A natural histone H2A variant lacking the Bub1 phosphorylation site and regulated depletion of centromeric histone CENP-A foster evolvability in Candida albicans.

Eukaryotes have evolved elaborate mechanisms to ensure that chromosomes segregate with high fidelity during mitosis and meiosis, and yet specific aneuploidies can be adaptive during environmental stress. Here, we identify a chromatin-based system required for inducible aneuploidy in a human pathogen. Candida albicans utilizes chromosome missegregation to acquire tolerance to antifungal drugs and for nonmeiotic ploidy reduction after mating. We discovered that the ancestor of C. albicans and 2 related pathogens evolved a variant of histone 2A (H2A) that lacks the conserved phosphorylation site for kinetochore-associated Bub1 kinase, a key regulator of chromosome segregation. Using engineered strains, we show that the relative gene dosage of this variant versus canonical H2A controls the fidelity of chromosome segregation and the rate of acquisition of tolerance to antifungal drugs via aneuploidy. Furthermore, whole-genome chromatin precipitation analysis reveals that Centromere Protein A/ Centromeric Histone H3-like Protein (CENP-A/Cse4), a centromeric histone H3 variant that forms the platform of the eukaryotic kinetochore, is depleted from tetraploid-mating products relative to diploid parents and is virtually eliminated from cells exposed to aneuploidy-promoting cues. We conclude that genetically programmed and environmentally induced changes in chromatin can confer the capacity for enhanced evolvability via chromosome missegregation

Exciting Times for Lipid Nanoparticles: How Canadian Discoveries Are Enabling Gene Therapies

In this brief perspective, we describe key events in the history of the lipid-based nanomedicine field, highlight Canadian contributions, and outline areas where lipid nanoparticle technology is poised to have a transformative effect on the future of medicine

Recording of DNA-binding events reveals the importance of a repurposed Candida albicans regulatory network for gut commensalism.

Candida albicans is a fungal component of the human gut microbiota and an opportunistic pathogen. C. albicans transcription factors (TFs), Wor1 and Efg1, are master regulators of an epigenetic switch required for fungal mating that also control colonization of the mammalian gut. We show that additional mating regulators, WOR2, WOR3, WOR4, AHR1, CZF1, and SSN6, also influence gut commensalism. Using Calling Card-seq to record Candida TF DNA-binding events in the host, we examine the role and relationships of these regulators during murine gut colonization. By comparing in-host transcriptomes of regulatory mutants with enhanced versus diminished commensal fitness, we also identify a set of candidate commensalism effectors. These include Cht2, a GPI-linked chitinase whose gene is bound by Wor1, Czf1, and Efg1 in vivo, that we show promotes commensalism. Thus, the network required for a C. albicans sexual switch is biochemically active in the host intestine and repurposed to direct commensalism

A natural histone H2A variant lacking the Bub1 phosphorylation site and regulated depletion of centromeric histone CENP-A foster evolvability in Candida albicans.

Eukaryotes have evolved elaborate mechanisms to ensure that chromosomes segregate with high fidelity during mitosis and meiosis, and yet specific aneuploidies can be adaptive during environmental stress. Here, we identify a chromatin-based system required for inducible aneuploidy in a human pathogen. Candida albicans utilizes chromosome missegregation to acquire tolerance to antifungal drugs and for nonmeiotic ploidy reduction after mating. We discovered that the ancestor of C. albicans and 2 related pathogens evolved a variant of histone 2A (H2A) that lacks the conserved phosphorylation site for kinetochore-associated Bub1 kinase, a key regulator of chromosome segregation. Using engineered strains, we show that the relative gene dosage of this variant versus canonical H2A controls the fidelity of chromosome segregation and the rate of acquisition of tolerance to antifungal drugs via aneuploidy. Furthermore, whole-genome chromatin precipitation analysis reveals that Centromere Protein A/ Centromeric Histone H3-like Protein (CENP-A/Cse4), a centromeric histone H3 variant that forms the platform of the eukaryotic kinetochore, is depleted from tetraploid-mating products relative to diploid parents and is virtually eliminated from cells exposed to aneuploidy-promoting cues. We conclude that genetically programmed and environmentally induced changes in chromatin can confer the capacity for enhanced evolvability via chromosome missegregation

Lipid nanoparticles to silence androgen receptor variants for prostate cancer therapy

Advanced-stage prostate cancer remains an incurable disease with poor patient prognosis. There is an unmet clinical need to target androgen receptor (AR) splice variants, which are key drivers of the disease. Some AR splice variants are insensitive to conventional hormonal or androgen deprivation therapy due to loss of the androgen ligand binding domain at the C-terminus and are constitutively active. Here we explore the use of RNA interference (RNAi) to target a universally conserved region of all AR splice variants for cleavage and degradation, thereby eliminating protein level resistance mechanisms. To this end, we tested five siRNA sequences designed against exon 1 of the AR mRNA and identified several that induced potent knockdown of full-length and truncated variant ARs in the 22Rv1 human prostate cancer cell line. We then demonstrated that 2′O methyl modification of the top candidate siRNA (siARvm) enhanced AR and AR-V7 mRNA silencing potency in both 22Rv1 and LNCaP cells, which represent two different prostate cancer models. For downstream in vivo delivery, we formulated siARvm-LNPs and functionally validated these in vitro by demonstrating knockdown of AR and AR-V7 mRNA in prostate cancer cells and loss of AR-mediated transcriptional activation of the PSA gene in both cell lines following treatment. We also observed that siARvm-LNP induced cell viability inhibition was more potent compared to LNP containing siRNA targeting full-length AR mRNA (siARfl-LNP) in 22Rv1 cells as their proliferation is more dependent on AR splice variants than LNCaP and PC3 cells. The in vivo biodistribution of siARvm-LNPs was determined in 22Rv1 tumor-bearing mice by incorporating 14C-radiolabelled DSPC in LNP formulation, and we observed a 4.4% ID/g tumor accumulation following intravenous administration. Finally, treatment of 22Rv1 tumor bearing mice with siARvm-LNP resulted in significant tumor growth inhibition and survival benefit compared to siARfl-LNP or the siLUC-LNP control. To best of our knowledge, this is the first report demonstrating therapeutic effects of LNP-siRNA targeting AR splice variants in prostate cancer