SANT domain-containing proteins modulate gene transcription and negatively regulate plant thermotolerance by coordinating histone H3KAc and Khib modifications [RNA-seq]

Histone post-translational modifications (PTMs), such as acetylation and recently identified lysine 2-hydroxyisobutyrylation (Khib), act as active epigenomic marks in plants. SANT domain-containing proteins (SANT1/2/3/4), derived from PIF/Harbinger transposases, form a novel complex with histone deacetylase 6 (HDA6) to regulate gene expression via histone deacetylation. However, whether SANT1/2/3/4 coordinate different types of PTMs to regulate transcription and mediate responses to specific stresses in plants remain unclear. Here, in addition to modulating histone deacetylation, we found that SANT1/2/3/4 proteins acted like HDA6 or HDA9, in regulating the removal of histone Khib. H3KAc and histone Khib coordinated by SANT1/2/3/4 act in concert to regulate gene expression, with H3KAc playing a predominant role and Khib acting complementarily to H3KAc. SANT1/2/3/4 mutation significantly increased the expression of heat-inducible genes with concurrent change of H3KAc levels under normal and heat stress conditions, resulting in enhanced thermotolerance. Interestingly, hda6 mutant plants exhibited a reduced thermotolerance, suggesting that SANT1/2/3/4 negatively regulate plant thermotolerance independently of HDA6. This study revealed the critical roles of Harbinger transposon-derived SANT domain-containing proteins in transcriptional regulation by coordinating different types of histone PTMs and in the regulation of plant thermotolerance. Overall design: To obtain further insights into the role of SANT1/2/3/4-mediated transcriptional regulation in response to high temperature, we performed RNA-seq on triplicate wild type (Col-0), sant-null and hda6 seedlings grown under normal condition (22 ?) or exposed to 37 ? for 1 h. Total RNA was extracted from 7-day-old Arabidopsis seedlings growing on 1/2 MS medium plates with and without 37 ? treatment using TRIzol reagent (Invitrogen). mRNA-seq libraries were built according to the manufacturer's protocol (NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA)) and paired-end sequenced on an Illumina NovaSeq 6000 platform.