Abstract
Chromatin is a DNA-protein complex, composed largely of histone proteins. Changes in chromatin structure and composition influence gene expression throughout the genome. These chromatin and transcriptional changes are largely regulated by epigenetic factors including histone modification. In mice, histone variant H3.3 is encoded by two genes, H3f3a and H3f3b. Unlike canonical histones, H3.3 is expressed and deposited onto chromatin throughout the cell cycle and is associated with both transcriptionally active and poised genes. Replacement of canonical histone H3 with H3.3 may lead to functional differences in cell behavior. For instance, H3.3 is required for normal murine development, as studies have shown that loss of H3.3 can cause strong deleterious phenotypes. Furthermore, mutations of H3.3 particularly in cells during development of the central nervous system lead to childhood brain tumors. Better treatments for neurological diseases and these pediatric brain cancers represent a current unmet clinical need. Stem cell research is a promising field in regenerative medicine that may provide new treatments for developmentally related diseases, including neurological disorders. Stem cells are unique because they are able to self renew and differentiate into different cell types. H3.3 has been found to regulate stem cell characteristics and we hypothesize it serves a key role in neural stem cell (NSC) differentiation. A better understanding of the mechanisms of H3.3 function in regulating gene expression is necessary in order to develop safe stem cell therapies including the use of NSC, which could facilitate the development of new brain cancer therapies. In learning more about H3.3, we studied both of it’s genes, H3f3a and H3f3b, which encode for the same protein. To test our hypothesis and determine the role of H3.3 in NSC, we examined the effect of H3f3a and H3f3b mouse NSC knockouts both in naïve cells and during differentiation. Differentiation towards glial cells is generally favored in both knockout lines, implicating a strong role for H3.3 in cell fate control. We also found evidence of H3.3 coding gene compensation during late differentiation in H3f3b knockout NSCs in the form of elevated H3f3a. Increased protein levels of H3.3 relative to total H3, H3K4me3 and H3K27me3 in differentiated NSCs could indicate a role for H3.3 in chromatin and epigenetic cellular functions. We theorize that cleavage of H3.3 protein is initiated during differentiation, leading potentially to cellular senescence and impaired development. Finally we generated a potential double loss of function approach for H3f3a and H3f3b in NSCs and plan to further study the effect of H3.3 on differentiation, without the confounding effects of H3.3 coding gene compensation.