Abstract
Barrier-to-autointegration factor (BAF) is a conserved metazoan protein that plays a critical role in retrovirus infection. To elucidate its role in uninfected cells, we first examined the localization of BAF in both mortal and immortal or cancerous human cell lines. In mortal cell lines (e.g. TIG-1, WI-38 and IMR-90 cells) BAF localization depended on the age of the cell, localizing primarily in the nucleus of >90% of young proliferating cells but only 20-25% of aged senescent cells. In immortal cell lines (e.g. HeLa, SiHa and HT1080 cells) BAF showed heterogeneous localization between the nucleus and cytoplasm. This heterogeneity was lost when the cells were synchronized in S phase. In S-phase-synchronized populations, the percentage of cells with predominantly nuclear BAF increased from 30% (asynchronous controls) to approximately 80%. In HeLa cells, RNAi-induced downregulation of BAF significantly increased the proportion of early S-phase cells that retained high levels of cyclin D3 and cyclin E expression and slowed progression through early S phase. BAF downregulation also caused lamin A to mislocalize away from the nuclear envelope. These results indicate that BAF is required for the integrity of the nuclear lamina and normal progression of S phase in human cells.
Highlights
Barrier-to-autointegration factor (BAF), a ~10 kDa dsDNAbinding protein, was first discovered as a host component of retroviral pre-integration complexes that facilitated integrasemediated integration into target DNA in vitro (Lee and Craigie, 1994)
BAF localization pattern is different in various cell types To better understand BAF localization, we used indirect immunofluorescence staining in various human cell lines using affinity-purified antibodies raised against a synthetic BAF peptide
Mostly cancer-derived cell lines, BAF localization differed from cell to cell, as shown above for HeLa cells: some cells showed mostly nuclear localization (Fig. 1C, arrows), others showed mostly cytoplasmic localization (Fig. 1C, arrowheads) and yet others showed BAF evenly distributed between the nucleus and cytoplasm (Fig. 1C, asterisks). These results suggest that BAF localization may be differently regulated in mortal and immortal cell lines that possess different proliferating ability
Summary
Barrier-to-autointegration factor (BAF), a ~10 kDa dsDNAbinding protein, was first discovered as a host component of retroviral pre-integration complexes that facilitated integrasemediated integration into target DNA in vitro (Lee and Craigie, 1994) (reviewed by Segura-Totten and Wilson, 2004). BAF exists as a dimer in solution (Cai et al, 1998) and binds dsDNA in vitro in a sequence-non-specific manner through a helixhairpin-helix motif common to other DNA-binding proteins (Zheng et al, 2000; Umland et al, 2000; Bradley et al, 2005). In the presence of 21-bp dsDNA, BAF dimers can associate as hexamers of dimers in vitro (Zheng et al, 2000). This activity is exploited by retroviruses to compact the retroviral DNA prior to integration (Zheng et al, 2000), but whether BAF has similar activity in uninfected cells remains unknown
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