end of N-tubulin expression in the medial stripe on each side of the embryo. (I) Reduction of Gem protein level in P19 cells by RNAi. (J) Reduction of Gem sensitized P19 cells to differentiate into neurons at a subthreshold level (50 ng) of NeuroD2. P19 cells were transfected as indicated and probed with TuJ1 antibody to detect neurons. GFP expression plasmid was cotransfected to trace transfected cells.

Gem blocks the association of Brg1 with proneural bHLH proteins and prevents transcriptional activation of their target genes. (A) Wild-type Gem can block the association of Brg1 and Ngnr1/NeuroD. HEK293 cells were transfected as indicated. Lysates were applied for co-IP assays with anti-Myc antibody. Brg1 binding-defective Gem lost the activity to block the Brg1–bHLH interaction. (B) Overexpression of Gem decreases reporter gene expression driven by Ngn3. E1X3-luciferase reporter plasmid contains triple E-boxes, which is a consensus binding site for Ngn3/bHLH class proteins. (C) Model for Gem's activity on neural progenitor cells and during neuronal differentiation. Currently, it is unknown whether proneural bHLH proteins or the SWI/SNF complex is initially recruited to E-box target sites. In progenitor cells or committed neural precursor cells, Gem is highly expressed and blocks the association of the SWI/SNF complex with proneural bHLH proteins, resulting in suppression of neuron-specific gene expression. In differentiating neurons, Gem levels diminish and SWI/SNF complexes and proneural bHLH proteins can associate to activate the transcription of neuron-specific target genes.