(expression, we constructed expression vectors encoding either GFP and wild-type human Brm (wBrm) or GFP and a dominant-negative form of human Brm (mutBrm), which has an inactive ATPase domain (Muchardt and Yaniv 1993; Muchardt et al

(expression, we constructed expression vectors encoding either GFP and wild-type human Brm (wBrm) or GFP and a dominant-negative form of human Brm (mutBrm), which has an inactive ATPase domain (Muchardt and Yaniv 1993; Muchardt et al. which is the catalytic subunit in a subset of SWI/SNF chromatin-remodeling complexes. Finally, we show that the conversion of OPCs to NSLCs is usually associated with the recruitment of Brca1 and Brm to an enhancer in the promoter and that histone H3 in the enhancer is usually modified during the conversion to an active form, which is usually methylated at Lys 4 (K4) and acetylated at Lys 9 (K9). These findings suggest that the conversion is associated with extensive chromatin remodeling, which is usually mediated in part by Brca1 and Brm. Results 2As express several NSC markers We first addressed whether the BMP-induced conversion of OPCs to 2As is usually associated with the expression of antigens that are expressed by at least some NSCs. We purified OPCs from postnatal day 6 (P6) rat optic nerve and expanded them for 4 wk in PDGF, in the absence of TH to avoid their differentiation into oligodendrocytes (Barres et al. 1994). We then recultured the cells for 2 d on PDL-coated slide flasks, either Hydroflumethiazide in PDGF alone or in PDGF plus BMP2, and then fixed and immunolabeled them with the A2B5 monoclonal antibody (Eisenbarth et al. 1979), which labels rat OPCs (Raff et al. 1983); anti-GFAP antibodies, which label astrocytes (Bignami et al. 1972) and some NSCs (Doetsch et al. 2002); and antibodies against Nestin, PSA-NCAM, Hydroflumethiazide LeX/SSEA1, or Sox2, all of which are expressed by at least some NSCs (Lendahl et al. 1990; Seki and Arai 1991; Li et al. 1998; Zappone et al. 2000; Capela and Temple 2002). Whereas 90% of both OPCs and 2As expressed A2B5 and Nestin, no OPCs expressed GFAP, PSA-NCAM, SSEA1, or Sox2; 90% of the BMP-induced 2As, however, expressed GFAP, 80% expressed SSEA1, 6% expressed PPP1R53 PSA-NCAM, and 60% expressed Sox2 (Fig. 1A; data not shown). Thus, BMP2 rapidly induces many OPCs to express antigens that are characteristic of at least some NSCs. Open in a separate window Physique 1. Expression of NSC-characteristic molecules in OPCs, 2As, NSCs, and NSLCs. (mRNAs, all of which have been shown to be expressed by at least some NSCs or by more restricted neural precursors. encode basic helix-loop-helix (bHLH) transcription factors (Akazawa et al. 1992; Sasai et al. 1992; Lu et al. 2000; Takebayashi et al. 2000; Zhou et al. 2000); encodes an RNA-binding protein (Nakamura et al. 1994); encodes a polycomb family transcription factor (van Lohuizen et al. 1991); encode HLH proteins that inhibit cell differentiation (Benezra et al. 1990; Christy et al. 1991; Sun et al. 1991; Biggs et al. 1992; Riechmann et al. 1994); and encodes an ABC transporter expressed Hydroflumethiazide by various types of stem cells (Doyle et al. 1999). OPCs were cultured in PDGF alone. We prepared 2As by culturing OPCs in PDGF plus BMP2 for 2 d, and we prepared NSLCs by culturing the 2As in bFGF alone for 1 wk. We prepared NSCs from embryonic day 14.5 (E14.5) mouse telencephalon and expanded them in bFGF for 2 wk (Nakashima et Hydroflumethiazide al. 1999). As shown in Physique 1B, NSCs expressed all of the examined mRNAs except mRNAs. (We showed previously that freshly isolated P0 rat OPCs express all four known mammalian mRNAs but that rapidly declines as OPCs proliferate in vitro and in vivo [Kondo and Raff 2000b]; the present results indicate that this other three mRNAs also eventually decrease as OPCs proliferate in culture and are no longer detectable after 4 wk.) The 2As expressed all but Hydroflumethiazide two of the examined mRNAs (and mRNA decreased significantly compared to its expression in OPCs. When the 2As were cultured in bFGF for a week (to become NSLCs), they re-expressed both and mRNAs.

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