Assessment of Nucleosomal Binding using a High-throughput Interactions by Fluorescent Intensity Assay

Open Access
Cognetti, Daniel Joseph
Area of Honors:
Biochemistry and Molecular Biology
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Song Tan, Thesis Supervisor
  • David Scott Gilmour, Honors Advisor
  • Gene regulation
  • Epigenetic
  • Chromatin
  • Nucleosome
  • HI-FI
  • RCC1
  • Ran
  • Histone
  • Biochemistry
Genetic information, DNA, encodes for cellular products that play important roles in function and development. However, properly functioning cells are not unrestrained factories. They are finely tuned through differential expression of various genes. Appropriate gene regulation is essential for healthy organisms, with many common diseases, such as cancer, resulting from malfunctions of gene expression. The organizational structure targeted for gene regulation is the nucleosome, consisting of DNA wrapped around proteins called histones. These basic units of DNA packaging are organized further into higher order chromatin where associated changes affect transcription and gene expression. Gene expression is regulated by a number of mechanisms, but our laboratory’s focus is chromatin epigenetics. Simply, epigenetics is the study of heritable changes that turn genes “on” or “off”, without altering the DNA sequence. By interrogating the biochemical and structural relationships between binding proteins, DNA and the organizational structure of the nucleosome the laboratory’s ultimate goal is to facilitate downstream clinical therapies. X-ray crystallography is the principal technique used to determine the molecular structure of proteins, however a relatively new technique, the HI-FI assay (High-Throughput Interactions by Fluorescent Intensity) can provide a quantitative characterization of the structural interactions occurring on the nucleosomal architecture. My project was to create four histone mutant analogs to be used in the HI-FI binding assay. By introducing cysteine mutations at particular locations in a histone protein, fluorescent probes can be covalently attached in a site-specific manner. Upon fluorescent dye attachment, binding factors of interest can be assayed for variation in fluorescent intensity. Alteration in fluorescent intensity then provides insight into the location and binding of the protein based on the specific location of the fluorescent probe. The creation of these four histone mutants provides the laboratory with an optimized and robust technique to study how chromatin enzymes interact with their nucleosome substrate.