Steiner, Florian A et al. (2011) International Worm Meeting "A method for purifying nuclei from different tissues."

Henikoff, Steven, Priess, James R, Steiner, Florian A

[International Worm Meeting, 2011]

Obtaining tissue- or cell-type specific expression profile and chromatin profiles is one of the challenges in creating complete pictures of the genome landscapes. Our group recently published a simple method for the cell-type specific purification of nuclei from Arabidopsis roots1. The method, termed INTACT (Isolation of Nuclei TAgged in specific Cell Types), relies on a nuclear envelope tag that is biotinylated in vivo, which allows for affinity purification of the biotin-tagged nuclei. We have adapted the INTACT method to C. elegans tissues. In a first proof of principle strain, an outer nuclear pore complex protein is fused to mCherry for visualization and a BLRP tag (Biotin Ligase Recognition Peptide), and its expression is driven by the muscle-specific myo-3 promoter. The E. coli biotin ligase BirA is expressed ubiquitously, mediating in vivo biotinylation. Using this approach, muscle nuclei can be efficiently purified. Specificity of the pull-down is shown by an enrichment of mRNAs from muscle-specific genes. The system will allow purification of nuclei from different tissues at different developmental stages from embryos to adults. 1) A Simple Method for Gene Expression and Chromatin Profiling of Individual Cell Types within a Tissue. Deal RB, Henikoff S. Dev Cell. 2010 18(6) 1030-40.

Steiner, Florian A. et al. (2013) International Worm Meeting "Holocentromeres are dispersed point centromeres localized at transcription factor hotspots."

Steiner, Florian A., Henikoff, Steven

[International Worm Meeting, 2013]

Centromeres are universally marked by the conserved variant histone cenH3 (also called HCP-3 or CENP-A), but vary greatly in size and sequence composition. They range from 'point' centromeres with a single cenH3-containing nucleosome to 'regional' centromeres embedded in tandemly repeated sequences to holocentromeres that extend along the length of entire chromosomes. However, the precise distribution of centromeric nucleosomes in regional and holocentromeres has remained elusive. We used high-resolution mapping of cenH3-associated DNA to show that Caenorhabditids elegans holocentromeres are organized as dispersed but discretely localized point centromeres, each forming a single cenH3-containing nucleosome. These centromeric sites co-localize with kinetochore components, and their occupancy is dependent on the cenH3 loading machinery. The sites coincide with non-specific binding sites for multiple transcription factors ('HOT' sites), which become occupied when cenH3 is lost, suggesting that HOT site factors act as centromere 'placeholders'. Our results provide the first insight into the precise organization of centromeric nucleosomes outside of budding yeast. We show that the point centromere is the basic unit of holocentromeres and provide a basis for understanding how centromeric chromatin is maintained.

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.

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.

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.

Meng, J. et al. (2017) International Worm Meeting "A two-pore potassium channel regulates C. elegans locomotor behaviors."

Boulin, T., Mercier, M., Zhen, M., Hung, W., Mouridi, S., Meng, J.

[International Worm Meeting, 2017]

The resting membrane potential (RMP) is a key determinant of neuronal excitability1. The two-pore potassium (K2P) channel family has been shown to affect the RMP by conducting the background potassium current2. While many subfamilies of K2P channels have been identified molecularly, less is known about their modulation and physiological functions. C. elegans genome has 47 encoding K2P channel genes, providing an excellent experimental platform to examine and compare the diversity of cellular and physiological functions of K2P family channels. We have identified loss-of-function mutations in a putative two-pore potassium channel, TWK-40, that lead to exaggerated body bends, and increased reversals during locomotion. We find that TWK-40 is expressed and functionally required primarily in premotor interneurons to regulate the motor patterns. We further discover that optogenetical activation of the premotor interneurons may only partially mimic the effect of the endogenous changes of the RMP. I will present ongoing studying on how TWK-40 regulates locomotor behaviors in C. elegans. Reference 1. Hille, Bertil. Ion channels of excitable membranes. Vol. 507. Sunderland, MA: Sinauer, 2001. 2. Lesage, Florian, and Michel Lazdunski. "Molecular and functional properties of two-pore-domain potassium channels." American Journal of Physiology-Renal Physiology 279.5 (2000): F793-F801.