PNAS: The study reveals the molecular basis of H3K9 methylation specific to the catalytic site of plant SUVH6 enzymes

The Proceedings of the National Academy of Sciences (PNAS) published online a research paper
entitled Molecular Basis of Locus-specific H3K9 Methylation Catalyzed by SUVH6 in Plants, completed by the Duan Chengguo Research Group of the Center for Excellence in Molecular Plant Sciences of the Chinese Academy of Sciences and the Du Jiamu Research Group of Southern University of Science and Technology.
The study revealed a novel mechanism
of conserved SUVH6 histone methyltransferase family catalytic site-specific H3K9 methylation in plants.
In this mechanism, an unresolved peptide structure at the N-terminus of the SUVH6 family can be specifically recognized
by the BAH domain of the chromatin regulator ASI1.
The SUVH6-ASI1 module controls the deposition of H3K9me2 on most SUVH6 targets and produces different regulatory patterns for gene expression depending on the location of the target site, including transcriptional silencing or post-transcriptional regulation
.
What's more, this mechanism and its key amino acid sites persist in plants, suggesting a co-evolution
between SUVH6 and ASI1.

As a marker of eukaryotic heterochromatin, histone H3K9 methylation also plays a regulatory role
in the autochromatin region.
H3K9me2 in the model Arabidopsis thaliana was mainly catalyzed by SUVH4 (KYP), SUVH5 and SUVH6, and there was a positive feedback regulation with DNA methylation to enhance heterochromatinization
in this region.
Among them, SUHV4 is the main catalytic enzyme, and SUVH5 and SUVH6 have certain functional redundancy
as coenzymes.
However, apart from DNA methylation-mediated mechanisms of SUVH recruitment, it is unclear
whether there are other mechanisms in plants that more finely determine the specificity of the site of SUVH catalysis.

The team identified ASI1
, an interaction protein of SUVH6 by immunoprecipitation mass spectrometry in Arabidopsis thaliana and rice, respectively.
ASI1 is a key component of the AAE (Asi1-Aipp1-Edm2) complex discovered by Duan Chengguo's group to identify H3K9me2 and control RNA processing (Duan et al.
, 2017; You et al.
, 2021; Zhang et al.
, 2021).

The protein truncation interaction experiment localized the interaction to an unreported conserved peptide at the N-terminus of the SUVH6 family protein, and biochemical evidence showed that this peptide was necessary
for the interaction between SUVH6 and ASI1 。 Duan Chengguo's research group cooperated with Du Jiamu's research group to analyze the high-resolution crystal structure of ASI1-BAH domain and SUVH-N-terminal polypeptide, and found that ASI1-BAH has a classical aromatic cage that can specifically identify arginine residues at the N-terminus of SUVH6 through cation-π interactions, and this single arginine residue has a decisive effect
on ASI1-SUVH6 interaction 。 Chromatin immunoprecipitation sequencing (ChIP-seq) analysis showed that most of the chromatin loci bound by SUVH6 were covered by ASI1, and the localization of ASI1 and SUVH6 on the common target depended on this interaction, and the mutation of key arginine could directly affect the chromatin localization
of SUVH6 。 H3K9me2 ChIP-seq analysis showed that the ASI1-SUVH6 interaction module facilitated the deposition of H3K9me2 at the target site and affected gene expression in a position-dependent manner: facilitating transcriptional silencing or full-length transcript processing
of post-transcriptional mRNA.
The N-terminal polypeptides of ASI and SUVH6 exist only in plants and are expressed simultaneously in most plants, suggesting evolutionary consistency
.
In conclusion, the study found a conserved H3K9me2 self-reinforcing positive feedback loop
in plants.
This pathway interacts with the DNA methylation-mediated SUVH histone methyltransferase recruitment mechanism to enhance the deposition
of H3K9me2 at specific chromatin sites.

The research work is supported
by the Strategic Leading Science and Technology Project of the Chinese Academy of Sciences, the Shenzhen Science and Technology Plan Project, and the Key Laboratory of Molecular Design of Plant Cell Factories in Guangdong Province.

Figure 1.
A.
Schematic diagram of the structure of the N-terminal conserved peptide of SUVH6 family and the BAH of ASI1 family; Schematic diagram of the interaction structure and key amino acids of B-C, ASI1-BAH and SUVH-N peptides; Colocalization and interdependence of D-F, ASI1 and SUVH6 on chromatin; G.
APA site controlled by ASI1-SUVH6 module in rice; H.
Schematic diagram
of the co-evolution of the SUVH family of plant population.