Molecular characterization of the histone demethylase PHF2 in maintaining proliferation, homeostasis and genomic integrity of neural progenitors

Abstract

[eng] The histone demethylase PHF2, previously studied in the laboratory, has been shown to be crucial for the expansion of neural progenitors by maintaining low levels of H3K9me2/3 at the promoters of cell cycle and developmental genes. It was also discovered that the absence of PHF2 induces the expression of repetitive elements such as Major Satellite, and that PHF2 interacts with components of heterochromatin. The thesis focuses on the interaction of PHF2 with constitutive pericentromeric heterochromatin and its role during mitosis. Experiments were conducted on neural stem cells (NSCs) both in control conditions and depleted of PHF2 through shRNA. The effects were analyzed across three areas of chromatin: euchromatin, pericentromeric heterochromatin (PcH), and the borders of PcH. In euchromatin, PHF2 was observed to localize at the promoters of cell cycle and developmental genes. Depletion of PHF2 resulted in increased levels of H3K9me3 and decreased accessibility at these promoters. In heterochromatin, PHF2 was enriched in satellite repeat sequences, such as GSAT_MM. Depletion of PHF2 increased the accessibility of heterochromatin and decreased H3K9me3 levels, leading to increased transcription of repetitive sequences. Live imaging experiments confirmed these changes in methylation.

Upon observing the disorganization of heterochromatin and considering its nucleation and extension mechanisms described in the literature, the study examined the boundary region between heterochromatin and euchromatin to elucidate what was causing this disruption of the heterochromatin and further genome instability. A significant enrichment of PHF2 was observed at the borders of pericentromeric heterochromatin (from megabase 3 to 3.5 on each chromosome). Depletion of PHF2 in these regions led to an increase in H3K9me3 and a decrease in accessibility, suggesting that PHF2 balances H3K9me3 across the genome. Eliminating PHF2 causes an imbalance between euchromatin and heterochromatin, silencing euchromatic regions and de-repressing previously repressed genes that were not targeted by PHF2. The integrity of heterochromatin can be affected either by the lack of transcriptional activity in the boundary regions or by the dilution of constitutive heterochromatin components, which are limited. This increases the expression of repetitive sequences, causing genomic instability and DNA damage. PHF2 requires both its PHD and JmjC domains to maintain the balance of H3K9me3 and genomic integrity. This was confirmed through overexpression and rescue experiments with mutants for each of these domains in shPHF2 neural stem cells. The positively charged amino acid-rich domain, important for phase separation and gene expression, is not essential for the integrity of heterochromatin.

In collaboration with Amanda Fisher's laboratory at the University of Oxford, the role of PHF2 during mitosis was studied. It was observed that PHF2 binds to mitotic chromosomes, affecting chromosomal size: depletion of PHF2 reduces chromosome size and alters methylation marks such as H3K9me3 and H3K27me3. An increase in H3K27me3 in the body of the chromosome and a reduction in H3K9me3 levels in the chromosome centromere were observed. Temporal transcriptomics revealed that post- mitotic transcription reactivation was delayed when PHF2 was depleted, especially in histone genes.

This doctoral thesis has significantly advanced the understanding of PHF2's role in balancing H3K9me3 and maintaining genomic stability. The research has demonstrated the importance of PHF2 in chromatin regulation and the proliferation of neural progenitor cells, highlighting its critical function in both heterochromatin and mitosis.