RING-containing proteins regulate various stages of plant development such as seed germination, vegetative phase transition, and flowering. Among the 469 predicted RING proteins in Arabidopsis, many of them have been shown to function as single subunit E3 ubiquitin ligases or as components of E3 ubiquitin ligase complexes. Monoubiquitylations or polyubiquitylations by E3 ubiquitin ligases are markers for protein degradation, protein activation, transcriptional regulation, DNA repair, or intracellular sorting. Several RING proteins also participate in protein-protein interactions in which their molecular functions are still unclear.
Here, I studied four Arabidopsis RING domain proteins which have been previously identified as BO-TRYTIS SUSCEPTIBLE1 INTERACTOR (BOI), BOI-RELATED GENE1 (BRG1), BRG2, and BRG3 (collec-tively referred to as BOIs). A boi quadruple mutant (boi brg1 brg2 brg3) showed enhanced seed germination frequency in the presence of paclobutrazol (PAC), precocious vegetative development, and early flowering. On the contrary, the BOI-overexpressing lines displayed reduced germination frequency on the PAC-containing me-dium, late juvenile-to-adult phase transition, and late flowering. Phenotypes of the boi quadruple mutant and BOI-overexpressing lines suggest that BOIs act as repressors in Arabidopsis development. Overexpression of a RINGless BOI did not exhibit the phenotypes observed in the overexpressing lines of full-length BOI, indicating that the RING motif is required for the function of BOI. In yeast two-hybrid, in vitro pull-down and in vivo co-immunoprecipitation assays, BOIs was shown to interact with DELLAs, key negative regulators in the phyto-hormonal gibberellin (GA) signaling. Together with DELLAs, BOIs inhibited a subset of GA-responses such as seed germination, chlorophyll accumulation, vegetative phase transition and flowering; however, BOIs did not regulate DELLA-mediated hypocotyl and root elongation. Further studies with a gai-1 boiQ mutant (a gain-of-function mutant of GAI, a DELLA protein, in the boiQ background) showed that BOIs are necessary for the function of DELLAs in the inhibition of a subset of GA responses. Interestingly, BOIs did not significantly alter the protein stability of REPRESSOR OF GA (RGA), another DELLA protein, since endogenous RGA levels were similar between the wild-type and boiQ plants. Instead, BOI and RGA are targeted to the same promoters of several GA-responsive genes to repress their expression. Moreover, DELLAs are required for the BOI to be associated to the target promoters since the removal of DELLA proteins by the treatment of GA reduced the binding of BOI to the target genes.
In addition to DELLA-dependent actions, BOIs also function via DELLA-independent mechanisms in repressing flowering in Arabidopsis. BOIs were found to bind to CONSTANS (CO), a transcription factor acti-vating FLOWERING LOCUS T (FT) and SUPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) to trigger flo-wering initiation. Both BOI and CO proteins are targeted to the promoters of FT and SOC1 genes; however, they act antagonistically in the regulation of these two target genes. Similar to the DELLA case, BOIs did not influ-ence CO protein level as the CO levels were not different between the CO-overexpressing plants in the wil-type and BOI-overexpressing backgrounds. However, a ChIP analysis showed that BOIs possibly affect CO function by decreasing the binding of CO to the promoter fragments of FT and SOC1, hence inhibit CO in the activation of their expression. In this study, FLOWERING LOCUS C (FLC) was also observed to interact with BOIs in yeast and pull-down binding assays. FLC is a transcription factor targeting the promoters of FT and SOC1 and inhibiting their expression. The binding of BOIs and FLC suggests another molecular function of BOIs in re-pressing flowering.
Further investigations showed that BOIs interacted with CURLY LEAF (CLF), MULTICOPY OF INRA1 (MSI1), two core components of the chromatin modifying complex Polycomb Repressive Complex2 (PRC2). Also, BOIs bound to the core subunits of the chromatin remodeling complex Switch/Sucrose Non-Fermenting (SWI/SNF) called SWI3C and SNF5, implying the function of BOIs in the epigenetic regulation. Analyses of the H3K27me3 and H3Ac levels of the SOC1 promoters indicated that H3K27me3 levels were decreased by boiQ mutations and increased by BOI overexpression, whereas the H3Ac levels were elevated by boiQ mutations and declined by BOI overexpression. Taken together, my study has found that BOIs repress gene expression by inte-racting with several transcription factors, PRC2 and SWI/SNF complexes.
본 연구는 애기장대에서 씨발아, 잎의 발달, 개화등의 다양한 식물 발달 과정을 억제하는 RING domain 단백질의 기능을 규명하고 있다. 이전에 BOI (BOTRYTIS SUSCEPTIBLE1 INTE-RACTOR), BOI-RELATED GENE1 (BRG1), BRG2, BRG3 (BOIs)라고 알려진 이 단백질은 C-말단에 RING domain과 잘 보존된 중간 부위를 가지고 있다. RING domain을 통해 이들이 단백질 분해 혹은 단백질간의 상호작용에 관여함을 추측할 수 있다. BOI 유전자는 고른 발현 양상을 보이지만 씨에서 상대적으로 더 많이 발현된다. 먼저, BOIs는 이스트, in vitro 풀다운, in vivo coIP의 방법을 통해 지베렐린 (GA) 시그널링의 주요 억제인자인 DELLAs와 붙으며, 이들과 같이 작용하여 발아, 엽록소 축적, 잎 발달, 개화와 같은 GA 반응 억제함을 보았다. boi 4중 돌연변이 (boiQ)와 della5중 돌연변이는 PAC (paclobutrazol) 을 극복하는 씨발아, 이른 잎 발달, 이른 개화를 보였고, BOI 과발현체는 반대로 PAC에 저해되는 씨발아, 느린 잎 발달, 느린 개화를 보였다. BOIs는 유비퀴틴 E3 ligase일 것으로 추측되지만, BOIs가 DELLAs 단백질 안정성을 조절하지는 않는 다는 사실을 boiQ에서 DELLAs 단백질량을 측정하여 밝혔다. BOIs-DELLAs 단백질 상호작용이 빛 시그널링에 관여하는 PIF3의 DNA 부착에는 영향을 주지 않았지만, 대신 BOIs와 DELLAs는 GA 반응 유전자의 프로모터에 붙어 이들의 발현을 억제하였다. BOIs는 또한 DELLAs와는 다른 기능을 가지고 있는데, 예를 들어, BOIs는 개화를 촉진하는 CONSTANS (CO) 와 상호작용하여 FLOWERING LOCUS T (FT), SUPPERSSOR OF OVEREXPRESSION OF CO1 (SOC1) 의 발현조절에 CO와 반대작용을 한다. BOIs는 CO의 단백질 레벨에는 영향을 주지 않지만, 이 두 유전자의 프로모터에 CO가 붙는 것을 저해하여 이들의 유전자 발현을 조절한다. 이에 더해, BOIs는 FT와 SOC1발현을 조절하는 FLOWERING LOCUS C (FLC) 와도 상호작용한다는 것을 밝혔으며, 이를 통해 BOIs가 개화조절에 또 다른 방식으로 관여함을 알았다. 흥미롭게도, Polycomb Repressive Complex2 (PRC2) 다수체의 주요 구성인자인 MULTICOPY OF INRA1 (MSI1) 와 CURLY LEAF (CLF), 그리고, Switch/Sucrose Non-Fermenting (SWI/SNF)의 주요 구성인자인 SWI3C와 SNF5가 BOIs와 각각 상호작용함을 이스트와 풀다운 실험을 통해 확인하였는데, 이는 크로마틴 조절에 BOIs가 관여함을 보여준다. 이와 연관성 있게, boiQ에서 SOC1 프로모터의 H3K27me3가 감소하고, H3Ac가 증가하며, BOI 과발현체에서는 이와 반대 양상로 나타남을 확인했다. 이러한 결과들은 BOIs가 PRC2, SWI/SNF와 상호작용하여 유전자발현 억제에 관여함을 제시하고 있다.
