The regulation of virulence gene expression is controlled by phase separation of heterochromatin protein 1 (HP1) in Plasmodium falciparum

Mamatharani, D. V.; Purkayastha, D.; Niederwieser, I.; Rai, S. K.; Gaur, P.; Ganji, M.; Mukhopadhyay, S.; Voss, T. S.; Karmodiya, K.

doi:10.1101/2025.09.23.678141

bioRxiv · 10.1101/2025.09.23.678141

The regulation of virulence gene expression is controlled by phase separation of heterochromatin protein 1 (HP1) in Plasmodium falciparum

Mamatharani, D. V.·Purkayastha, D.·Niederwieser, I.·Rai, S. K.·Gaur, P.·Ganji, M.·Mukhopadhyay, S.·Voss, T. S.·Karmodiya, K.

View original source ↗Export metadata ↓

Abstract

Malaria caused by Plasmodium falciparum involves antigenic variation on the infected red blood cell surface by a mutually exclusive expression of virulence (var) genes. The repressed var genes have a distinct genome organization where they localize as a cluster near the nuclear periphery and are bound by PfHP1 (P. falciparum Heterochromatin Protein 1). However, the mode of regulation of var genes by PfHP1 remains unclear. In this study, we show that PfHP1 undergoes phase separation in vitro in an RNA and DNA-dependent manner. Single-molecule DNA tethering experiments further revealed that AT-rich DNA sequences act as nucleation sites for the assembly and compaction of PfHP1-mediated heterochromatinization. We have also identified point mutations in the IDRs (intrinsically disordered regions) of PfHP1 that disrupt its phase separation as well as DNA compaction in vitro. To assess the dynamic properties of PfHP1 condensates in vivo, we performed fluorescence recovery after photobleaching (FRAP) in GFP tagged PfHP1 parasites, which revealed rapid fluorescence recovery, supporting their fluidity and phase separation behaviour. Ectopic expression of PfHP1 phase separation and DNA compaction mutants led to dispersed nuclear localization of PfHP1, in contrast to the punctate appearance of the wild-type protein and altered chromatin binding at var genes. These results were corroborated with DiCre/loxP-based conditional expression of the same PfHP1 mutants, which also led to the de-repression of multiple var genes (as many as 54 out of 60 var genes), mimicking the phenotype of PfHP1 depletion mutants. Hi-C sequencing of PfHP1 mutants revealed loss of interactions in the heterochromatic regions, indicating that PfHP1 phase separation is an essential mechanism for repressive cluster formation. In conclusion, our study demonstrates the role of PfHP1-mediated phase separation in heterochromatin formation and var gene silencing; unveiling a fundamental mechanism that drives antigenic variation in P. falciparum.

Explore related subjects

molecular biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Mamatharani, D. V., Purkayastha, D., Niederwieser, I., Rai, S. K., Gaur, P., Ganji, M., Mukhopadhyay, S., Voss, T. S., Karmodiya, K.. 2025-09-25. The regulation of virulence gene expression is controlled by phase separation of heterochromatin protein 1 (HP1) in Plasmodium falciparum. https://doi.org/10.1101/2025.09.23.678141

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Msp1-dependent extraction promotes ubiquitylation of translocation-stalled mitochondrial precursor proteins

The translocase of the outer membrane (TOM complex) imports more than 1,000 proteins into mitochondria. Clogging of the TOM pore with a precursor protein causes proteotoxic stress and eventually cell death. Two quality control pathways remove translocation-stalled precursor proteins. In the mitochondrial protein translocation-associated degradation (mitoTAD), Ubx2 recruits the cytosolic AAA-ATPase Cdc48 to clear precursor proteins from the TOM complex. In the mitochondrial compromised protein import response (mitoCPR), the stress-induced Cis1 recruits the AAA-ATPase Msp1 to Tom70. The role of Msp1 for the removal of mitochondrial precursor proteins remains unknown. Here, we demonstrate that parallel loss of Msp1 and Ubx2 strongly affects removal of precursor proteins and cell viability. Msp1 and Ubx2 bind independently of import stress and Cis1 to the TOM complex to remove a large variety of precursor proteins. Msp1-dependent extraction promotes ubiquitylation of precursor proteins, which in turn allows Ubx2-recruited Cdc48 to transfer the substrates to proteasomal degradation. We conclude that two AAA-ATPases cooperate in mitochondrial precursor quality control. Msp1-dependent extraction from the TOM complex facilitates precursor ubiquitylation and Cdc48-mediated transfer to proteasomal degradation.

molecular biology↗

bioRxiv2026

Dietary selenium deficiency drives sex-specific circadian disturbance through redox imbalance and causes early systolic dysfunction in mice

Background: Selenium is a vital trace element involved in antioxidant defence and cardiovascular health. Although selenium deficiency is implicated in cardiomyopathies, its early cardiac effects and underlying mechanisms remain poorly defined. Methods: C57BL6/Njr mice were fed either a selenium deficient or control diet for 12 weeks. Systemic selenium status, cardiac function by echocardiography, left ventricular (LV) transcriptomic profiles, redox balance, and circadian pathway markers were assessed, including sex-specific analyses. Results: Selenium deficiency reduced plasma selenium levels without inducing overt cardiac hypertrophy or fibrosis. Echocardiography showed preserved ejection fraction and fractional shortening but reduced global longitudinal strain, indicating early systolic dysfunction. Cardiac stress markers were increased predominantly in male mice. Left ventricular RNA sequencing revealed enrichment of pathways related to cardiac remodelling, redox regulation, mitochondrial function, and circadian rhythm. Additional protein and metabolic analyses supported sex-specific redox circadian alterations, with males showing a more pronounced stress response profile. Conclusions: Dietary selenium deficiency induces early myocardial dysfunction and molecular remodelling before overt cardiac failure. These changes are associated with redox and circadian pathway disruption and show sex specific features, suggesting that selenium contributes to cardiac homeostasis through sex dependent redox circadian regulation.

molecular biology↗

bioRxiv2026

Dysregulation of FMR1 Splicing in Human Fragile X Syndrome

Fragile X Syndrome (FXS) is a neuro-developmental disorder caused by a CGG expansion in FMR1, leading to transcriptional silencing and loss of the encoded protein FMRP. Surprisingly, ~70% of FXS individuals express FMR1, but the RNA is mis-spliced to isoform FMR1-217, composed of exon 1 spliced to a pseudo-exon in intron 1 and cannot produce FMRP. Splice-switching ASOs rescue proper FMR1 splicing and restore FMRP. FMR1-217 mis-splicing increases with CGG repeat length and is negatively correlated with patient IQ. FMR1-217 is associated with ribosome footprints, indicating it is translated into a polypeptide that may impair cognition. R-loops form at the FMR1 locus and extend into the pseudo-exon, but splice-switching ASOs reduce FMR1-217 and elevate FMRP independently of R-loop formation. DRB-based transcription analysis identified impaired Pol II elongation at the 5 prime region of FMR1 in FXS cells, indicated by accumulation of hypophosphorylated Pol II at the transcription start site. Consistent with this, camptothecin-induced Pol II stalling increased FMR1-217 pseudo-exon inclusion. The splicing factors PTBP1 and PTBP2 regulate FMR1-217 splicing in a differentiation stage-dependent manner. Together, these findings indicate that FMR1-217 mis-splicing in FXS is associated with CGG repeat expansion, R-loop formation, impaired co-transcriptional Pol II elongation and context-dependent regulation by PTBP1/PTBP2.

molecular biology↗