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Despite the availability of several large-scale proteomics studies aiming to identify protein interactions on a global scale, little is known about how proteins interact and are organized within macromolecular complexes. Here, we describe a technique that consists of a combination of biochemistry approaches, quantitative proteomics and computational methods using wild-type and deletion strains to investigate the organization of proteins within macromolecular protein complexes. We applied this technique to determine the organization of two well-studied complexes, Spt-Ada-Gcn5 histone acetyltransferase (SAGA) and ADA, for which no comprehensive high-resolution structures exist. This approach revealed that SAGA/ADA is composed of five distinct functional modules, which can persist separately. Furthermore, we identified a novel subunit of the ADA complex, termed Ahc2, and characterized Sgf29 as an ADA family protein present in all Gcn5 histone acetyltransferase complexes. Finally, we propose a model for the architecture of the SAGA and ADA complexes, which predicts novel functional associations within the SAGA complex and provides mechanistic insights into phenotypical observations in SAGA mutants.

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.

Immunoglobulin heavy chain (IgH) class switch recombination and regulation of IgH expression levels are processes suggested to be controlled by the IgH 3' enhancer. Here we demonstrate that CD40 or IgM receptor stimulation of primary B cells results in transactivation of this enhancer. 4-Hydroxy-3-nitrophenylacetyl (NIP)-BSA induction of a K46 B cell line expressing a chimeric NIP-specific CD40 single chain receptor results in a ligand receptor-dependent response of a 3' enhancer ETS/AP-1 minimal promoter construct. Gel retardation analysis and genomic footprinting experiments reveal that CD40 or IgM induction recruits NFAB (nuclear factors of activated B cells) to the ETS/AP-1 motif. While IgM signalling recruits c-Fos, JunB and Elf-1 (NFAB-I), only JunB and Elf-1 were observed following CD40 signalling (NFAB-II). CD40 signalling, however, induces a Fos family-related partner for JunB, which may account for the transcriptional activity observed by NFAB-II in K46 cells. We propose a model whereby CD40 and IgM receptor-mediated signalling converge in the process of 3' enhancer activation in B lymphocytes. Our data provide a putative molecular explanation as to why CD40L-deficient mice, and possibly patients with hyper-IgM syndrome, are unable to undergo T cell-dependent class switch recombination but respond properly upon lipopolysaccharide-induced switch recombination.

Core histones isolated from normal and butyrate-treated HeLa cells have been reconstituted into nucleosome cores in order to analyze the role of histone acetylation in enhancing transcription factor binding to recognition sites in nucleosomal DNA. Moderate stimulation of nucleosome binding was observed for the basic helix-loop-helix factor USF and the Zn cluster DNA binding domain factor GAL4-AH using heterogeneously acetylated histones. However, by coupling novel immunoblotting techniques to a gel retardation assay, we observed that nucleosome cores containing the most highly acetylated forms of histone H4 have the highest affinity for these two transcription factors. Western analysis of gel-purified USF-nucleosome and GAL4-AH-nucleosome complexes demonstrated the predominant presence of acetylated histone H4 relative to acetylated histone H3. Immunoprecipitation of USF-nucleosome complexes with anti-USF antibodies also demonstrated that these complexes were enriched preferentially in acetylated histone H4. These data show that USF and GAL4-AH preferentially interact with nucleosome cores containing highly acetylated histone H4. Acetylation of histone H4 thus appears to play a primary role in the structural changes that mediate enhanced binding of transcription factors to their recognition sites within nucleosomes.

The function of the temporally regulated B lymphocyte-specific immunoglobulin heavy chain (IgH) 3' enhancer has been linked to the IgH class switch machinery, but the physiological mechanism(s) of activation has not been discerned. Following crosslinking of the IgM receptor, we demonstrate that the enhancer is transactivated in the B lymphoma cell line BAL-17. In both induced primary B lymphocytes and BAL-17 cells, the enhancer activation is concomitant with the recruitment of a novel DNA binding complex, nuclear factor of activated B cells (NFAB). NFAB contains the tissue-restricted Ets protein Elf-1 and the AP-1 factors Jun-B and c-Fos, which bind to a novel 3' enhancer ETS-AP-1 motif. IgM receptor-mediated activation or stimulation by phorbol-ester in BAL-17 cells demonstrates that the ETS-AP-1 motif, when linked to a heterologous gene, can confer a ligand/receptor-dependent response. In NIH 3T3 cells, Elf-1 expression is required for efficient ETS-AP-1 promoter activity in response to stimulation by 12-O-tetradecanylphorbol 13-acetate. Our results suggest a biological role for Elf-1 in the regulation of IgH gene expression, attribute a functional role for receptor-induced AP-1 proteins in B lymphocytes and provide evidence for a direct link between IgM receptor-mediated signalling and 3' enhancer activation.