SWI/SNF-type chromatin remodelers, such as BRAHMA (BRM), and H3K27 demethylases both have active roles in regulating gene expression at the chromatin level1–5, but how they are recruited to specific genomic sites remains largely unknown. Here we show that RELATIVE OF EARLY FLOWERING 6 (REF6), a plant-unique H3K27 demethylase6, targets genomic loci containing a CTCTGYTY motif via its zinc-finger (ZnF) domains and facilitates the recruitment of BRM. Genome-wide analyses showed that REF6 colocalizes with BRM at many genomic sites with the CTCTGYTY motif. Loss of REF6 results in decreased BRM occupancy at BRM–REF6 co-targets. Furthermore, REF6 directly binds to the CTCTGYTY motif in vitro, and deletion of the motif from a target gene renders it inaccessible to REF6 in vivo. Finally, we show that, when its ZnF domains are deleted, REF6 loses its genomic targeting ability. Thus, our work identifies a new genomic targeting mechanism for an H3K27 demethylase and demonstrates its key role in recruiting the BRM chromatin remodeler.

We have synthesized a novel molecule, 3,6-bis(1-methyl-4-vinylpyridium)carbazole diiodide (BMVC), for recognizing specific quadruplex structures, particularly the quadruplex of human telomeric sequence d(T(2)AG(3))(4). The fluorescence intensity of the BMVC molecule increases from 1 to almost 2 orders of magnitude upon interacting with various DNAs. At a concentration of BMVC of 10 microM, fluorescence bands with different colors of BMVC in electrophoresis gels of various DNAs can be observed. The fluorescence of BMVC can be used to discriminate between duplex and quadruplex DNAs. At the low concentration of 0.1 microM BMVC in prestained gels, the fluorescence is observed in the presence of quadruplexes with anti-anti-anti-anti and anti-anti-syn-syn arrangements. However, no fluorescence band is detected upon interacting with duplexes and quadruplexes with anti-syn-anti-syn arrangement. Moreover, the sensitivity assays show that as little as 0.2 pmol of quadruplex of d(T(2)AG(3))(4) can be revealed by BMVC.

We have introduced a G-quadruplex-binding ligand, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC), to verify the major structure of d(T2AG3)4 (H24) in potassium solution and examine the structural conversion of H24 in sodium solution upon potassium titration. The studies of circular dichroism, induced circular dichroism, spectral titration and gel competition have allowed us to determine the binding mode and binding ratio of BMVC to the H24 in solution and eliminate the parallel form as the major G-quadruplex structure. Although the mixed-type form could not be eliminated as a main component, the basket and chair forms are more likely the main components of H24 in potassium solution. In addition, the circular dichroism spectra and the job plots reveal that a longer telomeric sequence d(T2AG3)13 (H78) could form two units of G4 structure both in sodium or potassium solutions. Of particular interest is that no appreciable change on the induced circular dichroism spectra of BMVC is found during the change of the circular dichroism patterns of H24 upon potassium titration. Considering similar spectral conversion detected for H24 and a long sequence H78 together with the G4 structure stabilized by BMVC, it is therefore unlikely that the rapid spectral conversion of H24 and H78 is due to structural change between different types of the G4 structures. With reference to the circular dichroism spectra of d(GAA)7 and d(GAAA)5, we suggest that the spectral conversion of H24 upon potassium titration is attributed to fast ion exchange resulting in different loop base interaction and various hydrogen bonding effects.

Plant growth is controlled by integration of hormonal and light-signaling pathways. BZS1 is a B-box zinc finger protein previously characterized as a negative regulator in the brassinosteroid (BR) signaling pathway and positive regulator in the light-signaling pathway. However, the mechanisms by which BZS1/BBX20 integrates light and hormonal pathways are not fully understood. Here, using a quantitative proteomic workflow, we identified several BZS1-associated proteins including light signaling components COP1 and HY5. Direct interactions of BZS1 with COP1 and HY5 were verified by yeast two-hybrid and co-immunoprecipitation assays. Overexpression of BZS1 causes a dwarf phenotype that is suppressed by the hy5 mutation, while overexpression of BZS1 fused with the SRDX transcription repression domain (BZS1-SRDX) causes a long-hypocotyl phenotype similar to hy5, indicating that BZS1’s function requires HY5. BZS1 positively regulates HY5 expression, whereas HY5 negatively regulates BZS1 protein level, forming a feedback loop that potentially contributes to signaling dynamics. In contrast to BR, strigolactone (SL) increases BZS1 level, whereas the SL responses of hypocotyl elongation, chlorophyll and HY5 accumulation are diminished in the BZS1-SRDX seedlings, indicating that BZS1 is involved in these SL responses. These results demonstrate that BZS1 interacts with HY5 and plays a central role in integrating light and multiple hormone signals for photomorphogenesis in Arabidopsis.