Biology subjects

Zernia, S.

Publications and source records attributed to Zernia, S..

4 recordsLinked to original sources

bioRxiv2025

Single-Molecule Imaging Reveals DNA Shape Read-out by the INO80 Chromatin Remodeler

ATP-dependent chromatin remodeling enzymes act together to construct complex chromatin architectures around functionally important regulatory regions like promoters and origins of replication. The INO80 complex accurately positions nucleosomes bordering these regions, which is crucial for proper transcription start site selection and efficient replication. How INO80 localizes these nucleosomes and regulates its remodeling activity to produce specific positions mechanistically is unclear, although recent findings suggest a role for DNA shape features and barrier factors. Here, we use single-molecule DNA curtains to directly observe interactions of INO80 with DNA, nucleosomes and barrier factor Reb1. We show that INO80 recognizes DNA shape and strongly binds to shape features that are enriched in promoters and ORIs. Moreover, we find that INO80 performs 1D searches and intersegmental transfers to dynamically interact with nucleosomes and Reb1, but cannot bypass them, indicating that they could confine INO80 within promoters and thereby increase engagement with flanking nucleosomes. Our findings reveal how INO80s target-site search and remodeling activity are influenced by DNA shape recognition and molecular architecture and elucidate how remodelers can integrate different kinds of information to transform the chromatin landscape.

biochemistry↗

bioRxiv2024

LINE-1 ribonucleoprotein complexes bind DNA to enable nuclear entry during mitosis

Long Interspersed Nuclear Element-1 (LINE-1) is an autonomous retrotransposon that makes up a significant portion of the human genome, contributing to genetic diversity and genome evolution. LINE-1 encodes two proteins, ORF1p and ORF2p, both essential for successful retrotransposition. ORF2p has endonuclease and reverse transcription activity, while ORF1p binds RNA. Many copies of ORF1p assemble onto the LINE-1 RNA to form a ribonucleoprotein complex (RNP). However, the functional role of RNPs in the LINE-1 life cycle is unclear. Using reconstitution assays on DNA curtains, we show that L1 RNPs gain DNA binding activity only when RNA is super-saturated with ORF1p. In cells, L1 RNPs bind to chromosomes during mitosis. Mutational analysis reveals that DNA binding is crucial for nuclear entry and LINE-1 retrotransposition activity. Thus, a key function of ORF1p is to form an RNP that gains access to the genome through DNA binding upon nuclear envelope breakdown.

biophysics↗

bioRxiv2023

Single-molecule imaging reveals a direct role of CTCF's zinc fingers in SA interaction and cluster-dependent RNA recruitment

CTCF is a zinc finger protein associated with transcription regulation that also acts as a barrier factor for topologically associated domains (TADs) generated by cohesin via loop extrusion. These processes require different properties of CTCF-DNA interaction, and it is still unclear how CTCFs structural features may modulate its diverse roles. Here, we employ single-molecule imaging to study both full-length CTCF and truncation mutants. We show that CTCF enriches at CTCF binding sites (CBSs), displaying a longer lifetime than observed previously. We demonstrate that the zinc finger domains mediate CTCF clustering and that clustering enables RNA recruitment, possibly creating a scaffold for interaction with RNA-binding proteins like cohesins subunit SA. We further reveal a direct recruitment and an increase of SA residence time by CTCF bound at CBSs, suggesting that CTCF-SA interactions are crucial for cohesin stability on chromatin at TAD borders. Furthermore, we establish a single-molecule transcription assay and show that although a transcribing polymerase can remove CTCF from CBSs, transcription is impaired. Our study shows that context-dependent nucleic acid binding determines the multifaceted CTCF roles in genome organization and transcription regulation.

molecular biology↗

bioRxiv2021

The loader complex Scc2/4 forms co-condensates with DNA as loading sites for cohesin

The genome is organized by diverse packaging mechanisms like nucleosome formation, loop extrusion and phase separation, which all compact DNA in a dynamic manner. Phase separation additionally drives protein recruitment to condensed DNA sites and thus regulates gene transcription. The cohesin complex is a key player in chromosomal organization that extrudes loops to connect distant regions of the genome and ensures sister chromatid cohesion after S-phase. For stable loading onto the DNA and for activation, cohesin requires the loading complex Scc2/4. As the precise loading mechanism remains unclear, we investigated whether phase separation might be the initializer of the cohesin recruitment process. We found that, in absence of cohesin, budding yeast Scc2/4 forms phase separated co-condensates with DNA, which comprise liquid-like properties shown by droplet shape, fusion ability and reversibility. We reveal in DNA curtain and optical tweezer experiments that these condensates are built by DNA bridging and bending through Scc2/4. Importantly, Scc2/4-mediated condensates recruit cohesin efficiently and increase the stability of the cohesin complex. We conclude that phase separation properties of Scc2/4 enhance cohesin loading by molecular crowding, which might then provide a starting point for the recruitment of additional factors and proteins.

biophysics↗