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

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.

SWR1 Chromatin Remodeling Complex: A Key Transcriptional Regulator in Plants

“…These modified histones are recognized by MBD9 and NPX1 and thereby guide SWR1-C to these distinct sites to deposit H2A.Z, and finally H2A.Z recruits REPRESSOR OF SILENCING1 (ROS1) to prevent DNA methylation and gene silencing [9]. The preferential binding of MBD9 at the regions with active histone marks, and its interaction with SWR1-C, is also in agreement with the recent reports [9,56,57].…”

“…They also identified additional interacting partners associated with ARP6, including the plant homeodomain (PHD)-and Bromo domain-containing protein Methyl CpG-BINDING DOMAIN 9 (MBD9). The findings from both studies suggest that the MBD9 interacts with the Arabidopsis SWR1-C and plays a crucial role in H2A.Z deposition at active genes [56,57]. Consistently, MBD9 interaction with SWR1-C is also shown in a forward genetic screen for REPRESSOR OF SILENCING 1 (ROS1) mediated DNA demethylation and anti-silencing [9].…”

“…Additionally, PIE1 works as scaffold for other subunits to associate and form the active complex and it may be possible that an assembled but inactive complex in arp6 and swc6 mutants has a different impact on gene expression compared to a completely absent SWR1-C complex in a pie1 mutant. Recently, two groups independently identified the conserved subunits of yeast SWR1 and SRCAP complexes in Arabidopsis using ARP6 subunit as bait for IP-mass spec and Tandem Affinity Purification (TAP) [56,57]. They also identified additional interacting partners associated with ARP6, including the plant homeodomain (PHD)-and Bromo domain-containing protein Methyl CpG-BINDING DOMAIN 9 (MBD9).…”

Insights Into the Function of the NuA4 Complex in Plants

“…benthamiana leaves that demonstrated a physical interaction between AtHAM1 and AtYAF9A proteins in vivo (Crevillén et al, 2019). Intriguingly, AtTRA1 homologs were also pulled down in proteomic assays performed using either the SWR1 subunit ARP6 (Sijacic et al, 2019) or the SWR1-interacting protein MBD9 (Methyl-CpG-binding domain 9) (Potok et al, 2019) as baits, further supporting an intricate functional relationship between Arabidopsis SWR1 and NuA4 complexes. Altogether, these observations reinforce our hypothesis that a putative NuA4 exists in plants and may be closely linked with SWR1.…”

“…Most AP-MS approaches using subunits of SWR1 as baits reveal enrichments in SWR1 components and shared subunits with NuA4 among the co-immunoprecipitated proteins. In addition to these proteins, only homologs for the yeast Tra1, present in both NuA4 and SAGA HAT complexes, were recovered in these immunoprecipitation experiments when the SWR1 specific subunit ARP6 was used as bait, but not the NuA4 scaffold (EAF1) nor the catalytic subunit (HAM) (Potok et al, 2019;Sijacic et al, 2019). Based on these results, it is unlikely that these complexes may represent a merge of the Arabidopsis SWR1 and NuA4 complexes, similar to the mammalian TIP60 complex (Cai et al, 2003).…”