DNAJC9 prevents CENP-A mislocalization and chromosomal instability by maintaining the fidelity of histone supply chains.

The centromeric histone H3 variant CENP-A is overexpressed in many cancers. The mislocalization of CENP-A to noncentromeric regions contributes to chromosomal instability (CIN), a hallmark of cancer. However, pathways that promote or prevent CENP-A mislocalization remain poorly defined. Here, we performed a genome-wide RNAi screen for regulators of CENP-A localization which identified DNAJC9, a J-domain protein implicated in histone H3-H4 protein folding, as a factor restricting CENP-A mislocalization. Cells lacking DNAJC9 exhibit mislocalization of CENP-A throughout the genome, and CIN phenotypes. Global interactome analysis showed that DNAJC9 depletion promotes the interaction of CENP-A with the DNA-replication-associated histone chaperone MCM2. CENP-A mislocalization upon DNAJC9 depletion was dependent on MCM2, defining MCM2 as a driver of CENP-A deposition at ectopic sites when H3-H4 supply chains are disrupted. Cells depleted for histone H3.3, also exhibit CENP-A mislocalization. In summary, we have defined novel factors that prevent mislocalization of CENP-A, and demonstrated that the integrity of H3-H4 supply chains regulated by histone chaperones such as DNAJC9 restrict CENP-A mislocalization and CIN.

The histone H3/H4 chaperone CHAF1B prevents the mislocalization of CENP-A for chromosomal stability.

Restricting the localization of the evolutionarily conserved centromeric histone H3 variant CENP-A to centromeres prevents chromosomal instability (CIN). The mislocalization of CENP-A to non-centromeric regions contributes to CIN in yeasts, flies and human cells. Even though overexpression and mislocalization of CENP-A have been reported in cancers, the mechanisms responsible for its mislocalization remain poorly understood. Here, we used an imaging-based high-throughput RNAi screen to identify factors that prevent mislocalization of overexpressed YFP-tagged CENP-A (YFP-CENP-A) in HeLa cells. Among the top five candidates in the screen - the depletion of which showed increased nuclear YFP-CENP-A fluorescence - were the histone chaperones CHAF1B (or p60) and CHAF1A (or p150). Follow-up validation and characterization experiments showed that CHAF1B-depleted cells exhibited CENP-A mislocalization, CIN phenotypes and increased enrichment of CENP-A in chromatin fractions. The depletion of DAXX, a histone H3.3 chaperone, suppressed CENP-A mislocalization and CIN in CHAF1B-depleted cells. We propose that in CHAF1B-depleted cells, DAXX promotes mislocalization of the overexpressed CENP-A to non-centromeric regions, resulting in CIN. In summary, we identified regulators of CENP-A localization and defined a role for CHAF1B in preventing DAXX-dependent CENP-A mislocalization and CIN.

Author Correction: Ras hyperactivation versus overexpression: Lessons from Ras dynamics in Candida albicans.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Modulation of azole sensitivity and filamentation by GPI15, encoding a subunit of the first GPI biosynthetic enzyme, in Candida albicans.

Glycosylphosphatidylinositol (GPI)-anchored proteins are important for virulence of many pathogenic organisms including the human fungal pathogen, Candida albicans. GPI biosynthesis is initiated by a multi-subunit enzyme, GPI-N-acetylglucosaminyltransferase (GPI-GnT). We showed previously that two GPI-GnT subunits, encoded by CaGPI2 and CaGPI19, are mutually repressive. CaGPI19 also co-regulates CaERG11, the target of azoles while CaGPI2 controls Ras signaling and hyphal morphogenesis. Here, we investigated the role of a third subunit. We show that CaGpi15 is functionally homologous to Saccharomyces cerevisiae Gpi15. CaGPI15 is a master activator of CaGPI2 and CaGPI19. Hence, CaGPI15 mutants are azole-sensitive and hypofilamentous. Altering CaGPI19 or CaGPI2 expression in CaGPI15 mutant can elicit alterations in azole sensitivity via CaERG11 expression or hyphal morphogenesis, respectively. Thus, CaGPI2 and CaGPI19 function downstream of CaGPI15. One mode of regulation is via H3 acetylation of the respective GPI-GnT gene promoters by Rtt109. Azole sensitivity of GPI-GnT mutants is also due to decreased H3 acetylation at the CaERG11 promoter by Rtt109. Using double heterozygous mutants, we also show that CaGPI2 and CaGPI19 can independently activate CaGPI15. CaGPI15 mutant is more susceptible to killing by macrophages and epithelial cells and has reduced ability to damage either of these cell lines relative to the wild type strain, suggesting that it is attenuated in virulence.

Characterising N-acetylglucosaminylphosphatidylinositol de-N-acetylase (CaGpi12), the enzyme that catalyses the second step of GPI biosynthesis in Candida albicans.

Candida albicans N-acetylglucosaminylphosphatidylinositol de-N-acetylase (CaGpi12) recognises N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) from Saccharomyces cerevisiae and is able to complement ScGPI12 function. Both N- and C-terminal ends of CaGpi12 are important for its function. CaGpi12 was biochemically characterised using rough endoplasmic reticulum microsomes prepared from BWP17 strain of C. albicans. CaGpi12 is optimally active at 30°C and pH 7.5. It is a metal-dependent enzyme that is stimulated by divalent cations but shows no preference for Zn2+ unlike the mammalian homologue. It irreversibly loses activity upon incubation with a metal chelator. Two conserved motifs, HPDDE and HXXH, are both important for its function in the cell. CaGPI12 is essential for growth and viability of C. albicans. Its loss causes reduction of GlcNAc-PI de-N-acetylase activity, cell wall defects and filamentation defects. The filamentation defects could be specifically correlated to an upregulation of the HOG1 pathway.

Ras signaling activates glycosylphosphatidylinositol (GPI) anchor biosynthesis via the GPI-N-acetylglucosaminyltransferase (GPI-GnT) in Candida albicans.

The ability of Candida albicans to switch between yeast to hyphal form is a property that is primarily associated with the invasion and virulence of this human pathogenic fungus. Several glycosylphosphatidylinositol (GPI)-anchored proteins are expressed only during hyphal morphogenesis. One of the major pathways that controls hyphal morphogenesis is the Ras-signaling pathway. We examine the cross-talk between GPI anchor biosynthesis and Ras signaling in C. albicans. We show that the first step of GPI biosynthesis is activated by Ras in C. albicans This is diametrically opposite to what is reported in Saccharomyces cerevisiae Of the two C. albicans Ras proteins, CaRas1 alone activates GPI-GnT activity; activity is further stimulated by constitutively activated CaRas1. CaRas1 localized to the cytoplasm or endoplasmic reticulum (ER) is sufficient for GPI-GnT activation. Of the six subunits of the GPI-N-acetylglucosaminyltransferase (GPI-GnT) that catalyze the first step of GPI biosynthesis, CaGpi2 is the key player involved in activating Ras signaling and hyphal morphogenesis. Activation of Ras signaling is independent of the catalytic competence of GPI-GnT. This too is unlike what is observed in S. cerevisiae where multiple subunits were identified as inhibiting Ras2. Fluorescence resonance energy transfer (FRET) studies indicate a specific physical interaction between CaRas1 and CaGpi2 in the ER, which would explain the ability of CaRas1 to activate GPI-GnT. CaGpi2, in turn, promotes activation of the Ras-signaling pathway and hyphal morphogenesis. The Cagpi2 mutant is also more susceptible to macrophage-mediated killing, and macrophage cells show better survival when co-cultured with Cagpi2.

Ras hyperactivation versus overexpression: Lessons from Ras dynamics in Candida albicans.

Ras signaling in response to environmental cues is critical for cellular morphogenesis in eukaryotes. This signaling is tightly regulated and its activation involves multiple players. Sometimes Ras signaling may be hyperactivated. In C. albicans, a human pathogenic fungus, we demonstrate that dynamics of hyperactivated Ras1 (Ras1G13V or Ras1 in Hsp90 deficient strains) can be reliably differentiated from that of normal Ras1 at (near) single molecule level using fluorescence correlation spectroscopy (FCS). Ras1 hyperactivation results in significantly slower dynamics due to actin polymerization. Activating actin polymerization by jasplakinolide can produce hyperactivated Ras1 dynamics. In a sterol-deficient hyperfilamentous GPI mutant of C. albicans too, Ras1 hyperactivation results from Hsp90 downregulation and causes actin polymerization. Hyperactivated Ras1 co-localizes with G-actin at the plasma membrane rather than with F-actin. Depolymerizing actin with cytochalasin D results in faster Ras1 dynamics in these and other strains that show Ras1 hyperactivation. Further, ergosterol does not influence Ras1 dynamics.