Nucleosomes as carriers of epigenetic memory
A core assumption in chromatin biology is that nucleosomes store and transmit epigenetic memory of gene regulation through cell division. A requirement of this model is that nucleosomes – modified in place in one generation – continue to occupy the same DNA locus in the subsequent generation. In my graduate work, I asked whether nucleosomes remember their position through cell division. To execute my experiments, I developed a synthetic chromatin modifying enzyme that deposited high affinity covalent chromatin modification at a defined locus. The chromatin-modifying enzyme could be delocalized within minutes by the addition of a small molecule antibiotic, and the synthetic chromatin modification could be tracked for several hours after preventing new label deposition. Using this synthetic enzyme, I labeled nucleosomes at the GAL10 locus in S cerevisiae and tracked their position through DNA replication. I found that nucleosomes remained localized through DNA replication, suggesting that nucleosomes can transmit epigenetic. Furthermore, I found that in the absence of the fork associated nucleosome chaperones Mcm2 and Dpb3, nucleosomes did not remain localized, suggesting that mcm2 dpb3 mutants cannot transmit epigenetic memory through chromatin. In addition to exploring the fate of nucleosomes during DNA replication, I tracked nucleosomes through transcription and discovered that contrary to published models, nucleosomes are not translated linearly along an open reading frame during transcription.
In the thesis that follows, I discuss the context for our collective intuition about the role of chromatin in transmitting epigenetic memory, and I identify the points at which our collective understanding rests on thin or contradictory data. I then discuss my attempts to develop and apply technology to reach a satisfactory resolution of one of the central unanswered questions in chromatin biology: do nucleosomes remember their position? Lastly I discuss side projects adjacent to chromatin biology that occupied my attention during the slower moments of my core thesis research.