ABSTRACT
The diversity of dendritic patterns is one of the fundamental characteristics of neurons and is in part regulated by transcriptional programs initiated by electrical activity. We show that dendritic outgrowth requires a family of combinatorially assembled, neuron-specific chromatin remodeling complexes (nBAF complexes) distinguished by the actin-related protein BAF53b and based on the Brg/Brm ATPases. nBAF complexes bind tightly to the Ca(2+)-responsive dendritic regulator CREST and directly regulate genes essential for dendritic outgrowth. BAF53b is not required for nBAF complex assembly or the interaction with CREST, yet is required for their recruitment to the promoters of specific target genes. The highly homologous BAF53a protein, which is a component of neural progenitor and nonneural BAF complexes, cannot replace BAF53b's role in dendritic development. Remarkably, we find that this functional specificity is conferred by the actin fold subdomain 2 of BAF53b. These studies suggest that the genes encoding the individual subunits of BAF complexes function like letters in a ten-letter word to produce biologically specific meanings (in this case dendritic outgrowth) by combinatorial assembly of their products.
Subject(s)
Actins/genetics , Chromatin Assembly and Disassembly/physiology , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Dendrites/physiology , Neurons/cytology , Actins/deficiency , Animals , Calcium/metabolism , Cells, Cultured , Chromatin Assembly and Disassembly/genetics , Chromatin Immunoprecipitation/methods , Chromosomal Proteins, Non-Histone/deficiency , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/metabolism , Dendrites/genetics , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/metabolism , Hippocampus/cytology , Mice , Mice, Knockout , Models, Biological , Nerve Tissue Proteins/metabolismABSTRACT
Expression of the gamma-globin gene is silenced in adult humans. However, certain point mutations in the gamma-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T-->C point mutation at position -198 of the Agamma-globin gene promoter results in 4-10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH -198 gamma-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH -198 gamma-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of gamma-globin gene expression in the adult transcriptional environment is discussed.
Subject(s)
Fetal Hemoglobin/genetics , Gene Expression Regulation, Developmental , Globins/genetics , Promoter Regions, Genetic/physiology , Transcription Factors/metabolism , Adult , Animals , Antibody Specificity , Blotting, Western , Cell Fractionation , Cell Line, Tumor , Chromatography, Affinity , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/immunology , DNA (Cytosine-5-)-Methyltransferases/isolation & purification , DNA (Cytosine-5-)-Methyltransferases/metabolism , Humans , Leukemia, Erythroblastic, Acute , Mass Spectrometry , Mice , Mice, Transgenic , Nuclear Matrix-Associated Proteins/immunology , Nuclear Matrix-Associated Proteins/isolation & purification , Nuclear Matrix-Associated Proteins/metabolism , Point Mutation , Sp1 Transcription Factor/immunology , Sp1 Transcription Factor/isolation & purification , Sp1 Transcription Factor/metabolism , Transcription Factors/immunology , Transcription Factors/isolation & purification , Transcription Factors, TFII/immunology , Transcription Factors, TFII/isolation & purification , Transcription Factors, TFII/metabolism , Transcriptional ActivationABSTRACT
The existence and function of actin in the nucleus has been hotly debated for forty years. Recently, beta-actin was found to be a component of mammalian SWI/SNF-like BAF chromatin remodeling complexes and still more recently other SWI/SNF-related chromatin remodeling complexes in yeast, flies, and man. Although the function of actin in these chromatin remodeling complexes is only starting to be explored, the fact that actin is one of the most regulated proteins in the cell suggests that control of nuclear actin may be a critical regulatory point in the control of chromatin remodeling. Actin rapidly shuttles between the nucleus and the cytoplasm offering additional sites and modes of regulation. In addition, actin-related proteins (Arps) are also components of these chromatin remodeling complexes and have been implicated in transcriptional control in yeast. The observation that the BAF chromatin remodeling complex in which actin was originally identified, is also a human tumor suppressor complex necessary for the actions of the retinoblastoma protein indicates that the study of nuclear actin is likely to contribute to understanding cell growth control.
Subject(s)
Actins/metabolism , Chromatin/metabolism , Drosophila Proteins , Nuclear Proteins/metabolism , RNA-Binding Proteins , Animals , Calcium Signaling/physiology , Cell Nucleus/metabolism , Cell Transformation, Neoplastic , Fungal Proteins/metabolism , Insect Proteins/metabolism , Macromolecular Substances , Microfilament Proteins/metabolism , Nucleosomes/metabolism , Ribonucleoprotein, U1 Small Nuclear/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , rho GTP-Binding Proteins/metabolismABSTRACT
A highly conserved multisubunit enzymic complex, SWI/SNF, participates in the regulation of eukaryote gene expression through its ability to remodel chromatin. While a single component of SWI/SNF, Swi2 or a related protein, can perform this function in vitro, the other components appear to modulate the activity and specificity of the complex in vivo. Here we describe the cloning of hELD/OSA1, a 189 KDa human homologue of Drosophila Eld/Osa protein, a constituent of Drosophila SWI/SNF. By comparing conserved peptide sequences in Eld/Osa homologues we define three domains common to all family members. A putative DNA binding domain, or ARID (AT-rich DNA-interacting domain), may function in targetting SWI/SNF to chromatin. Two other domains unique to Eld/Osa proteins, EHD1 and EHD2, map to the C-terminus. We show that EHD2 mediates binding to Brahma-related gene 1 (BRG1), a human homologue of yeast Swi2. EHD1 and EHD2 also appear capable of interacting with each other. Using an antibody raised against EHD2 of hELD/OSA1, we detected Eld/Osa1 in endogenous SWI/SNF complexes derived from mouse brain.
