Histone 2B monoubiquitination complex integrates transcript elongation with RNA processing at circadian clock and flowering regulators.

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 <i>SPEN3</i> and <i>KHD1</i> had reduced rosette and leaf areas. Strikingly, in <i>spen3</i> mutants, the flowering time was slightly, but significantly, delayed, as opposed to the early flowering time in the <i>hub1-4</i> mutant. The mutant phenotypes in biomass and flowering time suggested a deregulation of their respective regulatory genes <i>CIRCADIAN CLOCK-ASSOCIATED1</i> (<i>CCA1</i>) and <i>FLOWERING LOCUS C</i> (<i>FLC</i>) that are known targets of the HUB1-mediated histone H2B monoubiquitination (H2Bub). Indeed, in the <i>spen3-1</i> and <i>hub1-4</i> mutants, the circadian clock period was shortened as observed by luciferase reporter assays, the levels of the <i>CCA1</i>α and <i>CCA1</i>β splice forms were altered, and the <i>CCA1</i> expression and H2Bub levels were reduced. In the <i>spen3-1</i> mutant, the delay in flowering time was correlated with an enhanced <i>FLC</i> expression, possibly due to an increased distal versus proximal ratio of its antisense <i>COOLAIR</i> 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 <i>CCA1</i> Furthermore, the presence of an intact HUB1 at the <i>FLC</i> is required for SPEN3 function in the formation of the <i>FLC</i>-derived antisense <i>COOLAIR</i> transcripts.