ABSTRACT
HISTONE MONOUBIQUITINATION1 (HUB1) and its paralog HUB2 act in a conserved heterotetrameric complex in the chromatin-mediated transcriptional modulation of developmental programs, such as flowering time, dormancy, and the circadian clock. The KHD1 and SPEN3 proteins were identified as interactors of the HUB1 and HUB2 proteins with in vitro RNA-binding activity. Mutants in SPEN3 and KHD1 had reduced rosette and leaf areas. Strikingly, in spen3 mutants, the flowering time was slightly, but significantly, delayed, as opposed to the early flowering time in the hub1-4 mutant. The mutant phenotypes in biomass and flowering time suggested a deregulation of their respective regulatory genes CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and FLOWERING LOCUS C (FLC) that are known targets of the HUB1-mediated histone H2B monoubiquitination (H2Bub). Indeed, in the spen3-1 and hub1-4 mutants, the circadian clock period was shortened as observed by luciferase reporter assays, the levels of the CCA1α and CCA1ß splice forms were altered, and the CCA1 expression and H2Bub levels were reduced. In the spen3-1 mutant, the delay in flowering time was correlated with an enhanced FLC expression, possibly due to an increased distal versus proximal ratio of its antisense COOLAIR transcript. Together with transcriptomic and double-mutant analyses, our data revealed that the HUB1 interaction with SPEN3 links H2Bub during transcript elongation with pre-mRNA processing at CCA1 Furthermore, the presence of an intact HUB1 at the FLC is required for SPEN3 function in the formation of the FLC-derived antisense COOLAIR transcripts.
Subject(s)
Arabidopsis Proteins , Gene Expression Regulation, Plant , Histones , RNA, Plant , Ubiquitin-Protein Ligases , Ubiquitination , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Circadian Clocks/genetics , Circadian Clocks/physiology , Flowers/genetics , Flowers/physiology , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Histones/genetics , Histones/metabolism , Protein Domains/genetics , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Plant/genetics , RNA, Plant/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/genetics , Ubiquitination/physiologyABSTRACT
Previously, we identified HISTONE MONOUBIQUITINATION1 (HUB1) as an unconventional ubiquitin E3 ligase that is not involved in protein degradation but in the histone H2B modification that is implicated in transcriptional activation in plants. HUB1-mediated regulation of gene expression played a role in periodic and inducible processes such as the cell cycle, dormancy, flowering time and defense responses. Here, we determined the effects of the hub1-1 mutation on expression of a set of diurnally induced circadian clock genes identified from a comparative microarray analysis between the hub1-1 mutant and an HUB1 over-expression line. The hub1-1 mutation reduced the amplitudes of a number of induced clock gene expression peaks, as well as the HUB1-mediated histone H2BUb and H3K4Me3 marks associated with the coding regions, suggesting a role for HUB1 in facilitating transcriptional elongation in plants. Furthermore, double mutants between hub1-1 and elongata (elo) showed an embryo-lethal phenotype, indicating a synergistic genetic interaction. The double mutant embryos arrested at the torpedo stage, implying that together histone ubiquitination and acetylation marks are essential to activate expression of target genes in multiple pathways.
