Our goal is to understand the functions of Barrier-to-Autointegration Factor (BAF) and its conserved binding partner emerin at the nuclear envelope. BAF is a highly conserved DNA-binding protein in metazoans; without BAF, dividing cells die. BAF binds a family of nuclear membrane proteins that includes LAP2[5 and emerin. Mutations in emerin cause a tissue-specific disease, Emery-Dreifuss muscular dystrophy (EDMD), which affects heart, skeletal muscle and tendons. We discovered that emerin binds directly to several transcription repressors, including GCL. Interestingly, BAF competes with GCL for binding to emerin in vitro, suggesting that BAF might antagonize repressor binding to emerin in vivo. We further discovered that BAF binds directly to histone H 1 and histone H3, suggesting that BAF interacts with nucleosomes and may either regulate or be influenced by chromatin structure in vivo. We propose that emerin and BAF form a variety of protein complexes at the inner nuclear membrane, which affect chromatin structure and gene regulation. We discovered a mammalian protein named BFL, which is 54% identical to BAF. BFL interacts with BAF and LAP2[3, but does not bind to DNA or emerin. BFL mRNA is expressed in most tissues, but its absence from heart and skeletal muscle (selectively affected by EDMD disease) suggests that BFL might protect other tissues from the loss of emerin. Our models for the mechanisms and functions of BAF, BFL and emerin will be tested using biochemistry, microscopy, cell-free Xenopus extracts, and in vivo analysis in human cell lines. We have four specific aims. (1) Determine which domains in histone H1 and H3 bind BAF; test the hypothesis that BAF interacts with nucleosomes or influences HI-mediate chromatin compaction; determine if BAF binding to histone H3 is regulated by posttranslational modifications of the H3 tail. (2) Determine how phosphorylation affects BAF binding to emerin and other partners, and dissect structural roles for BAF during nuclear assembly in vitro. (3) Test the hypothesis that emerin represses gene expression, using luciferase reporter assays in cultured mammalian cells; we will also use our large collection of biochemically-characterized mutations in emerin and BAF to understand the functions of different emerin complexes both in vitro and in living cells. (4) Use a proven transcriptional repressor, LAP2[3, in luciferase reporter assays to test our hypothesis that BFL or BAF regulate LAP2[5 repression in vivo; use biochemical competition assays to determine which LAP2[5- or emerin-binding proteins compete, or cooperate, with each other for binding to LAP2[$ or emerin. Our goal is determine which partners contribute to repressive LEM- domain protein complexes at the inner nuclear membrane, and which partners anchor these complexes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM048646-12S1
Application #
7495928
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Shapiro, Bert I
Project Start
1994-05-01
Project End
2008-06-30
Budget Start
2006-08-01
Budget End
2008-06-30
Support Year
12
Fiscal Year
2007
Total Cost
$168,503
Indirect Cost
Name
Johns Hopkins University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Berk, Jason M; Wilson, Katherine L (2016) Simple Separation of Functionally Distinct Populations of Lamin-Binding Proteins. Methods Enzymol 569:101-14
Mojica, Sergio A; Hovis, Kelley M; Frieman, Matthew B et al. (2015) SINC, a type III secreted protein of Chlamydia psittaci, targets the inner nuclear membrane of infected cells and uninfected neighbors. Mol Biol Cell 26:1918-34
Wilson, Katherine L; Weis, Karsten (2015) Editorial overview: Cell nucleus: Nuclear structure and organization—open frontiers in cell and genome biology. Curr Opin Cell Biol 34:v-vi
Bar, Daniel Z; Davidovich, Maya; Lamm, Ayelet T et al. (2014) BAF-1 mobility is regulated by environmental stresses. Mol Biol Cell 25:1127-36
Berk, Jason M; Simon, Dan N; Jenkins-Houk, Clifton R et al. (2014) The molecular basis of emerin-emerin and emerin-BAF interactions. J Cell Sci 127:3956-69
Berk, Jason M; Tifft, Kathryn E; Wilson, Katherine L (2013) The nuclear envelope LEM-domain protein emerin. Nucleus 4:298-314
Simon, Dan N; Wilson, Katherine L (2013) Partners and post-translational modifications of nuclear lamins. Chromosoma 122:13-31
Berk, Jason M; Maitra, Sushmit; Dawdy, Andrew W et al. (2013) O-Linked ?-N-acetylglucosamine (O-GlcNAc) regulates emerin binding to barrier to autointegration factor (BAF) in a chromatin- and lamin B-enriched ""niche"". J Biol Chem 288:30192-209
Gjerstorff, Morten F; Rosner, Heike I; Pedersen, Christina B et al. (2012) GAGE cancer-germline antigens are recruited to the nuclear envelope by germ cell-less (GCL). PLoS One 7:e45819
Barkan, Rachel; Zahand, Adam J; Sharabi, Kfir et al. (2012) Ce-emerin and LEM-2: essential roles in Caenorhabditis elegans development, muscle function, and mitosis. Mol Biol Cell 23:543-52

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