Project description:Sun-loving plants have the ability to detect and avoid shading through sensing of both blue and red light wavelengths. Higher plant cryptochromes (CRYs) control how plants modulate growth in response to changes in blue light. For growth under a canopy, where blue light is diminished, CRY1 and CRY2 perceive this change and respond by directly contacting two bHLH transcription factors, PIF4 and PIF5. These factors are also known to be controlled by phytochromes, the red/far-red photoreceptors; however, transcriptome analyses indicate that the gene regulatory programs induced by the different light wavelengths are distinct. Our results indicate that CRYs signal by modulating PIF activity genome-wide, and that these factors integrate binding of different plant photoreceptors to facilitate growth changes under different light conditions. We performed whole-genome chromatin immunoprecipitation with sequencing (ChIP-Seq) analysis on 5 day old Flash-CRY2, PIF4-Flash and PIF5-Flash treated in low blue-light for 16h.
Project description:We developed a proximity photo-crosslinking method (Spotlight) with a 4-azido-N-ethyl-1,8-naphthalimide (AzNP) moiety that can be converted to reactive aryl nitrene species using ambient blue light-emitting diode light. Using an AzNP-conjugated HaloTag ligand (VL1), blue light-induced photo-crosslinked products of various HaloTag-conjugated proteins of interest were detected in subcellular spaces in live cells. Using Spotlight, we further identified the host interactome of SARS-CoV-2 nucleocapsid (N) protein, which is essential for viral genome assembly. Mass analysis of the VL1-crosslinked product of N-HaloTag in HEK293T cells showed that RNA-binding proteins in stress granules were exclusively enriched in the cross-linked samples. These results tell that our method can reveal the interactome of protein of interest within a short distance in live cells.
Project description:Cryptochromes (CRYs) is known as the key blue light receptors that promote photomorphogenesis in Arabidopsis, but to date, the underlying mechanisms are still not fully understood. Through interrogating the CRY2 interactome, we identified MOS4-ASSOCIATED COMPLEX subunits 3A and 3B (MAC3A and MAC3B) as blue light-independent CRY2 interacting partners. MAC3A/B proteins could be assembled into liquid nuclear condensates of CRYs in a blue light-dependent manner. Hypocotyl elongation is markedly repressed in mac3ab double knock-out mutants under various light conditions, which uncovers a previously unknown role of MAC3A/B as negative regulators in plant photomorphogenesis. Our results also uncover the noncanonical activities of MAC3A as the DNA-binding proteins that regulate transcription. Genome-wide mapping of MAC3A-binding sites reveals that blue light facilitates the binding of MAC3A to its targets, which is weakened in cry1cry2 mutants, suggesting that CRYs may enhance MAC3A activities in blue light to negatively influence photomorphogenesis. Interestingly, we observe that the genomic binding sites of MAC3A and HY5 are largely overlapped, and physical interactions between MAC3A and HY5 are detected as well. In addition, the in vitro DNA-binding assay shows that both proteins compete for the same DNA probe. These results indicate that MAC3A may antagonize the function of HY5 by competing for the common binding sites across the genome. Taken together, we propose that cryptochromes may fine-tune Arabidopsis photomorphogenesis by balancing the positive and negative effects on HY5 activities.
Project description:Sun-loving plants have the ability to detect and avoid shading through sensing of both blue and red light wavelengths. Higher plant cryptochromes (CRYs) control how plants modulate growth in response to changes in blue light. For growth under a canopy, where blue light is diminished, CRY1 and CRY2 perceive this change and respond by directly contacting two bHLH transcription factors, PIF4 and PIF5. These factors are also known to be controlled by phytochromes, the red/far-red photoreceptors; however, transcriptome analyses indicate that the gene regulatory programs induced by the different light wavelengths are distinct. Our results indicate that CRYs signal by modulating PIF activity genome-wide, and that these factors integrate binding of different plant photoreceptors to facilitate growth changes under different light conditions.
Project description:Light is one of the main environmental cues that affects the physiology and behavior of many organisms. The effect of light on genome-wide transcriptional regulation has been well-studied in green algae and plants, but not in red algae. Cyanidioschyzon merolae is used as a model red algae, and is suitable for studies on transcriptomics because of its compact genome with a relatively small number of genes. In addition, complete genome sequences of the nucleus, mitochondrion, and chloroplast of this organism have been determined. Together, these attributes make C. merolae an ideal model organism to study the response to light stimuli at the transcriptional and the systems biology levels. Previous studies have shown that light significantly affects cell signaling in this organism, but there are no reports on its blue light- and red light-mediated transcriptional responses. We investigated the direct effects of blue and red light at the transcriptional level using RNA-seq. Blue and red light were found to regulate 35% of the total genes in C. merolae. Blue light affected the transcription of genes involved protein synthesis while red light specifically regulated the transcription of genes involved in photosynthesis and DNA repair. Blue or red light regulated genes involved in carbon metabolism and pigment biosynthesis. Overall, our data showed that red and blue light regulate the majority of the cellular, cell division, and repair processes in C. merolae.
Project description:Light is one of the main environmental cues that affects the physiology and behavior of many organisms. The effect of light on genome-wide transcriptional regulation has been well-studied in green algae and plants, but not in red algae. Cyanidioschyzon merolae is used as a model red algae, and is suitable for studies on transcriptomics because of its compact genome with a relatively small number of genes. In addition, complete genome sequences of the nucleus, mitochondrion, and chloroplast of this organism have been determined. Together, these attributes make C. merolae an ideal model organism to study the response to light stimuli at the transcriptional and the systems biology levels. Previous studies have shown that light significantly affects cell signaling in this organism, but there are no reports on its blue light- and red light-mediated transcriptional responses. We investigated the direct effects of blue and red light at the transcriptional level using RNA-seq. Blue and red light were found to regulate 35% of the total genes in C. merolae. Blue light affected the transcription of genes involved protein synthesis while red light specifically regulated the transcription of genes involved in photosynthesis and DNA repair. Blue or red light regulated genes involved in carbon metabolism and pigment biosynthesis. Overall, our data showed that red and blue light regulate the majority of the cellular, cell division, and repair processes in C. merolae.