PMC

A model for how Brca1- and Brm-containing SWI/SNF chromatin-remodeling complexes and histone H3 modifications may help activate sox2 transcription when OPCs are induced to convert to 2As and NSLCs.

Role of Brca1 in sox2 expression in NSLCs. (A) NSCs, OPCs, 2As, and NSLCs were immunostained for Brca1 (green) and for Nestin, A2B5, or GFAP (in red). (B) NSLCs were transfected either with a control vector encoding GFP alone or with brca1-siRNA, cultured in bFGF for 3 d, and then stained for GFP (green) and Brca1 (red). (C,D) NSLCs were transfected with the control vector, sox2-siRNA, or brca1-siRNA, cultured in bFGF for 3 d, and then stained for both GFP (green) and either Brca1 or Sox2 (in red). Arrows indicate the GFP-positive cells. Bar: A-D, 25 μm.

Expression of NSC-characteristic molecules in OPCs, 2As, NSCs, and NSLCs. (A) Purified P6 OPCs were expanded in PDGF without TH for 4 wk. They were then cultured in either PDGF (OPCs) or in PDGF plus BMP2 (2As) for 2 d and stained (in red) with the antibodies indicated. The cells were counterstained with Hoechst 33342 to visualize all nuclei (blue). Bar, 25 μm. (B) Purified OPCs were cultured as above for 4 wk and then in PDGF alone (OPCs), in PDGF plus BMP2 for 2 d (2As), or in PDGF plus BMP2 for 2 d and then in bFGF for 7 d (NSLCs). NSCs were prepared from E14.5 mouse brain by culturing the cells as floating neurospheres on uncoated dishes in bFGF for 2 wk. RNA was extracted and analyzed by RT-PCR, as described in Materials and Methods.

Recruitment of Brca1 and Brm and histone H3 modification on the sox2 R1 enhancer during the conversion of OPCs to 2As and NSLCs. (A) ChIP analysis showing that R1 coprecipitates with Brca1 or Brm. Extracts of formaldehyde-fixed OPCs, 2As, and NSLCs were precipitated with either anti-Brca1 antibodies, anti-Brm antibodies, or anti-Flag antibody and Protein-A-Sepharose. Precipitated genomic DNA fragments were analyzed by PCR with the primer set for the R1 sequence. (B) NSLCs were transfected with the wBrm or mutBrm vector, cultured in bFGF for 3 d, and then immunostained for both GFP (green) and Sox2 (in red). The Sox2-negative cells are indicated with arrowheads. Bar, 25 μm. (C) ChIP analysis showing the modifications of histone H3 associated with the R1 enhancer during OPC conversion. The cell extracts were precipitated with anti-dimethyl-H3-K4, anti-dimethyl-H3-K9, or anti-acethyl-H3-K9 antibodies and Protein-A-Sepharose, and the precipitated genomic DNA fragments were analyzed as in A.

Effect of sox2-siRNA. (A) NSLCs were transfected with either a control vector encoding GFP alone or sox2-siRNA, cultured in bFGF for 3 d, and then stained for both GFP (green) and Sox2 (red). (B) OPCs were transfected as above and cultured either in PDGF alone (OPCs) or in PDGF plus BMP2 for 2 d and then bFGF for 5 d (NSLCs); BrdU was then added for 8 h, and the cells were then fixed and immunolabeled for BrdU. The proportion of GFP-expressing cells that were BrdU⁺ is shown as the mean ± SD of three cultures. (C-E) OPCs were transfected and cultured in PDGF plus BMP2 and then bFGF for 5 d. The cells were then stained for both GFP (green) and the neural markers Nestin (C), GFAP (D), or NFs (E)—all in red. The cell nuclei were stained with Hoechst 33342 (blue). The GFP-positive cells are indicated with arrows. Bars: A,C-E, 25 μm.

Association of Brca1 and Brm on the R1 sox2 enhancer in NSCs. (A) NSCs or 293 cells were cotransfected with both pEF-Rluc, which encodes a sea pansy luciferase, and one of the sox2-enhancer-firefly-luciferase expression vectors containing the full-length or deleted versions of the sox2 promoter (shown on left). The activity of firefly luciferase was analyzed after 24 h, normalized to the sea pansy luciferase activity. (B) ChIP analysis to determine if Brca1 is bound to the R1 DNA sequence in the mouse sox2 promoter. Extracts of formaldehyde-fixed NSCs were immunoprecipitated with Brca1 antibodies and Protein-A-Sepharose, and genomic DNA fragments in the immunoprecipitate were analyzed by PCR with specific primer sets to amplify the R1, R2, or R3 sequences. The gel positions of the R1, R2, and R3 sequences are shown on the right (input DNA). (C) ChIP analysis to determine whether the R1 coprecipitates with Brm or Brg1 subunits of the SWI/SNF complex; anti-Brca1 antibodies and anti-Flag antibodies were used as positive and negative controls, respectively. The gel position of the R1 sequence is shown at the right (input DNA). (D) Coprecipitation of Brca1 and Brm proteins from extracts of NSCs. The extracts were immunoprecipitated with antibodies against Brca1, Brm, or Flag, and Protein-A-Sepharose, and the immunoprecipitates were analyzed by Western blotting using anti-Brm antibodies.