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

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.

Two distinct HIRA-dependent pathways handle H3.3de novodeposition and recycling during transcription

“…We next recorded the TMR signal retained in the nuclei of a population of cells at the different times and used this measure as a proxy to assess histone loss (for details of the methods see Torné et al 2018). In agreement with previous observations (Clément et al 2018), the signal loss for both H3.1-and H3.3-SNAP could not be explained simply by the two-fold dilution expected from cell divisions, occurring every 24 hours in these cells ( Figure 1a). Furthermore, signal intensity showed a rapid decrease of 17% for H3.1-SNAP and 36% for H3.3-SNAP in the first two hours, with kinetics that cannot be explained by a single turnover rate ( Figure 1a).…”

Active and repressed chromatin domains exhibit distinct nucleosome segregation during DNA replication

“…It is only through our technique that accurately scores for parental nucleosome position prior to DNA replication that the parental histone origin can be determined. Although ChIP-seq and imaging systems in conjunction with synchronization experiments can detect the restoration dynamics of newly synthesized chromatin (Clement et al, 2018;Reveron-Gomez et al, 2018), such genomewide approaches cannot ascertain the original locale of a parental nucleosome. Thus, our findings here can attribute the inheritance of a repressed state to both the local segregation of parental nucleosomes and to the "write and read" modules of the histone methyltransferase that deposit the repressive modification ( Figure 6).…”

“…In particular, the in vivo re-deposition of parental histones within the general vicinity of their original genomic position has not yet been examined through direct methods, but instead through proteomics, marking of newly replicated DNA and ChIP-sequencing techniques (Zee et al, 2012;Alabert et al, 2014;Clement et al, 2018;Reveron-Gomez et al, 2018). Although, the combination of these approaches provides insights into the "bulk" re-deposition of parental nucleosomes, these studies cannot determine the fidelity of such re-deposition at a given chromatin domain, key to tackling the mechanisms of epigenetic inheritance.…”

Tousled-like kinase activity is required for transcriptional silencing and suppression of innate immune signaling

“…ALT+ tumors are frequently characterized by inactivation of the ATRX-DAXX complex, which is involved in histone H3.3 deposition and the maintenance of pericentromeric and telomeric heterochromatin. ASF1 has been shown to facilitate H3.3 deposition through the HIRA or CAF1 chaperones, which may be required to compensate for the lack of ATRX-DAXX in ALT+ tumors (Clement et al, 2018;Lovejoy et al, 2012).…”