Functional Regulation of the Histone Chaperone Nucleoplasmin

Abstract

Vertebrate pre-blastula cells are transcriptionally quiescent and undergo accelerated cell replication. This unique cellular environment necessitates the use of the maternally stored proteins for replication, one of which is histones. Since histones are essential for replication, yet excess soluble histones are deleterious to cells, a regulated switch between storage of histones to its rapid release for the accelerated cell replication is essential for the proper assembly and maintenance of the zygotic epigenome. The frog Xenopus laevis is therefore a compelling experimental model to study histone storage and release due to its extreme store of histones, rapid initial cell cycles, and transcriptional quiescence.;Nucleoplasmin (N pm), a histone chaperone for histones H2A-H2B is abundantly expressed in the oocyte, laid egg, and throughout embryogenesis. The large amount of Npm in the Xenoupus nucleus during oogenesis suggested its function as a storage chaperone for histones. However, no direct evidence has been shown to support the function of Npm in histone storage. Npm is also known to be dynamically phosphorylated during development. Therefore, I hypothesized that the distinct post-translational modifications (PTMs) of Npm regulate its chaperoning functions.;The precise PTMs were identified employing mass spectrometry. Glutamylation and arginine methylation on the C-terminal tail and multiple phosphorylations on the Nand C-terminal tails were identified, while a higher degree of phosphorylation was observed on egg Npm (eNpm). Consistent with my hypothesis, in vitro oocyte Npm (oNpm) and eNpm exhibited distinct histone deposition patterns in in vitro chromatin assembly assays, where oNpm showed strong histone deposition, and eNpm showed sequestration of histones.;Through truncation analyses I found that the long acidic patch on the C-terminal tail is critical for histone deposition. Further analysis using Ser-to-Asp phospho-mimetic mutations and in vitro glutamylation by TTLL4 suggested that C-terminal tail PTMs cause a conformational change to expose the acidic patch, leading to strong histone deposition. Phospho-mimetic mutations on the N-terminus exhibited partial sequestration of histones and suggested a further conformational change. Consistent with our model that PTMs cause conformational changes, electron microscopy reconstructions of rNpm, oNpm, and eNpm revealed distinct conformations at each stage. Here, I present that the PTMs on Npm modulate the conformation of Npm leading to sequestration of histones in the egg.