| The nuclear envelope, composed of the inner and outer nuclear membranes, serves to compartmentalize the nucleus within the cell. LAP2, emerin and MAN1 are part of a family of proteins of the inner nuclear membrane that share a 43-residue LEM domain. The conserved LEM domain interacts with Barrier-to-autointegration factor (BAF), a small DNA-binding protein conserved in metazoans. Although BAF was first discovered as a cytosolic activity that prevented retroviral self-integration, its cellular function was unknown. To understand the role of BAF in the nucleus, I looked at the effect of BAF on nuclear assembly and chromatin decondensation in Xenopus nuclear extracts. This work showed that BAF has a potent effect on chromatin structure and nuclear assembly. I biochemically mapped the regions of interaction in BAF for emerin and DNA using a collection of 25 BAF mutants. The region of DNA interaction I determined coincided with structural predictions, while the region of emerin interaction in BAF complements the shape and hydrophobicity of the LEM domain. I also determined the null phenotype for BAF in C. elegans by RNA interference. Embryos depleted of BAF die by the 100-cell stage and have DNA segregation defects. Emerin and MAN1 are mislocalized in nuclei lacking BAF, while lamins appear disorganized. These findings suggest that BAF plays a role in the assembly and organization of LEM proteins and lamins. We also discovered a novel protein that is homologous to BAF, which we termed BAF-like (BAF-L). BAF-L is expressed in all tissues tested except for heart, skeletal muscle and kidney. BAF and BAF-L have distinct biochemical properties: BAF-L does not interact with emerin or DNA. However, BAF-L interacts with two regions in LAP2 and MAN1, with BAF, and with itself. A two-hybrid study with BAF-L as bait yielded a transcriptional regulator MLL3, as a potential partner. This discovery links BAF-L, and potentially BAF, to transcriptional regulatory complexes. |
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