Gaarenstroom, Tessa et al. (2017) International Worm Meeting "Heterochromatin factors collaborate with small RNA pathways to combat repetitive elements and germline genotoxic stress."

Huang, Ni, Wysolmerski, Brian, Ahringer, Julie, McMurchy, Alicia, Appert, Alex, Aussianikava, Darya, Miska, Eric, Sapetschnig, Alexandra, Gaarenstroom, Tessa, Dong, Yan, Stempor, Przemyslaw, Kolasinska-Zwierz, Paulina

[International Worm Meeting, 2017]

Repetitive sequences derived from transposons make up a large fraction of the genome and must be silenced to protect genome integrity. Silencing of these elements is especially important in the germ line, where the piRNA pathway has been shown to be involved. Repeats are often found in heterochromatin, which in C. elegans are regions dispersed over the chromosome arms, however the roles and interactions of heterochromatin proteins are poorly understood. We have shown that a diverse set of heterochromatin factors act together with the piRNA and nuclear RNAi pathways to silence repetitive elements and prevent genotoxic stress in the germ line. HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2 and the H2K9me2 histone methyltransferase MET-2/SETDB1 show genome-wide co-binding and enrichment at repetitive elements, and mutants show a derepression of a subset of transposons. Furthermore, heterochromatin mutants are characterized by functionally redundant and temperature sensitive sterility, and display increased germline apoptosis and activation of DNA damage signalling. Remarkably, fertility of heterochromatin mutants could be partially restored by inhibiting expression of MIRAGE1 DNA transposons or endogenous meiotic double strand breaks. Loss of CEP-1/p53 also ameliorates both their fertility and somatic defects, suggesting that DNA damage signalling contributes to the phenotypes observed. Through genetics and transcriptional profiling, we uncovered complex interactions between heterochromatin factors and the piRNA and nuclear RNAi pathways, including functional redundancy in repetitive element repression between let-418 and nrde-2. This redundancy underlies the importance of safeguarding the genome through multiple means. It is also becoming evident that heterochromatic silencing occurs through different mechanisms at different genomic elements. We are currently dissecting interactions between the various factors, as well as H3K9me2/3, to uncover their mechanisms of action at heterochromatic regions, as well as investigating their roles in development and adult homeostasis.

Frapporti, Andrea et al. (2019) International Worm Meeting "Heterochromatin formation in embryogenesis."

Sirinakis, George, Allgeyer, Edward, Butler, Richard, Frapporti, Andrea, Ahringer, Julie

[International Worm Meeting, 2019]

The formation and stable maintenance of heterochromatin is essential for the functionality of the genome. Heterochromatin restrains the activity of parasitic mobile elements, keeps repetitive sequences recombinationally inert, and contributes to the functionality of euchromatin. A range of widely conserved proteins and pathways have been implicated in different aspects of heterochromatin regulation in eukaryotes. Hallmarks of constitutive heterochromatin are the presence of HP1 proteins and di- or tri-methylation of H3K9. Recent work carried out in the host laboratory uncovered the existence of a functional network of heterochromatin protein factors and small RNA pathways collaborating in ensuring gene and transposable element repression and proper development1. However, how they contribute to heterochromatin formation and organisation remains to be understood. I am addressing these questions by employing super-resolution nanoscopy and high-throughput genome-wide analyses. Towards this goal, I have adapted one- and two-colour direct-Stochastic Optical Reconstruction Microscopy (dSTORM) to C. elegansto analyse heterochromatin formation during early embryogenesis. In preliminary experiments, I show that H3K9me2 and H3K9me3 heterochromatin puncta appear early during embryo development and form larger nanoclusters by the 32-cell stage. The heterochromatin proteins LIN-13 and LET-418, which extensively co-localize with H3K9me2 and HPL-2 by ChIP-seq in adult mixed tissues1, are also organised into similar puncta. By combining in situ analyses by dSTORM with genome-wide chromatin immunoprecipitation experiments, my specific aims are: 1 - investigate the interdependencies and role of heterochromatin proteins in regulating heterochromatin establishment and structure during embryogenesis. 2 - identify sites of de novo heterochromatin formation by genome-wide chromatin profiling and determine whether and how heterochromatin is altered in mutant backgrounds. 1. McMurchy, A. N. et al.A team of heterochromatin factors collaborates with small RNA pathways to combat repetitive elements and germline stress. eLife6, e21666 (2017).

Yang, B. et al. (2017) International Worm Meeting "SMRC-1, a putative annealing helicase, links chromatin regulation and DNA repair in the C. elegans germ line."

Xu, X., Sullenberger, M., Yang, B., Yanowitz, J., Maine, E., Russell, L.

[International Worm Meeting, 2017]

Histone post-translational modifications are implicated in transcriptional control and chromosomal events. One modification, histone H3 lysine 9 dimethylation (H3K9me2), accumulates at certain classes of repetitive sequence across the C. elegans genome and is enriched on unsynapsed chromosomes in meiosis (Guo et al. 2015, Zeller et al. 2016, McMurchy et al. 2017, Kelly et al. 2002). MET-2 is the histone methyltransferase responsible for H3K9me2 (Andersen & Horvitz 2007, Bessler et al. 2010, Towbin et al. 2012). To understand how MET-2 activity is targeted to particular genomic sites, we have recovered candidate MET-2 interactors using a co-immunoprecipitation (co-IP) approach. One candidate is SMRC-1, the C. elegans ortholog of mammalian SMARCAL1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein). Mammalian and Drosophila SMARCAL1 family members are annealing helicases that provide protection from DNA replication stress (Poole & Cortez 2016). Using CRISPR-Cas9 methods, we flag-tagged the endogenous smrc-1 gene and generated smrc-1 loss-of-function mutations. We confirmed the interaction with MET-2 by co-IP with 3xFLAG::SMRC-1. We detect 3xFLAG::SMRC-1 in mitotic and meiotic nuclei in XX and XO germ lines. Consistent with the proposed role of SMARCAL1 in repairing DNA damage at the replication fork, smrc-1 mutant animals have an impaired response to DNA damage. E.g., they are hypersensitive to replication stress introduced by hydroxyurea and have elevated germline apoptosis that is dependent on the DNA damage checkpoint protein, CEP-1. In addition, smrc-1 mutants display developmental defects, including reduced fertility and embryonic lethality. Interestingly, meiotic H3K9me2 is variably reduced in smrc-1 mutants after passage for multiple generations, suggesting that SMRC-1 might promote MET-2 activity in the germline. Moreover, meiotic recombination is elevated in smrc-1 mutants. We observe enhanced sensitivity to DNA damage and highly penetrant sterility in the met-2 smrc-1 double mutant. We propose that MET-2 and SMRC-1 activities function in parallel to limit DNA damage and recombination at repetitive regions.

Townley, Anna et al. (2021) International Worm Meeting "Defining the functional components of constitutive heterochromatin through genetic interaction screening"

Pradhan, Roopali, Ahringer, Julie, Townley, Anna

[International Worm Meeting, 2021]

Constitutive heterochromatin makes up 20-50% of animal genomes and is associated with the silencing of gene expression, including that of repetitive elements. It is enriched for repressive histone modifications H3K9me2 and/or H3K9me3, which act as a binding sites for chromodomain-containing proteins related to HP1. Heterochromatin-induced gene silencing is an important modulator of developmental transitions, and defects in heterochromatin have been linked to a variety of human diseases, including cancer. The mechanisms through which heterochromatin is established and maintained have been extensively studied in the single-celled yeast Saccharomyces pombe, but they are not well understood in animals. We seek to identify and functionally dissect components of constitutive heterochromatin in C. elegans. Previous work in our lab identified a network of five factors that genetically interact and co-localise with H3K9me2 at repetitive elements (met-2, set-25, let-418, lin-13 and hpl-2; McMurchy et al, 2017). These heterochromatin factors regulate transposable element repression, gene expression, DNA repair, fertility and growth in C. elegans, in close collaboration with small RNA pathways. In order to expand this network, we carried out multiple genetic interaction screens using RNAi to discover genes whose knockdown could enhance or suppress the phenotypes of heterochromatin-defective mutants. Strains we screened include mutants of H3K9 methyltransferases (met-2, set-25), HP1 orthologues (hpl-1, hpl-2), and HP1-interacting genes (lin-61, lin-13, tdp-1). We identified genetic interactions through RNAi, using a panel of 2288 RNAi clones that target genes known or predicted to encode nuclear localised proteins. The screens identified shared as well as unique enhancers and suppressors of the heterochromatin-defective mutants and highlighted components of ubiquitylation, SUMOylation, RNA splicing and chromatin remodelling pathways, with most hits having orthologues in humans. Our results expand knowledge of the network of functional players in heterochromatin formation and function in a metazoan organism and identify targets for in depth studies in C. elegans and human cells.