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Chromatin remodeling complexes play crucial roles in transcription and are implicated in processes including cell proliferation, differentiation and embryonic patterning. Brg1 is the catalytic subunit of the SWI/SNF chromatin remodeling complex and shows neural-enriched expression. Although early lethality of Brg1-null mice reflects its importance in embryogenesis, this phenotype precluded further study of specific Brg1-dependent developmental processes. Here, we have identified a requirement of Brg1 for both Xenopus primary neurogenesis and neuronal differentiation of mammalian P19 embryonic carcinoma cells. In Xenopus, loss of Brg1 function did not affect neural induction or neural cell fate determination. However, the Sox2-positive, proliferating neural progenitor cell population was expanded, and expression of a terminally differentiated neuronal marker, N-tubulin, was diminished upon loss of Brg1 activity, suggesting that Brg1 is required for neuronal differentiation. The ability of the bHLH transcription factors Ngnr1 and NeuroD to drive neuronal differentiation was also abolished by loss of Brg1 function, indicating that Brg1 is essential for the proneural activities of Ngnr1 and NeuroD. Consistent with this, dominant-negative interference with Brg1 function in P19 cells suppressed neuronal differentiation promoted by NeuroD2, showing the requirement of Brg1 for neuronal differentiation is conserved in mammalian cells. Finally, we discovered that Brg1 physically associates with both Ngnr1 and NeuroD and that interference with Brg1 function blocks Neurogenin3- and NeuroD2-mediated reporter gene transactivation. Together, our results demonstrate that Brg1 (and by inference the SWI/SNF complex) is required for neuronal differentiation by mediating the transcriptional activities of proneural bHLH proteins.
Fig. 1. Cloning and expression profile of xBrg1. (A) Structure of hBrg1 and xBrg1. Domains are labeled after (Khavari et al., 1993) with percent amino acid identity shown. Asterisks mark the ATP binding pocket targeted by mutagenesis to generate a dominant-negative form. Lines under xBrg1 indicate probes used for in situ hybridization. (B) Phylogenetic analysis of Brg1 and Brm orthologs. Units indicate the number of substitutions. Distance between any two sequences is the sum of horizontal branch length separating them. (C-I) Expression profile of xBrg1. (C) Stage 8 and (D) stage 12, side views (vegetal pole toward bottom). (E) Dorsal view, anterior towards the top (stage 16). (F) Anterior view (stage 22). (G) Stage 25/26 and (H) stage 33/34, lateral views. (IJ) Cross-sectional views of stage 14 embryos stained for xBrg1 (I) or N-tubulin (J).
Fig. 3. Embryos depleted of xBrg1 fail to produce primary neurons. Embryos were injected with DN-xBrg1 (A-C), xBrg1MO (D-F) or standard MO (G-I) in one blastomere at stage 2. β-galactosidase mRNA was co-injected and X-Gal staining (blue) was performed to reveal distribution of injected materials. Embryos were analyzed for expression of Sox2 (A,B,D,E,G,H,J) and N-tubulin (C,F,I,K; stage 15). Embryos in J and K were injected with xBrg1MO and xBrg1 mRNA. Dorsal views with injected side facing rightwards.
Fig. 5. Brg1 is required for the proneural activities of xNgnr1 and xNeuroD. Embryos were injected with xNgnr1 (A), xNeuroD (B) or xMyT1 (C) alone or together with xBrg1MO (D-F) and analyzed for N-tubulin expression (stage 15). Views are dorsal with injected side (X-Gal, blue) facing rightwards.
Fig. 2. DN-xBrg1 and xBrg1MO injections have similar effects on embryonic morphology. (A-C) Morphology of tadpoles (stage 37/38) injected with DN-xBrg1, xBrg1MO or standard MO. Embryos were injected in both blastomeres at the two-cell stage and raised to tadpoles. (A) Bottom three embryos were injected with DN-xBrg1, while the top embryo was uninjected. (B) xBrg1MO (20 ng) injected tadpoles. xBrg1MO-injected tadpoles display similar defects to DN-xBrg1, while standard MO (C) injected ones do not show apparent defects. (D) Reduction of endogenous xBrg1 protein by xBrg1MO. Embryos were injected with 25 ng of xBrg1 or standard MO in both blastomeres at stage 2 and harvested at the indicated stages. Ten embryos were used per sample, with one embryo-equivalent of lysate loaded per lane for western blotting.
Fig. 4. Loss of Brg1 increases cell proliferation. (A-D) Whole-mount stage 14 embryos immunostained (brown) to detect phosphorylated histone H3 (PH3) after injection of standard MO (A) or xBrg1MO (B-D). (C,D) Lateral views of an xBrg1MO-injected embryo: (C) uninjected side; (D) xBrg1MO-injected side. (E) Transverse section of embryo in B, showing the injected side facing rightwards. Black arrowheads mark PH3-positive cells. n, notochord; s, somite. (F-H) Cell division was blocked by adding hydroxyurea (HU) from stage 12.5 until fixation at stage 15. (F) PH3-immunostained embryo showing cell cycle is efficiently blocked by HU treatment (xBrg1MO+β-galactosidase injection, blue; PH3 stain, brown). (G,H) xBrg1MO-injected embryos were raised in the presence of 30 mM HU from stage 12.5 and analyzed for Sox2 (G) and N-tubulin (H) expression. (I,J) Whole-mount TUNEL staining (brown) after injection of standard MO or xBrg1MO. A,B,F-J are dorsal views with injected side facing rightwards.
Smarca4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, anterior view, dorsal up.
