Mapping the invisible chromatin transactions of prophase chromosome remodelling

Abstract

We have used a combination of chemical genetics, chromatin proteomics and imaging to map the earliest chromatin transactions during vertebrate cell entry into mitosis. Chicken DT40 CDK1as cells undergo synchronous mitotic entry within 15 minutes following release from a 1NM-PP1-induced arrest in late G2. In addition to changes in chromatin association with nuclear pores and the nuclear envelope, earliest prophase is dominated by changes in the association of ribonucleoproteins with chromatin, particularly in the nucleolus, where pre-rRNA processing factors leave chromatin significantly before RNA polymerase I. Nuclear envelope barrier function is lost early in prophase and cytoplasmic proteins begin to accumulate on the chromatin. As a result, outer kinetochore assembly appears complete by nuclear envelope breakdown (NEBD). Most interphase chromatin proteins remain associated with chromatin until NEBD, after which their levels drop sharply. An interactive proteomic map of chromatin transactions during mitotic entry is available as a resource at https://mitoChEP.bio.ed.ac.uk.

Release of Histone H3K4-reading transcription factors from chromosomes in mitosis is independent of adjacent H3 phosphorylation

“…Removal of GFP-TAF5 from chromosomes began within 5 min of nuclear envelope breakdown, and recovery started approximately 5 min after anaphase onset (Figure 4B, C; Figure S4B). This finding was consistent with multiple reports of TAF protein dissociation from mitotic chromosomes using a variety of methods, both with and without the use of formaldehyde (Djeghloul et al, 2020;Ginno et al, 2018;Samejima et al, 2022;Segil et al, 1996;Varier et al, 2010). Importantly, there was no significant difference in the results in wild type or Haspin knockout cells (twoway mixed effects model Anova, p = 0.30).…”