Arabidopsis ATRX Modulates H3.3 Occupancy and Fine-Tunes Gene Expression

Abstract

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.

Replication‐coupled histone H3.1 deposition determines nucleosome composition and heterochromatin dynamics during Arabidopsis seedling development

“…S1a). To test H3.1 occupancy dynamics of another independent H3.1 gene product and to take into account potential changes in total H3.1 levels in the H3.1‐Myc tagged lines, we used a transgenic epitope tagged H3.1 (eH3.1) line expressing the HTR9 coding sequence fused to a short FLAG‐HA epitope under control of the endogenous promoter in a htr9 mutant background (Duc et al ., ). Immunofluorescence staining of cotyledon nuclei showed that HTR9‐FLAG‐HA is first enriched at the few visible conspicuous ‘pre‐CC’ structures at 2 dag and then in all mature CCs at 5 dag (Fig.…”

“…The uncoupling of DNA synthesis and chromatin assembly in the absence of CAF‐1 increases the time between replication fork passage and chromatin assembly. Setting of H3K9me2 methylation is then delayed until compensatory complexes such as HIR and ALPHA THALASSEMIA‐MENTAL RETARDATION X‐LINKED (ATRX) (Nie et al ., ; Duc et al ., , ) have restored nucleosome occupancy to allow for subsequent deposition of histone post‐translational modifications.…”

“…Transgenic plants expressing Myc‐tagged H3.1 and H3.3 (Stroud et al ., ) were kindly provided by C. Gutierrez. Transgenic plants expressing H3.1 (HTR9) with a FLAG‐HA tag, termed here epitope tagged H3.1 (eH3.1) under control of its endogenous promoter were described previously (Duc et al ., ). T 3 monolocus homozygous lines were crossed with fas2‐5 mutants to obtain eH3.1 fas2‐5 lines.…”

“…Indeed, H2A.W has been shown in vitro to promote long-range chromatin fiber-to-fiber interactions through its conserved C-terminal tail and to increase higher-order chromatin condensation in vivo (Yelagandula et al, 2014). Histone H3 variant H3.3 correlates with active gene expression (Stroud et al, 2012;Wollmann et al, 2012Wollmann et al, , 2017Shu et al, 2014;Duc et al, 2017), its incorporation results in more unstable nucleosomes (Jin et al, 2009;Thakar et al, 2009) and a transcription-permissive chromatin state, whereas H3.1 is enriched at transcriptionally inactive chromatin (Stroud et al, 2012;Wollmann et al, 2012). Another feature of heterochromatin is the presence of linker histones H1 that bind to DNA both at the entry and exit site of the nucleosome particle and which are enriched at CCs (She et al, 2013;Fyodorov et al, 2018).…”

Disruption ofNAP1genes supresses thefas1mutant phenotype and enhances genome stability

“…Coordinated interplay between histone eviction, re-loading and de novo nucleosome assembly is required for the maintenance of genome stability, in which histone chaperones play an essential role (Lankenau et al, 2003; Mozgova et al, 2010; Zhu et al, 2011; Dewari and Bhargava, 2014). In Arabidopsis thaliana , dysfunctions of CHROMATIN ASSEMBLY FACTOR 1 (CAF-1), ANTI-SILENCING FUNCTION 1 (ASF1) or FACILITATING CHROMATIN TRANSCRIPTION (FACT) lead to severe phenotypes, while disruption of HISTONE REGULATOR A (HIRA) or NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) are much better tolerated (Exner et al, 2006; Kirik et al, 2006; Liu et al, 2009; Zhu et al, 2011; Duc et al, 2017; Pfab et al, 2018). This suggests that functions of some histone chaperones can be replaced, and that their mutual interactions represent a survival­determining factor.…”