The functions of nuclear membrane proteins and A-type lamin filaments, linked to a growing number of signaling pathways, human diseases (`laminopathies') and human aging, are an important open frontier in biology. We propose to focus on emerin, a conserved lamin-binding nuclear membrane LEM-domain protein, and its conserved essential histone-binding chromatin partner Barrier-to-Autointegration Factor (BAF). Loss of emerin in human males causes X-linked recessive Emery-Dreifuss muscular dystrophy (EDMD). However, emerin is expressed in nearly all tissues, suggesting wider roles in cell physiology. Our recent purification and characterization of six distinct emerin-containing complexes from HeLa cell nuclei support the hypothesis that emerin `scaffolds'a variety of multi-protein complexes at the nuclear envelope, with functions that include signaling, chromatin silencing, gene regulation and nuclear architecture. Interestingly, emerin interacts with the DNA damage-response kinase, DNA-PK, in vivo, and emerin-downregulated HeLa cells have reduced response to DNA damage, as assayed by the formation of phosphorylated-H2AX foci. Our previous studies of BAF, the shared partner for emerin and all other LEM-domain nuclear proteins, demonstrated essential and broad-ranging roles in chromatin structure, mitotic chromosome segregation, nuclear assembly and gene regulation in specific tissues including muscle. Our supporting results show that BAF interacts with nucleosomes, influences several different histone posttranslational modifications in vivo, and associates in vivo with at least two histone-modifying proteins including poly(ADP-ribose) polymerase 1 (PARP1) and Cul4 of the Cul4-DDB-ROC E3 ligase complex. Both PARP1 and the Cul4-DDB- ROC complex modify nucleosomal histones in response to DNA damage. These findings implicate BAF as a regulator of muscle gene expression (and therefore relevant to the EDMD disease mechanism), and suggest broad physiological roles for BAF and emerin during cellular responses to DNA damage.
Aim 1 will test the hypothesis that BAF compacts nucleosomes by characterizing the effects of BAF on mono-nucleosomes and reconstituted 12-mer nucleosome arrays, and by determining the effect of BAF on PARP1-mediated polynucleosome folding in vitro.
Aim 2 will characterize roles for emerin and BAF in DNA damage response pathways in cells downregulated for emerin or BAF.
Aim 3 is a whole-genome and promoter-specific analysis of BAF function in C. elegans, which is expected to identify genes relevant to its roles in muscle and DNA- damage responses.
The proposed work is relevant to understanding the molecular mechanisms of Emery- Dreifuss muscular dystrophy, a syndrome that affects three tissues including skeletal muscle, tendons and the cardiac conduction system. The proposed investigation of BAF's interactions with chromatin and histone-modifying enzymes are also relevant to understanding why BAF is essential for human immunodeficiency virus type 1 (HIV- 1) to integrate into human chromosomes. The proposed studies of emerin and BAF in cellular responses to DNA damage are relevant to cancer and human aging.
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