Abstract
The SWR1 chromatin remodeling complex, which deposits the histone variant H2A.Z into nucleosomes, has been well characterized in yeast and animals, but its composition in plants has remained uncertain. We used the conserved SWR1 subunit ACTIN RELATED PROTEIN 6 (ARP6) as bait in tandem affinity purification experiments to isolate associated proteins from Arabidopsis thaliana. We identified all 11 subunits found in yeast SWR1 and the homologous mammalian SRCAP complexes, demonstrating that this complex is conserved in plants. We also identified several additional proteins not previously associated with SWR1, including Methyl-CpG-BINDING DOMAIN 9 (MBD9) and three members of the Alfin1-like protein family, all of which have been shown to bind modified histone tails. Since mbd9 mutant plants were phenotypically similar to arp6 mutants, we explored a potential role for MBD9 in H2A.Z deposition. We found that MBD9 is required for proper H2A.Z incorporation at thousands of discrete sites, which represent a subset of the genomic regions normally enriched with H2A.Z. We also discovered that MBD9 preferentially interacts with acetylated histone H4 peptides, as well as those carrying mono- or dimethylated H3 lysine 4, or dimethylated H3 arginine 2 or 8. Considering that MBD9-dependent H2A.Z sites show a distinct histone modification profile, we propose that MBD9 recognizes particular nucleosome modifications via its PHD- and Bromo-domains and thereby guides SWR1 to these sites for H2A.Z deposition. Our data establish the SWR1 complex as being conserved across eukaryotes and suggest that MBD9 may be involved in targeting the complex to specific genomic sites through nucleosomal interactions. The finding that MBD9 does not appear to be a core subunit of the Arabidopsis SWR1 complex, along with the synergistic phenotype of arp6;mbd9 double mutants, suggests that MBD9 also has important roles beyond H2A.Z deposition.
Highlights
Nucleosomes, the fundamental units of chromatin that consist of ~147 bp of DNA wrapped around a histone octamer, efficiently condense large eukaryotic DNA molecules inside the nucleus
Whether this complex exists in the same form in plants, and how the complex is targeted to specific genomic locations have remained open questions
In this study we demonstrate that plants do utilize a complex analogous to those of fungi and animals to deposit H2A.Z, and we identify several new proteins that interact with this complex
Summary
Nucleosomes, the fundamental units of chromatin that consist of ~147 bp of DNA wrapped around a histone octamer, efficiently condense large eukaryotic DNA molecules inside the nucleus. Nucleosomes present a physical barrier that restricts the access of DNA-binding proteins to regulatory sequences. This physical constraint imposed by nucleosomes on DNA can be modulated to expose or occlude regulatory DNA sequences, and is thereby used as a mechanism to control processes such as transcription that rely on sequencespecific DNA binding proteins. Enzymatic complexes that can remodel chromatin structure by manipulating the position and/or composition of nucleosomes are essential for proper transcriptional regulation and the execution of key developmental programs. One member of the INO80 CRC subfamily is the SWI2/SNF2-related 1 (SWR1) chromatin remodeler, a multisubunit protein complex required for incorporation of the H2A variant, H2A.Z, into chromatin [3, 4]. H2A.Z-deficient plants are viable but display many developmental abnormalities such as early flowering, reduced plant size, altered leaf morphology, and reduced fertility [18,19,20]
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