SUMMARYChromatin 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.

Histones are essential components of the nucleosome, the major chromatin subunit that structures linear DNA molecules and regulates access of other proteins to DNA. Specific histone chaperone complexes control the correct deposition of canonical histones and their variants to modulate nucleosome structure and stability. In this study, we characterize the Alpha Thalassemia-mental Retardation X-linked (ATRX) ortholog and show that ATRX is involved in histone H3 deposition. Arabidopsis ATRX mutant alleles are viable, but show developmental defects and reduced fertility. Their combination with mutants of the histone H3.3 chaperone HIRA (Histone Regulator A) results in impaired plant survival, suggesting that HIRA and ATRX function in complementary histone deposition pathways. Indeed, ATRX loss of function alters cellular histone H3.3 pools and in consequence modulates the H3.1/H3.3 balance in the cell. H3.3 levels are affected especially at genes characterized by elevated H3.3 occupancy, including the 45S ribosomal DNA (45S rDNA) loci, where loss of ATRX results in altered expression of specific 45S rDNA sequence variants. At the genome-wide scale, our data indicate that ATRX modifies gene expression concomitantly to H3.3 deposition at a set of genes characterized both by elevated H3.3 occupancy and high expression. Together, our results show that ATRX is involved in H3.3 deposition and emphasize the role of histone chaperones in adjusting genome expression.