Our model suggests that H3.3 plays a role in the opening of the chromatin around origins, allowing the replication fork to progress. Whether this defect, the accumulation of replication stress and the embryonic lethality observed at 25°C in the strain lacking H3.3 is due to the lack of this specific histone variant, or due to the lower abundance of nucleosomes on chromatin, remains an open question. To discriminate between these two possibilities, different experiments could be performed. First, we could detect nucleosome occupancy around origins in presence or in absence of H3.3. This could be done using MNase-seq, where MNase cuts the linker DNA while the nucleosomal DNA remains protected, and the nucleosome positions can be determined by sequencing these protected fragments (Chereji et al., 2019). The majority of fragments obtained by this method reflects nucleosome footprints, but it is important to be aware that DNA fragments can also be protected by non-nucleosomal DNA-binding proteins, such as replication initiation factors or transcription factors. This method would reveal if there are less nucleosomes flanking the replication origins in the strain lacking H3.3, which would indicate that a dilution of nucleosomes is the main driver of the observed defects. If the nucleosome occupancy is unchanged in the H3.3 null mutant strains, we would conclude that the loss of H3.3 is compensated by other histones (probably H3), and that the effects that we observe are likely due to the lack of this specific histone. The global compensation of the loss of H3.3 by H3 has been observed in Drosophila, where H3 became upregulated upon mutation of the two genes encoding for H3.3, and transgenic expression of H3 could rescue the lethality observed upon loss of H3.3 (Sakai et al., 2009). We can envision a similar experiment in C. elegans, where we would generate a new
strain with all H3.3 genes altered to express H3. This strain would produce the same number of histone molecules as the wild-type strain, but would only contain H3 nucleosomes. Therefore, nucleosome occupancy should be unaltered, and any observed phenotypes could be attributed specifically to the loss of H3.3.
DNA replication and transcription are well coordinated to avoid damage caused by collisions of the replication forks with the transcription machinery (Brambati et al., 2015; Gros et al., 2015). If the absence of H3.3 results in altered progression of DNA replication forks, this could affect the transcription of genes. Alternatively, as H3.3 is associated with active chromatin and could aid the transcription of genes, its absence could lead to alterations in genes expression that could lead to increased collisions with replication forks and replication stress, as observed in the H3.3 null mutant. To analyze the interplay between gene transcription and DNA replication, we performed RNA-seq experiments on embryos isolated from wild-type and H3.3 null mutant strains grown at 25°C. We found no global differences in transcription and thus no indication that H3.3 is an important regulator of transcription, in line with previous finding from our lab (Delaney et al., 2018). Very few genes showed significantly altered expression under temperature stress conditions. The most significantly upregulated genes encode uncharacterised proteins (ZK1025.3 and T05B4.9). These two proteins were not found as upregulated at permissive temperatures in a previous study from our lab (Delaney et al., 2018). Interestingly, ZK1025.3 contains a methyltransferase domain and could be involved in the regulation of nucleosomes or DNA replication through methylation under temperature stress conditions.
Our current analysis focused on DNA replication during embryogenesis, and we analyzed cells from mixed-stage embryos that likely include a variety of cell
types. Besides embryogenesis, DNA replication is also required to proliferate germ cells in the distal part of the adult germ line. It would be interesting to investigate if there are differences in the replication domain organization or origin usage between proliferating germ cells and proliferating embryonic cells. We hypothesize that this might be the case, as the former proliferate to generate a single cell type whereas the latter proliferate to produce a variety of cell types that will result in the formation of a complete larva. Profiling germ cells would require further adaptations of our methods, as the C. elegans germ line forms a syncytium with a shared cytoplasm that prevents the dissociation into individual cells (Hubbard and Greenstein, 2005). This makes it impossible to obtain synchronized germ cell populations that are required for EdU-seq, and Repli-seq experiments would have to rely on the isolation and sorting of EdU-treated nuclei.
The analysis of DNA replication at specific stages of embryogenesis, or in specific cell types is more feasible. Populations of embryos could be staged and harvested at specific developmental times. Additionally, specific cell types could be labelled with fluorescent markers and enriched by FACS. Both experiments would require refinement of our methods so that they could be applied to small populations of embryos or even single embryos or single cells.
Our data shows that loss of H3.3 affects the replication fork most around late and dormant origins, which are present in regions of lower transcriptional activity and more closed chromatin. Chromatin structure depends in part on transcriptional activity, and as some genes are sex-specific in C. elegans, the transcription patterns are different between males and hermaphrodites (Ebbing et al., 2018). If transcription influences DNA replication, we might expect to observe differences in origins usage and fork progression between males and hermaphrodites,
especially in absence of H3.3. To be able to separate male from hermaphrodite embryos, we have generated a strain with transgenic GFP expression driven by the male specific xol-1 gene promoter. In this strain, male (X0) embryos are green (Fig. 22), which allows to sort them with FACS to obtain a pure male population.
Figure 22. Strategy for enrichment of male embryos. Male (X0), but not hermaphrodites (XX) embryos are GFP positive, as they express GFP from the male specific xol-1 promoter. Images are shown for GFP and DIC channels. Scale bar is 50µm.
This method still requires adaptations because our mapping strategies necessitate a large number of nuclei or cells, and thus far embryos sorting has been successful only at low throughput.
Recent years provided much evidence for the importance of chromatin context in DNA replication. With my thesis, I have shown that H3.3 is an important player under stress condition for the fork progression around origins. More work is
needed to better understand how the presence of histone modifications or variants impacts origin definition, fork progression and more generally the relationship between chromatin context and DNA replication. Furthermore, is it now widely accepted that the genomes of most species are partitioned into domains that replicate at specific time during S phase, but the reasons and the regulation of this process are not well understood, and more research is needed in this field.
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