HIR complex

Abstract: Summary Chromatin organization is essential for coordinated gene expression, genome stability, and inheritance of epigenetic information. The main components involved in chromatin assembly are specific complexes such as Chromatin Assembly Factor 1 (CAF-1) and Histone Regulator (HIR), which deposit histones in a DNA synthesis-dependent or -independent manner, respectively. Here, we characterize the role of the plant orthologs Histone Regulator A (HIRA), Ubinuclein (UBN) and Calcineurin Binding protein 1 (CABIN1), which constitute the HIR complex. Arabidopsis loss-of-function mutants for the various subunits of the complex are viable, but hira mutants show reduced fertility. We show that loss of HIRA reduces extractable histone H3 protein levels and decreases nucleosome occupancy at both actively transcribed genes and heterochromatic regions. Concomitantly, HIRA contributes to maintenance of silencing of pericentromeric repeats and certain transposons. A genetic analysis based on crosses between mutants deficient in subunits of the CAF-1 and HIR complexes showed that simultaneous loss of both the CAF-1 and HIR histone H3 chaperone complexes severely affects plant survival, growth and reproductive development. Our results suggest that HIRA partially rescues impaired histone deposition in fas mutants to preserve nucleosome occupancy, implying plasticity in histone variant interaction and deposition.

Abstract: CAF-1, Hir proteins, and Asf1 are histone H3/H4 binding proteins important for chromatin-mediated transcriptional silencing. We explored genetic and physical interactions between these proteins and S-phase/DNA damage checkpoint kinases in the budding yeast Saccharomyces cerevisiae. Although cells lacking checkpoint kinase Mec1 do not display defects in telomeric gene silencing, silencing was dramatically reduced in cells lacking both Mec1 and the Cac1 subunit of CAF-1. Silencing was restored in cac1Δ and cac1Δ mec1Δ cells upon deletion of Rad53, the kinase downstream of Mec1. Restoration of silencing to cac1Δ cells required both Hir1 and Asf1, suggesting that Mec1 counteracts functional sequestration of the Asf1/Hir1 complex by Rad53. Consistent with this idea, the degree of suppression of silencing defects by rad53 alleles correlated with effects on Asf1 binding. Furthermore, deletion of the Dun1 kinase, a downstream target of Rad53, also suppressed the silencing defects of cac1Δ cells and reduced the levels of Asf1 associated with Rad53 in vivo. Loss of Mec1 and Rad53 did not alter telomere lengths or Asf1 protein levels, nuclear localization, or chromosome association. We conclude that the Mec1 and Dun1 checkpoint kinases regulate the Asf1-Rad53 interaction and therefore affect the activity of the Asf1/Hir complex in vivo.

Abstract: CAF-1 and HIR are highly conserved histone chaperone protein complexes that function in the assembly of nucleosomes onto chromatin. CAF-1 is characterized as having replication-coupled nucleosome activity, whereas the HIR complex can assemble nucleosomes independent of replication. Histone H3K56 acetylation, controlled by the acetyltransferase Rtt109 and deacetylase Hst3, also plays a significant role in nucleosome assembly. In this study, we generated a set of deletion mutants to genetically characterize pathway-specific and overlapping functions of CAF-1 and HIR in C. albicans. Their roles in epigenetic maintenance of cell type were examined by using the white-opaque switching system in C. albicans. We show that CAF-1 and HIR play conserved roles in UV radiation recovery, repression of histone gene expression, correct chromosome segregation, and stress responses. Unique to C. albicans, the cac2Δ/Δ mutant shows increased sensitivity to the Hst3 inhibitor nicotinamide, while the rtt109Δ/Δ cac2Δ/Δ and hir1Δ/Δ cac2Δ/Δ mutants are resistant to nicotinamide. CAF-1 plays a major role in maintaining cell types, as the cac2Δ/Δ mutant exhibited increased switching frequencies in both directions and switched at a high frequency to opaque in response to nicotinamide. Like the rtt109Δ/Δ mutant, the hir1Δ/Δ cac2Δ/Δ double mutant is defective in maintaining the opaque cell fate and blocks nicotinamide-induced opaque formation, and the defects are suppressed by ectopic expression of the master white-opaque regulator Wor1. Our data suggest an overlapping function of CAF-1 and HIR in epigenetic regulation of cell fate determination in an H3K56 acetylation-associated manner.

Abstract: The mammalian genome encodes multiple variants of histone H3 including H3.1/H3.2 and H3.3. In contrast to H3.1/H3.2, H3.3 is enriched in the actively transcribed euchromatin and the telomeric heterochromatins. However, the mechanism for H3.3 to incorporate into the different domains of chromatin is not known. Here, taking the advantage of well-defined transcription analysis system of yeast, we attempted to understand the molecular mechanism of selective deposition of human H3.3 into actively transcribed genes. We show that there are systemic H3 substrate-selection mechanisms operating even in yeasts, which encode a single type of H3. Yeast HIR complex mediated H3-specific recognition specificity for deposition of H3.3 in the transcribed genes. A critical component of this process was the H3 A-IG code composed of amino acids 87, 89 and 90. The preference toward H3.3 was completely lost when HIR subunits were absent and partially suppressed by human HIRA. Asf1 allows the influx of H3, regardless of H3 type. We propose that H3.3 is introduced into the active euchromatin by targeting the recycling pathway that is mediated by HIRA (or HIR), and this H3-selection mechanism is highly conserved through the evolution. These results also uncover an unexpected role of RI chaperones in evolution of variant H3s.