Phosphorylation Regulates Barrier-to-Autointegration Factor (BAF) Function during Nuclear Assembly

            Jason Lei,  Biology (on left in photo)             

Faculty Mentor: Miriam Segura-Totten

              During cell division, the nucleus is initially disassembled (taken apart), and later reassembled (put back together). Barrier-to-Autointegration Factor (BAF) is a DNA-binding protein that is involved in the process of nuclear assembly. BAF also binds to the LEM domain family of inner nuclear membrane proteins. Thus, BAF mediates the interaction between our genome and the nuclear periphery. A phosphate group is attached to BAF at three different amino acids (threonines 2 and 3 and serine 4) by a protein called VRK-1. Phosphorylation blocks BAF’s interaction with DNA and weakens its binding to LEM proteins.

              We hypothesize that BAF is phosphorylated at the start of mitosis, resulting in the release of both chromatin (the complex of DNA and the proteins that package it) and LEM proteins. Furthermore, we propose that release of DNA and LEM proteins by BAF facilitates the dismantling of the nucleus. We hypothesize that during reassembly of the nucleus, phosphates are removed from amino acids 2, 3, and 4 in BAF, which allows it to bind DNA and LEM proteins. This, in turn, leads BAF to recruit membranes (through the LEM proteins) to chromosomes. To test these hypotheses, we added mutant BAF proteins that could not be properly phosphorylated to Xenopus laevis nuclear assembly extracts. Using this system, nuclei can be assembled independently without the need for a cell. We found that nuclei assembled in the presence of a BAF protein in which all three phosphorylation sites are blocked (MAAAQ) did not grow to full size and had condensed chromatin. These results suggest that phosphorylation of BAF is necessary to allow nuclei to grow and chromatin to decondense (or open up) during nuclear assembly. Assembly of nuclei in the presence of low concentrations of wild type (normal) BAF leads to enhanced nuclear growth and chromatin decondensation. Blocking BAF phosphorylation at threonines 2 and 3 results in a loss of these activities, suggesting that modification of these amino acids is important in the regulation of BAF activity during nuclear assembly. Currently, we are using transmission electron microscopy to examine the effects of assembling nuclei in the presence of our BAF mutants in more detail.

Personal Statement

              The summer undergraduate research program was a fantastic experience because it combined the excitement of science, discovery, and lab work with the enjoyment of exchanging ideas with researchers from other disciplines. For instance, I enjoyed the social aspect of SURP because these activities gave me the chance to interact with other students and faculty from my department as well as from other departments. The SURP research program broadened my horizons and made me a better research student. The most rewarding aspect of the program was working with my faculty advisor full time because I learned so much in such a short period of time. For example, I learned many techniques commonly used in molecular biology including PCR, mutagenesis, and SDS-PAGE. For all of the above reasons, I highly recommend SURP to TCNJ students.