The HIR complex incorporates H3 histones in a DNA synthesis-independent manner throughout the cell cycle or in resting cells. The CAF-1 (Chromatin Assembly Factor 1) complex deposits H3 in a DNA-synthesis-coupled manner during DNA replication and repair ( Tagami et al., 2004 Smith and Stillman, 1989) and is composed of the subunits FASCIATA1 (FAS1), FAS2, and MULTICOPY SUPPRESSOR OF IRA1 in Arabidopsis ( Kaya et al., 2001).
For the assembly of H3-H4 dimers into nucleosomes, several chaperone complexes have been characterized in eukaryotes. These histone chaperones operate in a coordinated network. To ensure the incorporation of the adequate histone type at the right time and genomic location, specialized proteins called histone chaperones associate with histones during their shuttling from cytoplasm to nucleoplasm and deposit histones on DNA. Genome-wide studies revealed preferential enrichment of H3.1 at heterochromatic regions and of H3.3 at active genes, promoters, and telomeric repeats ( Stroud et al., 2012 Wollmann et al., 2012 Vaquero-Sedas and Vega-Palas, 2013 Shu et al., 2014). The plant model Arabidopsis thaliana also encodes H3.1 and H3.3 proteins that differ by only four amino acids, as well as H3.3-like variants expressed in specific reproductive tissues ( Ingouff et al., 2010 Okada et al., 2005). In mammals, H3.3 deposition is associated with dynamic chromatin regions such as transcriptionally active genes and regulatory regions, with high nucleosome turnover, and DNA accessibility. Most eukaryotes express variants of the canonical histone H3.1, such as the replacement variant H3.3, as well as tissue-specific H3 variants ( Talbert et al., 2012). These canonical histones can then be replaced with specific histone variants to modify nucleosome composition, stability, higher-order chromatin organization, and DNA accessibility in a site-specific manner ( Talbert and Henikoff, 2010). Canonical histones are deposited on newly synthesized DNA to maintain nucleosomal density following passage of the replication fork. Within nucleosomes, the basic building blocks of chromatin, the double-stranded DNA helix wraps around octamers of histone proteins.
Gene regulation in the eukaryotic genome requires a controlled balance between packaging the large linear DNA molecules and permitting regulated access to protein complexes involved in DNA transcription, replication, and repair. Together, our results show that ATRX is involved in H3.3 deposition and emphasize the role of histone chaperones in adjusting genome expression. 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.
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. Indeed, ATRX loss of function alters cellular histone H3.3 pools and in consequence modulates the H3.1/H3.3 balance in the cell. 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. Arabidopsis ATRX mutant alleles are viable, but show developmental defects and reduced fertility. In this study, we characterize the Arabidopsis thaliana Alpha Thalassemia-mental Retardation X-linked (ATRX) ortholog and show that ATRX is involved in histone H3 deposition. Specific histone chaperone complexes control the correct deposition of canonical histones and their variants to modulate nucleosome structure and stability. Histones are essential components of the nucleosome, the major chromatin subunit that structures linear DNA molecules and regulates access of other proteins to DNA.
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