Gaarenstroom, Tessa et al. (2021) International Worm Meeting "Proliferation/differentiation control by the SWI/SNF nucleosome remodeler in vivo"

Blackwell, Alexander, van der Vlist, Ewan, Gaarenstroom, Tessa, Van den Heuvel, Sander, Godfrey, Molly

[International Worm Meeting, 2021]

During development cells undergo well-controlled fate changes, passing through proliferating precursor states before exiting the cell cycle and permanently differentiating. Changes in gene expression underlie these cellular decisions, ensuring tight control over this dynamic process. We have combined genetics with cell type-specific next-generation sequencing and phenotypic analyses to study the divisions of the mesoblast lineage. Using low-input RNA sequencing technology we characterized changes in gene expression over time, and found that more than a quarter of transcripts change as cells exit quiescence to first proliferate, and then differentiate into muscle cells. We show that a major chromatin remodeler, the SWI/SNF complex, is required to promote cell cycle exit and cell fate changes. Tissue-specific loss of function of an essential complex component results in delayed downregulation of cell cycle genes, coinciding with cellular overproliferation. Furthermore, depletion of SWI/SNF results in a failure of mesoblast descendants to differentiate. Current work is focused on the mechanism and collaborating factors by which SWI/SNF controls the proliferation-differentiation transition. Several key transcription factors were found to be misregulated following SWI/SNF loss, and we investigated effects of loss-of function of homeobox factors mls-2 and lin-39, as well as hlh-1 CeMyoD. We show that loss of hlh-1 also causes cells to overproliferate, suggesting a potential collaboration with SWI/SNF to regulate cell cycle genes. Finally, to discover additional contributing factors, we are employing a forward and reverse genetic screening approach. We tested an RNAi library containing 350 chromatin factors, and found that depletion of putative H3K27 demethylases partially suppress the SWI/SNF overproliferation phenotype. Future experiments will be aimed at identifying the mechanism of action of SWI/SNF on the chromatin, using cell-type specific and low input genomic profiling methods.

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.

Blackwell, Alexander et al. (2021) International Worm Meeting "Studying chromatin regulation at single cell resolution during C. elegans postembryonic development"

Gaarenstroom, Tessa, Van den Heuvel, Sander, Blackwell, Alexander

[International Worm Meeting, 2021]

Despite containing identical genomes, developing cells differentiate into a plethora of diverse cell types. Distinct patterns of gene expression now form the basis for classifying different cell fates. How the combinatorial activity of transcription factors, chromatin regulators and histone modifications achieve the proper spatiotemporal patterns of gene expression is a major question in developmental biology. Biologists increasingly appreciate the need to investigate gene expression regulation at the single-cell level because much heterogeneity and complexity is lost when averaging across populations of cells. However, profiling chromatin at the single cell level is challenging due to limited input material. Chromatin immunocleavage with sequencing (ChIC-seq) is an efficient method to study chromatin modifications from low input samples. ChIC-seq utilises antibody targeted micrococcal nucleases, leading to controlled, binding-dependent enzymatic digestion of DNA. This releases short fragments which become preferentially incorporated during library preparation and enables high resolution mapping of genomic positions. Crucially, the absence of crosslinking and immunoprecipitation steps, required in less sensitive techniques such as ChIP-seq, leads to minimal material loss. Recently, ChIC-seq was used to profile histone modifications in single human cells [1]. Adapting ChIC-seq to profile histone modifications in C. elegans will provide a powerful tool for studying the epigenetic regulation of development. Here, we present progress in optimising ChIC-seq for profiling chromatin modifications at single-cell level across a developmental time-course in C. elegans. Specifically, we combine Cre/Lox lineage tracing with cell isolation and FACS procedures in order to isolate postembryonic mesoderm cells. Following prolonged quiescence, the mesoblast precursor resumes proliferation and produces fourteen muscle cells, two scavenger cells, and two migratory bipotent myoblasts over 24-hours. By profiling chromatin modifications at high temporal resolution, we aim to reveal regulatory processes controlling cellular proliferation and differentiation. This work will shed light on how epigenetic modifications contribute to cellular decision making in a living animal. [1] Ku WL, Nakamura K, Gao W, Cui K, Hu G, Tang Q, Ni B, Zhao K. (2019) Single-cell chromatin immunocleavage sequencing (scChIC-seq) to profile histone modification. Nature Methods, vol. 16, pages 323-325.