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 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 CCA1β 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.

Chromatin Dynamics and Transcriptional Control of Circadian Rhythms in Arabidopsis

“…The study focused on histone H2B monoubiquitination (H2Bub), which in Arabidopsis is controlled by HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2 E3 ubiquitin ligases together with the UBIQUITIN-CONJUGATING ENZYME 1 (UBC1) and UBC2 E2-conjugating enzymes [ 88 ]. HUB proteins interact with the previously uncharacterized RNA-binding motif-containing proteins, SPEN3 and KHD1 [ 88 ]. In the spen3-1 and hub1-4 mutants, H2Bub accumulation was reduced and CCA1α and CCA1β splice isoforms were altered.…”

“…The mutant plants showed short circadian period length phenotypes in agreement with the reduced expression of CCA1 . Overall, the study showed that H2Bub deposition associated with CCA1 transcript elongation and pre-mRNA processing are two processes that are facilitated by the HUB1/HUB2 complex [ 88 ].…”

“…Thus, the study involves a histone deacetylase and histone methylation readers to shape the photoperiodic-dependent waveform of FT expression. H2B monoubiquitination and SPEN3 function are not only important for CCA1 transcript elongation and pre-mRNA processing as mentioned above [ 88 ], but are also important in the regulation of the flowering [ 88 ]. Indeed, the spen3-1 mutant plants showed a delay in flowering time that correlated with an enhanced expression of the flowering-related gene FLOWERING LOCUS C ( FLC ), most likely due to an increased distal versus proximal ratio of its antisense COOLAIR transcript [ 88 ].…”

Visualization of Protein Coding, Long Noncoding, and Nuclear RNAs by Fluorescence in Situ Hybridization in Sections of Shoot Apical Meristems and Developing Flowers

“…This signal amplification process also enables the sensitive detection of transcripts with relatively low abundance. The TSA system has been applied in wholemount ISH to detect gene expression in Arabidopsis seedlings, embryos, and shoot apices (Bruno et al, 2011;Himanen et al, 2012;Rozier et al, 2014;Woloszynska et al, 2019). By incorporating TSA into standard RNA ISH procedures using sectioned material to allow highresolution visualization even deep in tissues, we constructed a pipeline for investigating gene expression and coexpression (Fig.…”

“…In plants, FISH has been used to visualize entire chromosomes or individual genetic loci (Lamb et al, 2007;Tirichine et al, 2009;Jiang, 2019) as well as pathogen DNA (Shargil et al, 2015). However, although there are notable examples using whole-mounted tissues (Bruno et al, 2011(Bruno et al, , 2015Himanen et al, 2012;Rozier et al, 2014;Bleckmann and Dresselhaus, 2016;Woloszynska et al, 2019), the use of FISH to investigate plant RNA localization at high resolution and deep in tissues remains to be fully exploited. We have established RNA FISH in the Arabidopsis shoot apex based on the tyramide signal amplification (TSA) system applied to sectioned tissue (Yang et al, 2017).…”