Please use this identifier to cite or link to this item:
|Title:||Synthesis of H2BK120ub in Mammalian Nuclei Using a Novel Method to Chemically Modify Chromatin|
|Abstract:||Histone post-translational modifications (PTMs) play a key regulatory role in the eukaryotic cell. Consequently, aberrant PTM levels are implicated in developmental disorders, neurodegenerative diseases, and carcinogenesis. However, there are considerable barriers to studying molecular interactions and functions of histone PTMs with traditional biological tools. In vitro analyses using chemically-defined nucleosomes have provided exceptional insight into the mechanistic functions of histone PTMs, partially filling the methodological void by isolating specific biochemical phenomena for study. Nonetheless, this approach is limited due to the inherent reduction of biological complexity. Indeed, there is a need for techniques providing the precise control of chemistry within complex biological systems. In this study, we have developed one such approach that uses protein trans-splicing (PTS) to chemically join expressed and synthetic histone fragments together on native chromatin via the autocatalytic activity of naturally split inteins. Specifically, we demonstrate that PTS can be used to generate lysine-120 ubiquitinated histone H2B (H2Bub) in isolated mammalian nuclei in a time- controlled manner. This PTM is intimately involved in active gene transcription, DNA damage repair, and DNA replication, rendering it an important focus of epigenetic inquiry. As a preliminary study, we have attempted to interrogate known histone crosstalk pathways using semisynthetic H2Bub in nucleo. This work provides a model for the molecular interrogation of histone PTM interactions within biochemically complex systems, a useful technology for epigenetics research using chemical tools.|
|Type of Material:||Princeton University Senior Theses|
|Appears in Collections:||Molecular Biology, 1954-2016|
Files in This Item:
|Verma_Shivam.pdf||2.41 MB||Adobe PDF||Request a copy|
Items in Dataspace are protected by copyright, with all rights reserved, unless otherwise indicated.