The role of satellite DNA organization in nuclear mechanostability
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2025
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Doctoral Thesis
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Abstract
Cells frequently experience mechanical forces that influence their physiological function. The nucleus must withstand these stresses while preserving structural integrity, yet, the strategies by which nuclei resist mechanical load are only partially understood. Heterochromatin organization is increasingly recognized as a key factor mediating nuclear mechanostability. However, the contribution of satellite DNA, a major constituent of constitutive heterochromatin, remained elusive. These non-coding repeats, present as tandem-repeated sequences in vast tracks of 10^5-10^7 bp at the pericentromeric heterochromatin of eukaryotic chromosomes, have traditionally been dismissed as ‘junk DNA’, a view largely driven by the lack of well-characterized functions. In this study, we demonstrate that higher order organization of satellite DNA repeats into chromocenters contributes to the mechanical stability of the nucleus. Our work focuses on the conserved satellite DNA-binding protein D1, which is required for chromocenter formation in the Drosophila male germline.
We show that chromocenter disruption upon D1 loss leads to increased nuclear deformation in adult germ cells. This phenotype is absent in larval testes, where chromocenters are intact and only baseline levels of nuclear deformation are observed. Interestingly, these tissues differ in their mechanical load: The adult testis is encased in a contractile muscle sheath, while the larval testis is embedded in a fat body and exhibits minimal movement. We reveal that nuclear deformations in D1 mutant germ cells arise from increased sensitivity to mechanical force, as artificial compression of larval testes induces chromocenter disruption, nuclear deformation and de novo formation of interphase micronuclei, a phenomenon previously only observed in adult tissue. These findings suggest that chromocenter disruption compromises nuclear integrity under mechanical stress, leading to micronucleus formation. Importantly, we found that loss of D1 does not alter the expression of known nuclear mechanobiology factors such as Lamin B or HP1. In contrast, blocking the transmission of cytoskeletal forces to the nucleus by mutating components of the LINC complex rescues nuclear deformations and reduces micronucleus formation in D1 mutants. These findings suggest that while force transmission leading to nuclear deformation occurs through canonical pathways (e.g. via the LINC complex), the mechanostability provided by chromocenters represents a novel mechanism to safeguard nuclear integrity independent of classical contributors such as the nuclear lamina or global heterochromatin compaction.
To gain mechanistic insight, we developed an in silico polymer model which suggests that chromocenters help distribute mechanical tension more evenly across the nuclear envelope. In our system, these chromocenters form via D1, which contains 11 AT-hook motifs that bind AATAT satellite repeats. We show that D1 binds AT-rich DNA in vitro and forms phase separated droplets both on its own and in association with DNA. Structural analysis of this intrinsically disordered protein revealed a modular organization of four DNA binding modules containing AT-hooks interspersed with four negatively charged regions. Using a CRISPR-based mutagenesis screen, we generated a library of D1 variants with altered charge distribution and DNA-binding domain composition. The variant proteins differ in chromocenter clustering efficiency in cell culture, with low numbers of AT-hooks and increased positively charged residues being especially detrimental to chromocenter formation. Introducing selected D1 variants into D1 mutant testes rescued nuclear deformation in correlation with their capacity to form chromocenters. Furthermore, we show that nuclear mechanostability can be improved by enhancing chromocenter clustering, indicating that chromocenters play a key role in establishing nuclear mechanical resistance.
Finally, preliminary data suggests that nuclear deformations in D1 mutant germ cells correlate with the positioning of a specialized organelle called the fusome, likely due to its role in organizing the cytoskeleton. In addition, transcriptome analyses reveal upregulation of genes associated with the cellular periphery, plasma membrane, and extracellular space, potentially indicating a tissue-level response to mechanical stress. This response may also be reflected in the elevated numbers of cytoplasmic lipid droplets observed in these germ cells, suggesting a possible dysregulation of lipid metabolism pathways following D1 loss. Lastly, expanding our scope beyond Drosophila, the phenotypes resulting from chromocenter disruption show remarkable parallels to features observed in human diseases such as laminopathies, offering new possibilities to investigate the complex interplay of nuclear components in this context. Taken together, our data establish a novel function for the clustering of satellite DNA into chromocenters in maintaining nuclear mechanostability. We propose that modulating chromocenter formation may serve as a general strategy by which cells adapt to context-specific mechanical challenges, and our preliminary findings point to further promising directions for uncovering the broader roles of satellite DNA organization in eukaryotic genome function.
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09669 - Jagannathan, Madhav / Jagannathan, Madhav