Wenda, Joanna et al. (2021) International Worm Meeting "Linking centromeric factors to chromosome condensation in C. elegans embryos"

Wenda, Joanna, Steiner, Florian, Prosee, Reinier, Gabus, Caroline

[International Worm Meeting, 2021]

The centromere is a chromosomal region that recruits the kinetochore, a large multi-subunit complex that binds spindle microtubules enabling chromosome segregation. Centromeric chromatin is therefore placed on the surface of condensed chromosomes, and specifically adapted to withstand the spindle pulling forces. The cross-talk between centromeres and condensation machinery is an area of active study. In contrast to other commonly studied model organisms, C. elegans is a holocentric species: its centromeres are placed along the chromosome axis, which makes it an attractive model to study centromere organisation. Depletion of the centromeric histone H3 variant CENP-A (HCP-3 in C. elegans) and its main loading factor KNL-2 in embryos results in defective chromosome formation. However, little is known about the mechanism of their action and the temporal regulation. Using an immunoprecipitation followed by mass spectrometry, we identified regulatory phosphosites on KNL-2 that are targeted by CDK-1 kinase. Mutation of these residues to alanines to prevent their phosphorylation leads to defects in cell division and an increase in embryonic lethality. We show that compromised chromosome formation is underlying these phenotypes. Mutant embryos exhibit delayed prophase condensation, and the prometaphase chromosomes lack rigidity and twist around their axis. Interestingly, centromeric chromatin seems properly maintained, as evidenced by unchanged CENP-A genomic distribution and proper kinetochore recruitment in the mutant strain. We further found that condensin II levels are reduced on metaphase chromosomes in the mutant embryos, indicating that that KNL-2 may act through condensin II complex recruitment. We conclude that KNL-2 orchestrates centromeric chromatin maintenance as well as the establishment of the proper chromosome structure, and that these processes are independently regulated.

Ozdemir, Isa et al. (2021) International Worm Meeting "Regulation of transgenerational epigenetic H3K27me3 inheritance"

Steiner, Florian, Delaney, Kamila, Wenda, Joanna, Ozdemir, Isa

[International Worm Meeting, 2021]

A crucial process in life is the ability of cells to pass on information to their descendants. This transmission can occur at several levels from genetics to epigenetics. Epigenetic inheritance refers to the heritable phenotypes affected by gene expression without altering the DNA sequence and occurs through various factors including histone modifications. Interruption of this transmission process can lead to severe developmental defects and fertility diseases. Methylation of histone H3 at position 27 (H3K27me3) is an epigenetic mark that is associated with heterochromatin and repressed gene expression. Several studies have demonstrated that the genomic patterns of H3K27me3 can be inherited from one generation to the next by depositing H3K27me3 histones and PRC21. However, the limits of this inheritance are difficult to test because PRC2 mutants are maternal-effect sterile in C. elegans. We have recently shown that the expression of an H3.3K27M mutant acts in a dominant-negative manner and alters genomic H3K27me3 patterns and distribution of PRC2 and results in infertility phenotypes in C. elegans2. We used this strain to follow the trans-generational inheritance of the H3.3K27M-induced phenotypes. Strikingly, we observed that the altered patterning of H3K27me3 and the fertility defects are heritable for multiple generations after the H3.3K27M mutation is lost. These findings were also validated in a tetracycline-inducible system where we can abruptly switch on and off the H3.3K27M expression. We performed a targeted RNAi screen in our tetracycline-inducible system and detected several enhancer and suppressor modifiers of H3.3K27M defects such as chromatin remodeling complexes, the nuclear RNAi pathway, and histone writer and reader complexes. Overall, our results revealed that the transmission of H3K27me3 is an epigenetically programmed event that can last for generations and that several biological pathways seem to be involved in the regulation of transmission of H3K27me3 patterns across generations. 1. L. J. Gaydos, W. Wang, S. Strome. H3K27me and PRC2 transmit a memory of repression across generations and during development. Science 345, 1515-1518 American Association for the Advancement of Science (AAAS), 2014. 2. Kamila Delaney, Maude Strobino, Joanna M. Wenda, Andrzej Pankowski, Florian A. Steiner. H3.3K27M-induced chromatin changes drive ectopic replication through misregulation of the JNK pathway in C. elegans. Nature Communications 10 Springer Science and Business Media LLC, 2019.

Parham C et al. (1994) Worm Breeder's Gazette "Tc4 and Tc5: What Makes Them Move and Why It Matters"

Beinhorn J, Collins JJ, Butze K, Parham C

[Worm Breeder's Gazette, 1994]

Tc4 and Tc5: what makes them move and why it matters Christi Parham, Kristie Butze, Joanna Beinhorn and John Collins. Dept. of Biochemistry and Molecular Biology, University of New Hampshire. Durham, NH 03824

Delaney, Kamila et al. (2019) International Worm Meeting "H3.3K27M-induced chromatin changes drive ectopic replication through misregulation of the JNK pathway."

Pankowski, Andrzej, Delaney, Kamila, Strobino, Maude, Wenda, Joanna, Steiner, Florian

[International Worm Meeting, 2019]

Substitution of lysine 27 with methionine in histone H3.3 is a recently discovered driver mutation of pediatric high-grade gliomas. Tumor cells carrying this oncohistone show decreased levels of polycomb silencing as a result of physical inhibition of PRC2 methyltransferase activity, but how these chromatin changes lead to tumorigenisis remains unclear. To gain insight into the interplay between PRC2 activity and H3.3K27M incorporation, we introduced this mutation to the C. elegans H3.3 homologue his-72 and examined its genomic and phenotypic consequences in the context of a whole organism. Worms carrying the H3.3K27M mutation suffer from ectopic replication and cell cycle progression in the proximal germline. Our detailed chromatin analysis provides mechanistic insight into how the H3.3K27M remodels polycomb silencing genome-wide. Our data supports a model where the PRC2 activity is locally both inhibited by H3.3K27M incorporation and stimulated by pre-existing H3K27me3 in a concentration-dependent way. Through unbiased genetic screening, we identified the JNK pathway as a key driver of H3.3K27-induced DNA replication and cell cycle progression. We show that the replicative cell fate is reversible by targeting JNK in both nematodes and human tumor-derived H3.3K27M cells, and thus establish C. elegans as a powerful model for the identification of potential drug targets for treatment of H3.3K27M tumors.