Barrier-to-autointegration factor plays crucial roles in cell cycle progression and nuclear organization in Drosophila.

Department of Chemistry, Faculty of Science, Niigata University, Niigata 950-2181, Japan.
Journal of Cell Science (Impact Factor: 5.88). 10/2003; 116(Pt 18):3811-23. DOI: 10.1242/jcs.00682
Source: PubMed

ABSTRACT Barrier-to-autointegration factor (BAF) is potentially a DNA-bridging protein, which directly associates with inner nuclear membrane proteins carrying LEM domains. These features point to a key role in regulation of nuclear function and organization, dependent on interactions between the nuclear envelope and chromatin. To understand the functions of BAF in vivo, Drosophila baf null mutants generated by P-element-mediated imprecise excision were analyzed. Homozygous null mutants showed a typical mitotic mutant phenotype: lethality at the larval-pupal transition with small brains and missing imaginal discs. Mitotic figures were decreased but a defined anaphase defect as reported for C. elegans RNAi experiments was not observed in these small brains, suggesting a different phase or phases of cell cycle arrest. Specific abnormalities in interphase nuclear structure were frequently found upon electron microscopic examination of baf null mutants, with partial clumping of chromatin and convolution of nuclear shape. At the light microscopic level, grossly aberrant nuclear lamina structure and B-type lamin distribution correlated well with the loss of detectable amounts of BAF protein from nuclei. Together, these data represent evidence of BAF's anticipated function in mediating interactions between the nuclear envelope and interphase chromosomes. We thus conclude that BAF plays essential roles in nuclear organization and that these BAF functions are required in both M phase and interphase of the cell cycle.

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    ABSTRACT: BAF (Barrier to Autointegration Factor) is a highly conserved DNA binding protein that senses poxviral DNA in the cytoplasm and tightly binds to the viral genome to interfere with DNA replication and transcription. To counteract BAF, a poxviral-encoded protein kinase phosphorylates BAF, which renders BAF unable to bind DNA and allows efficient viral replication to occur. Herein, we examined how BAF phosphorylation is affected by herpes simplex virus type 1 (HSV-1) infection and tested the ability of BAF to interfere with HSV-1 productive infection. Interestingly, we found that BAF phosphorylation decreases markedly following HSV-1 infection. To determine whether dephosphorylated BAF impacts HSV-1 productive infection, we employed cell lines stably expressing a constitutively unphosphorylated form of BAF (BAF-MAAAQ) and cells overexpressing wild type (wt) BAF for comparison. Although HSV-1 production in cells overexpressing wtBAF was similar to that in cells expressing no additional BAF, viral growth was reduced approximately 80% in the presence of BAF-MAAAQ. Experiments were also performed to determine the mechanism of the antiviral activity of BAF with the following results. BAF-MAAAQ was localized to the nucleus, whereas wtBAF was dispersed throughout cells prior to infection. Following infection, wtBAF becomes dephosphorylated and relocalized to the nucleus. Additionally, BAF was associated with the HSV-1 genome during infection, with BAF-MAAAQ associated to a greater extent than wtBAF. Importantly, unphosphorylated BAF inhibited both viral DNA replication and gene expression. For example, expression of two regulatory proteins, ICP0 and VP16, were substantially reduced in cells expressing BAF-MAAAQ. However, other viral genes were not dramatically affected suggesting that expression of certain viral genes can be differentially regulated by unphosphorylated BAF. Collectively, these results suggest that BAF can act in a phosphorylation-regulated manner to impair HSV-1 transcription and/or DNA replication, which is similar to the antiviral activity of BAF during vaccinia infection.
    PLoS ONE 01/2014; 9(6):e100511. · 3.73 Impact Factor
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    ABSTRACT: BAF, which is encoded by the BANF1 gene, binds with high-affinity to dsDNA and LEM domain-containing proteins at the nuclear periphery. A BANF1 mutation has recently been associated with a novel human progeria syndrome, and cells from these patients have aberrant nuclear envelopes. The interactions of BAF with its DNA and protein binding partners are known to be regulated by phosphorylation, and previously we validated BAF as a highly efficient substrate for the VRK1 protein kinase. Here we show that depletion of VRK1 in MCF10a and MDA-MB-231 cells resulted in aberrant nuclear architecture. The immobile fraction of GFP-BAF at the nuclear envelope (NE) was elevated, suggesting that prolonged interactions of BAF with its binding partners was likely responsible for the aberrant NE architecture. Since the detachment of BAF from its binding partners is associated with NE disassembly, we performed live imaging analysis of control and VRK1-depleted cells to visualize GFP-BAF dynamics during mitosis. In the absence of VRK1, BAF did not disperse but instead remained chromosome-bound from the onset of mitosis. VRK1 depletion also increased the number of anaphase bridges and multipolar spindles. Thus, phosphorylation of BAF by VRK1 is essential both for normal NE architecture and for the proper dynamics of BAF-chromosome interactions during mitosis. These results are consistent with previous studies of the VRK/BAF signaling axis in C. elegans and D. melanogaster and validate VRK1 as a key regulator of NE architecture and mitotic chromosome dynamics in mammalian cells.
    Molecular biology of the cell 01/2014; · 5.98 Impact Factor
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    ABSTRACT: Barrier to autointegration factor (BAF) is an essential component of the nuclear lamina that binds lamins, LEM-domain proteins, histones and DNA. Under normal conditions, BAF protein is highly mobile when assayed by Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss in Photobleaching (FLIP). We report that C. elegans BAF-1 mobility is regulated by caloric restriction, food deprivation and heat shock. This was not a general response of chromatin-associated proteins, since food deprivation did not affect the mobility of Heterochromatin Protein HPL-1 or HPL-2. Heat shock also increased the level of BAF-1 Ser4 phosphorylation. By using missense mutations that affect BAF-1 binding to different partners we find that overall, the ability of BAF-1 mutants to be immobilized by heat shock in intestine cells correlated with normal or increased affinity for emerin in vitro. These results show BAF-1 localization and mobility at the nuclear lamina are regulated by stress, and unexpectedly reveal BAF-1 immobilization as a specific response to caloric restriction in C. elegans intestinal cells.
    Molecular biology of the cell 02/2014; · 5.98 Impact Factor

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