Mapping the Binding Region for Histone H3 on Barrier-to-Autointegration Factor (BAF)

Kimberly Parks,  Biology (on right in photo)

Faculty Mentor: Dr. Segura-Totten, collaborating with Dr. Hirsh, Chemistry Department

              A cell’s genome, or its DNA, is organized within the nucleus to determine what genes will be “expressed” to produce proteins, the molecules that perform most of the work inside the cell. The complex formed by DNA and the proteins that attach to it is called chromatin. Barrier-to-Autointegration Factor (BAF) is a DNA-binding protein that helps to organize chromatin and regulate gene expression. BAF binds to histone H3, one of the proteins that bind to DNA and compact it to form chromatin. There are five types of histones: H2A, H2B, H3 and H4, which work together to wrap the DNA into a structure called a nucleosome, and H1, which “links” different nucleosomes to form the thick DNA fibers that make up chromatin. The specific region on BAF where histone H3 binds has not been clearly determined. Mapping where on BAF histone H3 attaches will shed light into the nature of the interaction between these two proteins, and will help us form hypotheses about the implications of BAF binding to histones on chromatin structure and gene expression.

              To determine where on BAF histone H3 binds, a marker called a spin label is attached to the amino acid cysteine in particular regions of BAF. This spin label rotates freely in solution but its movement is restricted if it is found near a protein. If the spin label is located in a region of BAF where histone H3 binds, then the spin label rotation will be slower than that of the free label in solution. To ensure that BAF only contains one site for spin label attachment, the DNA encoding for BAF must be changed (or mutated) to remove all the endogenous cysteines. Then, the BAF DNA is mutated once more to insert a cysteine amino acid at a particular region. This process is repeated to produce DNA encoding for different BAF proteins, each with a single cysteine in a different region of the protein. We then introduce each DNA into bacterial cells, which are induced to produce BAF protein. The BAF protein purified from bacteria is used in spin-label studies of BAF binding to histone H3. This summer, I designed the mutations to remove endogenous cysteine amino acids in BAF and to introduce new cysteines. I also purified wild type (non-mutant) BAF for preliminary spin labeling experiments done by Dr. Hirsh’s research group in the Chemistry Department. I am currently creating the mutant BAF DNA for protein expression. Once purified, this protein will be used in spin-labeling experiments to determine the binding region on BAF of histone H3.

Personal Statement

              Though textbooks are irrefutably one of the most important tools in education, the skills and knowledge essential in the laboratory can only be grasped through experience. I have learned lab techniques that I will be using throughout my career, from electrophoresis, to mutagenesis, to chromatography. Faculty and students alike have also enlightened the path that an undergraduate needs to take to prepare for graduate school. The summer research program has undoubtedly enabled me to familiarize myself with laboratory protocols and interact with faculty and peers in a manner far removed from the strict guidelines and limitations of a classroom.