High-resolution visualization of H3 variants during replication reveals their controlled recycling

Geneviève Almouzni,Alberto Gatto,Jean-Pierre Quivy,Zachary A Gurard-Levin,Ekaterina Boyarchuk,Audrey Forest,Mickaël Garnier,Judith Miné-Hattab,Bassam Hajj,Guillermo A Orsi,Camille Clément

doi:10.1038/s41467-018-05697-1

Abstract

DNA replication is a challenge for the faithful transmission of parental information to daughter cells, as both DNA and chromatin organization must be duplicated. Replication stress further complicates the safeguard of epigenome integrity. Here, we investigate the transmission of the histone variants H3.3 and H3.1 during replication. We follow their distribution relative to replication timing, first in the genome and, second, in 3D using super-resolution microscopy. We find that H3.3 and H3.1 mark early- and late-replicating chromatin, respectively. In the nucleus, H3.3 forms domains, which decrease in density throughout replication, while H3.1 domains increase in density. Hydroxyurea impairs local recycling of parental histones at replication sites. Similarly, depleting the histone chaperone ASF1 affects recycling, leading to an impaired histone variant landscape. We discuss how faithful transmission of histone variants involves ASF1 and can be impacted by replication stress, with ensuing consequences for cell fate and tumorigenesis.

Highlights

DNA replication is a challenge for the faithful transmission of parental information to daughter cells, as both DNA and chromatin organization must be duplicated
H3.1 in the genome; (ii) shows that H3.3 is enriched in earlyreplicating chromatin, while H3.1 is enriched in late-replicating chromatin; and (iii) shows that differences in transcription do not fully account for the opposite patterns of H3 variants. How do these findings translate into 3D spatial distribution and nuclear geography? Are they valid at the level of single cells? What are the dynamics of H3.3 and H3.1 that establish and maintain this histone variant landscape, in particular in S phase? To address these questions, we developed an assay to visualize histone variants and regions of replicated DNA using super-resolution microscopy
We focused on a 48 h chase for the following experiments for two reasons: (i) in our experimental conditions, it proved the minimum amount of time required for efficient small interfering RNA (siRNA) depletion, and (ii) a 48 h chase ensured that we examined parental histones without bias linked to cell cycle variation, for both variants H3.1 and H3.3

Summary

Introduction

DNA replication is a challenge for the faithful transmission of parental information to daughter cells, as both DNA and chromatin organization must be duplicated. While nucleosomes ahead of the replication fork are disrupted, the corresponding parental histones along with their modifications are recycled on newly synthesized DNA This process ensures the transmission of parental histone variants with their post-translational modifications[5]. Upon hydroxyurea treatment, replication arrest is concomitant with an accumulation of ASF1 loaded with histones carrying post-translational modifications characteristic of parental histones[31,37] These might be redeposited at unscheduled sites upon replication restart[37]. It remains unclear how replication stress affects the spatial distribution of parental histones, the importance of a tight control of histone recycling emerges as a central mechanism for the integrity of the epigenome[8]. The current model places ASF1 in a key position to recycle parental H3.3 and H3.1, yet its exact role and its contribution to maintaining a histone variant landscape need to be elucidated

Methods

Results

Conclusion