Biology subjects

Her, C.

Publications and source records attributed to Her, C..

4 recordsLinked to original sources

bioRxiv2026

The role of electrostatic interactions in the phase separation of HP1α and its protein binding partners

Heterochromatin protein 1 (HP1) is an intrinsic component of heterochromatin domains where it is involved in a diverse set of functions including heterochromatin spreading and organization, chromatin compaction and transcriptional silencing. It has been suggested that HP1 functions through a phase separation mechanism, a process that has been observed in vitro in the presence of N-terminal phosphorylation, nucleic acids and nucleosome arrays. HP1 can also interact with numerous binding partners that contain a specific motif called an HP1 access code (HAC). HACs recognize and bind to an interface formed by the chromoshadow (CSD) domains in the HP1 homodimer, the functional form of the protein. It has been shown that some HP1 binding partners can enhance its phase separation ability while others disrupt the process. Here, we focus on the interactions between HP1 and three binding partners, namely the p150 subunit of the chromatin assembly factor 1 (CAF-1), the N-terminal domain of the lamin B receptor (LBR), and the mitotic protein Shugoshin 1 (Sgo1). Using phase separation assays, we show that CAF-1 prevents HP1 phase separation while LBR and Sgo1 enhance it. Binding assays, mutational studies, NMR spectroscopy and computational analysis allow us to dissect the contributions of the HAC motifs, the charge patterns of the binding partner sequences and the role of N-terminal phosphorylation on HP1 in condensate formation. Our results demonstrate that each binding partner uniquely balances these contributions to modulate the properties of HP1, while electrostatic interactions dominate the regulation of phosphorylated HP1. These results suggest that HP1s binding partners play an important role in the modulation of its properties and the regulation of its functions in distinct biological contexts. Statement of significanceCellular nuclei are organized into euchromatin and heterochromatin domains, encompassing actively transcribed genes or transcriptionally silent regions, respectively. The regulation of these domains is an active area of study with vast consequences for human health and disease. Here, we focus on the HP1 protein, a key component of heterochromatin compartments that is believed to function through a phase separation mechanism. HP1 interacts with a multitude of binding partners that recognize a specific interaction surface on the protein. While these specific interactions are important, our results suggest that the overall electrostatic network that forms between the proteins has a profound effect on the properties of HP1. These results have important implications for understanding how HP1 function is regulated in heterochromatin environments.

biophysics↗

bioRxiv2023

The Immune-Evasive Proline 283 Substitution in Influenza Nucleoprotein Increases Aggregation Propensity Without Altering the Native Structure

Nucleoprotein (NP) is a key structural protein of influenza ribonucleoprotein complexes and is central to viral RNA packing and trafficking. In human cells, the interferon induced Myxovirus resistance protein 1 (MxA) binds to NP and restricts influenza replication. This selection pressure has caused NP to evolve a few critical MxA-resistant mutations, particularly the highly conserved Pro283 substitution. Previous work showed that this essential Pro283 substitution impairs influenza growth, and the fitness defect becomes particularly prominent at febrile temperature (39 {degrees}C) when host chaperones are depleted. Here, we biophysically characterize Pro283 NP and Ser283 NP to test if the fitness defect is owing to Pro283 substitution introducing folding defects. We show that the Pro283 substitution changes the folding pathway of NP without altering the native structure, making NP more aggregation prone during folding. These findings suggest that influenza has evolved to hijack host chaperones to promote the folding of otherwise biophysically incompetent viral proteins that enable innate immune system escape. TeaserPro283 substitution in flu nucleoprotein introduces folding defects, and makes influenza uniquely dependent on host chaperones.

biophysics↗

bioRxiv2022

Molecular interactions underlying the phase separation of HP1: Role of phosphorylation, ligand and nucleic acid binding

Heterochromatin protein 1 (HP1) is a crucial component for the proper maintenance of chromatin structure and function. It has been proposed that HP1 functions through liquid-liquid phase separation (LLPS), which allows it to sequester and compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1 can form phase separated liquid droplets upon phosphorylation of its N-terminus extension (NTE) and/or through interactions with DNA and chromatin. While it is known that LLPS requires homodimerization of HP1 and that it involves interactions between the positively charged hinge region of HP1 and the negatively charged phosphorylated NTE or nucleic acid, the precise molecular details of this process and its regulation are still unclear. Here, we combine computational modeling and experimental approaches to elucidate the phase separation properties of HP1 under phosphorylation-driven and DNA-driven LLPS conditions. We also tune these properties using peptides from four HP1 binding partners (Sgo1, CAF-1, LBR, and H3). In phosphorylation-driven LLPS, HP1 can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1 dimer conformation. This process can be enhanced by the presence of positively charged peptide ligands such as Sgo1 and H3 and disrupted by the addition of negatively charged or neutral peptides such as LBR and CAF-1. In DNA-driven LLPS, both positively and negatively charged peptide ligands can perturb phase separation. Our findings demonstrate the importance of electrostatic interactions in the LLPS of HP1 where binding partners can modulate the overall charge of the droplets and screen or enhance hinge region interactions through specific and non-specific effects. Our study illuminates the complex molecular framework that can fine tune the properties of HP1 and that can contribute to heterochromatin regulation and function.

biophysics↗

bioRxiv2020

Hepatocyte-specific deletion of XBP1 sensitizes mice to liver injury through hyperactivation of IRE1α

X-box binding protein-1 (XBP1) is a transcription factor that plays a central role in controlling cellular responses to endoplasmic reticulum (ER) stress. Under stress conditions, the transcriptionally active form of XBP1 is generated by unique splicing of Xbp1 mRNA by the ER-resident protein inositol-requiring enzyme-1 (IRE1). Genetic deletion of XBP1 has multiple consequences: some resulting from the loss of the transcription factor per se, and others related to compensatory upstream activation of IRE1. The objective of the current study was to investigate the effects of XBP1 deletion in adult mouse liver and determine to what extent they are direct or indirect. XBP1 was deleted specifically from hepatocytes in adult Xbp1fl/fl mice using AAV8-Transthyretin-Cre (Xbp1{Delta}hep). Xbp1{Delta}hep mice exhibited no liver disease at baseline, but developed acute biochemical and histologic liver injury in response to a dietary challenge with fructose for 4 wk. Fructose-mediated liver injury in Xbp1{Delta}hep mice coincided with heightened IRE1 activity, as demonstrated by cJun phosphorylation and regulated IRE1 -dependent RNA decay (RIDD). Activation of eIF2 was also evident, with associated up-regulation of the pro-apoptotic molecules CHOP, BIM and PUMA. To determine whether the adverse consequences of liver-specific XBP1 deletion were due to XBP1 loss or heightened IRE1 activity, we repeated a fructose challenge in mice with liver-specific deletion of both XBP1 and IRE1 (Xbp1{Delta}hep;IRE1{Delta}hep). Xbp1{Delta}hep;IRE1{Delta}hep mice were protected from fructose-mediated liver injury and failed to exhibit any of the signs of ER stress seen in mice lacking XBP1 alone. The protective effect of IRE1 deletion persisted even with long-term exposure to fructose. Xbp1{Delta}hep mice developed liver fibrosis at 16 wk, but Xbp1{Delta}hep;IRE1{Delta}hep mice did not. Overall, the results indicate that the deleterious effects of hepatocyte-specific XBP1 deletion are due primarily to hyperactivation of IRE1. They support further exploration of IRE1 as a contributor to acute and chronic liver diseases.

cell biology↗