Abstract
Glioblastoma is a type of brain tumor that accounts for 85% of the 17,000 new cases of malignant gliomas each year with a five-year survival rate of 4.7%. Eleven percent of glioblastoma cases have been found to contain histone H3.3 mutations, and nearly 80% of pediatric cases have been found to contain a histone H3.3 mutations. Histones wind DNA into nucleosomes, which in turn forms the 3-dimensional structure that make up chromatin and allows condensing of DNA. Histones play a major role in the epigenetic regulation of DNA, with post-translational effects determining how and which genes are turned on or off. Changes in histone H3.3 function may play a role during glioblastoma formation by preventing proper neural stem cell function and regulation that can lead to improper chromosomal segregation and aneuploidy. Histone H3.3 is constitutively expressed throughout the cell cycle, and is associated with both active and repressed gene states. H3.3 is coded by two genes, H3f3a and H3f3b. Knockout of either gene results in a loss of growth, viability, and fertility as well as chromosomal and karyotype abnormalities. Histone H3.3 can be present as both full length or cleaved, the cleaved forms are associated with several cell states including apoptosis, differentiation, and senescence. Cathepsin L, is an enzyme that can cleave histone H3.3 at the N-terminus producing known and unknown cleaved forms. H3.3cs is a cleaved form of Histone H3.3 between residues A22 and T23 that facilitates in gene silencing and the cellular senescence program; while embryonic stem cell differentiation produces a H3.3 N-terminally cleaved form at residues K27 and S28 are associated with cell growth and viability. Pinpointing how H3.3 modifications effect the epigenetic regulation of DNA is paramount to determining the cause of glioblastoma. The overall goal of Dr. Knoepfler’s lab is to determine the epigenetic controls of stem cells and particularly what causes this stem cell machinery to go awry. My project created a stepping stone by which cleaved histone h3.3 can be analyzed by creating a pBABE puro vector containing the cleaved histone #22 variant. The pBABE vector will be used to transduce neural stem cells and subsequent test of apoptosis, differentiation, and senescence will be done to conclude the effect of cleaved histone H3.3 #22. Our future hypothesis is that transduction of the cleaved histone H3.3 #22 will cause neural stem cells to differentiate into neural cells, glial cells, and astrocytes. Verification of cleaved histone H3.3 #22 role can be applied towards glioblastoma research by verifying whether it is expressed normally in glioblastoma cases.