Methyl-CpG-binding domain 9 (MBD9) is required for H2A.Z incorporation into chromatin at a subset of H2A.Z-enriched regions in the Arabidopsis genome

Author summary The histone H2A variant, H2A.Z, is found in all known eukaryotes and plays important roles in transcriptional regulation. H2A.Z is selectively incorporated into nucleosomes within many genes by the activity of a conserved ATP-dependent chromatin remodeling complex in yeast, insects, and mammals. 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 indeed utilize a complex analogous to those of fungi and animals to deposit H2A.Z, and we also identify several new proteins that interact with this complex. We found that one such interactor, Methyl-CpG-BINDING DOMAIN 9 (MBD9), is required for H2A.Z incorporation at thousands of genomic sites that share a distinct histone modification profile. The histone binding properties of MBD9 suggest that it may guide H2A.Z deposition to specific sites by interacting with modified nucleosomes and with the H2A.Z deposition complex. We hypothesize that this represents a general paradigm for the targeting of H2A.Z to specific sites. 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.