Identification of a polymorphic, neuron-specific chromatin remodeling complex

Abstract

A variety of chromatin remodeling complexes are thought to assist sequence-specific transcription factors. The complexes described to date are expressed ubiquitously, suggesting that they have general transcriptional functions. We show that vertebrate neurons have a specialized chromatin remodeling complex, bBAF, specifically containing the actin-related protein, BAF53b, which is first expressed in postmitotic neurons at about murine embryonic day 12.5 (E12.5). BAF53b is combinatorially assembled into polymorphic complexes with ubiquitous subunits including the two ATPases BRG1 and BRM. We speculate that bBAF complexes create neuronal-specific patterns of chromatin accessibility, thereby imparting new regulatory characteristics to ubiquitous sequence-specific transcription factors in neurons.

Figure 1

Protein sequence alignment of human BAF53 proteins and homologs in Drosophila and C. elegans. Human (Hu) BAF53 proteins have a single homolog in Drosophila (Dm) and in C.Elegans (Ce). Protein sequence alignment was done using the CLUSTAL method. Conserved amino acid residues are boxed in black.

Figure 2

BAF53b is a subunit of a brain-specific chromatin remodeling complex. (A) BAF53b mRNA expression is restricted to the nervous system. Expression patterns for BAF53b (top) and BAF53a (bottom) in different adult mice tissues using ribonuclease protection assay. Arrows, protected fragments for both RNAs. Note that the faint upper band in all lanes of the BAF53b gel is the undigested probe. (B) BAF53b protein is expressed exclusively in mouse brain nuclear extract and reciprocally immunoprecipitates with the BAF ATPase subunit BRG1. Western blots for BAF53b (top) and BRG1 (bottom) using anti-BAF53b or anti-BRG1 antibodies. (Lanes 1–3) Nuclear extracts (10 μg) prior to purification. Immunoprecipitations using the antibodies shown on top of each panel are in lanes 4–7. (C) The bBAF complex is unique. (Top) Silver-stained SDS-PAGE gel (7.5%) showing immunopurified chromatin remodeling complexes from mouse brain nuclear extracts (Brain, lane 1) or HeLa cells (lane 3) using anti-BRG1 antibodies. (Lane 4) Immunopurification control of brain extracts with 12CA5 antibodies. Molecular weight markers (MW, lane 2) are indicated at the right. BAF subunits are shown at the left. Asterisks indicate nonspecific proteins. IgG is immunoglobulin heavy chain. (Bottom) BAF53b and BAF53a are mutually exclusive subunits of BAF chromatin remodeling complexes. After immunopurification of BAF complexes with the indicated antibodies, blots were analyzed with BAF53b- and BAF53a-specific antibodies. (Lane 3) Control immunoprecipitation using 12CA5 antibody. (Lanes 4,5) Input nuclear extracts [HeLa (He); Brain (Br); 10 μg]. (D) bBAF has ATP-dependent mononucleosome disruption activity. bBAF complexes attached to beads were immunopurified as described above and assayed on mononucleosomes in the absence (lane 7) or presence (lane 8) of 1 mM ATP. As a control, BAF complexes from Jurkat cells were used (lanes 5,6). No disruption is observed in the absence of chromatin remodeling complexes (lanes 3,4). Arrows show the ATP-dependent disruption of mononucleosomes (cf. lanes 5–8 and 3,4). Lanes 1 and 2 show controls for naked DNA and mononucleosomes alone, respectively.

Figure 3

bBAF is a combinatorially assembled chromatin remodeling complex distinct from BAF and PBAF. (A) BAF complexes from Brain (Br) and Hela cells (He) were immunoprecipitated and blotted with antibodies to different BAF subunits. Brain (Br, lane 1) and HeLa-derived BAF complexes (He, lane 2). (Lane 3) Control immunopurification using 12CA5 antibodies. (Lanes 4,5) Input nuclear extracts (10 μg). (B) BAF53b containing bBAF complexes also associates with BRM ATPase subunit. Anti-BAF53b antibodies were used to immunoprecipitate BAF complexes from either HeLa cells or mouse brain nuclear extracts. Western blots with anti-hBRM antibodies (dil 1:1000) show that BRM ATPase coimmunoprecipitates with BAF53b in brain (lane 2) but not from HeLa nuclear extracts (lane 1). Lane 3 is a control immunoprecipitation using 12CA5 antibodies. Whole RIPA cell extracts (15 μg) of the BRG1/BRM-deficient cell line C33A transiently transfected (36 h) with Brg1 (lane 4) or hBrm (lane 5) expression vectors show the specificity of the anti-BRM antibody. (Lane 6) Input mouse brain nuclear extract (10 μg) used in the immunoprecipitation. (C) BAF180 is not a subunit of bBAF. Anti-BRG1 antibodies were used to immunoprecipitate BAF complexes from mouse brain or HeLa nuclear extracts and analyzed for the presence of BAF180 using specific antibodies. BAF180 is present in the HeLa (BAF)- but not brain (bBAF)-derived complex (lanes 1,2), although both input nuclear extracts (10 μg) contain 180 kD proteins (lanes 4,5). Note that the brain p180 runs at a different mobility, and at this time it is not certain that the band seen from the brain represents authentic p180 or a cross-reacting protein. (Lane 3) Control immunoprecipitation using normal rabbit serum (NRS).

Figure 4

BAF53b expression during murine development. (A) Western blots for BAF53b (top), BAF53a (middle), and BRG1 (bottom) expression in whole cell RIPA extracts (15 μg) from murine embryos of different developmental stages (lanes 1–4). Lanes 5 and 6 show control nuclear extracts (10 μg) from adult mouse brain and HeLa cells, respectively. (B) BAF53b is not expressed in endoderm-derived tissues of E12.5 mouse embryo. Western blots for BAF53b (top) and BRG1 (bottom) expression in liver, stomach, and brain RIPA extracts (30 μg; lanes 1–3). (Lane 4) Control nuclear extract (10 μg) from adult mice brains. (C–H) BAF53b is expressed in the nervous system. Whole-mount in situ hybridization using a specific antisense probe for BAF53b. Sense probe (data not shown) showed minimal staining. (C,D) Lateral view of E9.5 and E10.5 embryos, respectively. (E–H) E12.5 embryos showing lateral (E), frontal (F), dorsal (G), and dorsolateral (H) views. Dorsal root ganglia derived from the neural crest are clearly staining in H.

Figure 5

BAF53b is expressed in postmitotic neurons but not glia. (A,B) BAF53b is expressed in the nucleus of neurons but not glia cells. A mixed population of neurons and glia cells derived from P0 rat hippocampus were cultured for 10 d, and BAF53b detected by immunofluorescence. (A) Costaining of neurons with the neuronal-specific marker TUJ1 (red) and BAF53b (green) antibodies as indicated. (B) Costaining with the astrocyte-specific marker GFAP (red) and BAF53b (green) antibodies as indicated. Note the empty nuclei denoting no detectable BAF53b staining. (C–G) BAF53b is expressed in postmitotic neurons. Double immunohistochemistry on paraffin-embedded embryo sections using antibodies against BAF53b (black-labeled cells) and the proliferation markers Ki-67 (C, E–G) or BrdU (D, red-labeled cells). (C) Sagittal section of E12.5 embryo head. BAF53b is expressed in the postmitotic cells of the nervous system. (D) Transverse section of cervical spinal cord from E12.5 embryo pulse-labeled in utero with BrdU. (E) Sagittal section of spinal cord from E12.5 embryo. (F) Sagittal section of olfactory bulb from E15.5 embryo. (G) Sagittal section of retina from P0 mouse. Tel, telencephalon; Die, diencephalon; Met, metencephalon; Mye, myelencephalon; PF, pontine flexure; Sp C, spinal cord; VZ, ventricular zone; ManL, mantle layer; Olf B, olfactory bulb; SVZ, subventricular zone; GCL, granule cell layer; MCL, mitral cell layer; Ret, retina; ONL, outer neuroblastic layer; GCL, ganglia cell layer.