Regulation of nuclear envelope assembly and disassembly

Johns Hopkins University, Baltimore MD

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Abstract

The long-term goals are to understand how the nuclear envelope and lamin filaments support gene activity in the nucleus, an open frontier in biology. We will focus on Barrier-to-Autointegration Factor (BAF), a DNA-binding protein that is conserved and essential in multicellular animals. BAF has key structural roles during nuclear assembly that involve its major partners: chromatin, lamins and the abundant [unreadable]LEM-domain[unreadable] family of nuclear proteins including emerin, a nuclear membrane protein. Mutations in either emerin or A-type lamins cause Emery-Dreifuss muscular dystrophy. BAF also has roles in muscle: it is required to maintain adult muscles in C. elegans, and its over-expression perturbs differentiation of cultured mouse myoblasts. The least-understood aspects of BAF are its interactions with chromatin, particularly in somatic cells. BAF regulates gene expression in a highly tissue-specific manner. BAF binds directly to core histones H3 and H4, suggesting it interacts with nucleosomes, the basic unit of chromatin structure. Nucleosomes are targeted by numerous factors that regulate higherorder folding and transcriptional potential ([unreadable]active[unreadable] vs [unreadable]silenced[unreadable] chromatin). Histone H3 and H4 acetylation is reduced in cells that overexpress BAF, and increases in BAF-deficient cells, suggesting BAF might inhibit histone acetylation. Furthermore recombinant BAF inhibits histone H3 acetylation by purified Gcn5, a histone acetyltransferase (HAT), suggesting BAF might block enzyme access to histones. Whether BAF influences other histone posttranslational modifications is unknown. We hypothesize that BAF is a novel epigenetic regulator. We will test two nonexclusive mechanisms by which BAF might influence chromatin. In the [unreadable]nucleosome organizer[unreadable] model, BAF dimers are proposed to compact nucleosomes through binding to DNA, H3, H4 or combinations thereof. This compaction might be a [unreadable]generic[unreadable] function for BAF in organizing nuclear chromatin, with the potential to sterically block chromatin-modifying factors. In the [unreadable]tethered BAF[unreadable] model, BAF is proposed to target specific promoters by direct binding to specific transcription factors. Once tethered, BAF might block histone acetylation (or other modifications) sterically, or might recruit alternative chromatin-modifying factors. Aim 1 will test the hypothesis that BAF compacts nucleosomes, by structural analysis of the products formed by BAF plus either mononucleosomes, 12-mer arrays of nucleosomes, or 12-mer nucleosome arrays plus linker histones. Aim 2 will identify other epigenetic modifications regulated by BAF in cells, and characterize novel transcription factors and chromatin regulatory factors that affinitypurify with BAF. Aim 3 is the whole-genome and promoter-specific analysis of BAF function in C. elegans; BAF-1 mutants will be tested for functional rescue of baf-1 null phenotypes and their affects on histone modifications at a specific BAF-1-regulated promoter.