A CORRELATIVE CRYO-ELECTRON TOMOGRAPHY APPROACH TO STUDY BRD4-NUT CHROMATIN CONDENSATES ACROSS SCALES

Biomolecular condensates (BMCs) are key organizers of cellular functions, and dysregulation of these compartments has been linked to neurodegenerative disorders and cancer. One example is NUT carcinoma, in which the fusion oncoprotein BRD4-NUT leads to aberrant chromosomal megadomains with liquid-like behavior. While biochemical and genomic studies have investigated the condensation mechanisms and molecular functions of these BMCs, their architecture remains substantially unresolved. High-resolution structural investigation, however, is crucial for elucidating the material properties of these micro-compartments, their accessibility to the transcriptional machinery, and thus their compatibility with active transcription. Cryo-electron tomography (cryo-ET) is suitable for conducting such analyses, as it allows molecular-resolution imaging of cellular structures within their native context. However, methodological challenges limit the straightforward application of cryo-ET to these liquid-like compartments. Specifically, preserving the ultrastructure of BMCs during sample preparation requires high-pressure freezing (HPF), and preparing samples suitable for cryo-ET after HPF necessitates technically challenging and low-throughput lift-out procedures. Furthermore, the sparsity of BRD4-NUT condensates within the cell nucleus requires the development of advanced three-dimensional correlative pipelines for effective targeting in cryo-ET. To address the shortcomings of the current approach, this dissertation aims to develop a robust cryo-electron tomography workflow for the structural interrogation of BMCs in reconstituted systems and in cells. First, we introduced SOLIST, an optimized cryo-lift-out method that increases throughput and improves sample and data quality. We demonstrated SOLIST’s broad applicability by revealing native molecular landscapes from mouse brain, liver, and heart tissues. Furthermore, I combined SOLIST with fluorescent light microscopy, establishing the first 3D correlation workflow in high-pressure frozen samples. I applied this pipeline to in vitro BRD4-NUT chromatin condensates, acquiring tomograms that showed densely packed nucleosomes and a distinct phase boundary from the buffer, compatible with other observations of reconstituted heterochromatin. Lastly, I extended the cryo-ET investigations to BRD4-NUT biomolecular condensates in cells, using a tailored cryo-correlative and electron microscopy approach to study these oncogenic BMCs within their physiological nuclear context. Preliminary results suggested that, in contrast to the in vitro case, the chromatin architecture within these micro-compartments remains relatively accessible in cells and may be compatible with transcription. However, larger datasets are needed for quantitative accessibility analyses. Altogether, this multi-scale framework facilitates a comprehensive structural interrogation of BRD4-NUT chromatin condensates in vitro and in their native cellular environment. Moreover, it establishes a versatile platform generalizable to other membraneless organelles. In combination with SOLIST, this approach may be extended to patient-derived NUT midline carcinoma specimens, effectively enabling a “biopsy at the nanoscale” and offering a tool for direct molecular mapping of human physiopathology.