Analysis of COP1/SPA signalling events in plant developmental responses

Plants are able to perceive the light cues and adjust its development according to the prevalent conditions. An essential core component of the light signaling is the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) complex, that operates downstream of the photoreceptors to inhibit the light responses. Thus, the COP1/SPA complex is involved in the regulation of a variety of plant developmental processes, such as the tPlants are able to perceive the light cues and adjust its development according to the prevalent conditions. An essential core component of the light signaling is the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) complex, that operates downstream of the photoreceptors to inhibit the light responses. Thus, the COP1/SPA complex is involved in the regulation of a variety of plant developmental processes, such as the timing of flowering and seedling growth. At the molecular level, COP1/SPA acts as an E3 ubiquitin ligase that promotes the selective degradation of its substrates, which mainly consist of transcription factors, i.e. CONSTANS (CO/BBX1). Since the spanQ and cop1 mutants exhibit severe growth and developmental compromises that cannot be explained by the targets described so far, this study aims to identify and characterize a novel COP1/SPA substrate. In addition, COP1 and SPAs have been shown to act in concert to regulate post-embryonic development. However, the degree at which COP1 and SPAs act independently of each other was not addressed so far. Furthermore, it was unknown whether COP1/SPA plays a role in the regulation of embryogenesis. These novel aspects of the COP1/SPAs co-action are addressed in part II of this thesis. Previously, a screening for interactors of COP1/SPA in vitro suggested that several B-Box transcription factors (BBX), that share similarity with CO, interact with COP1 and SPA1. This study confirms that several BBX proteins bind to COP1/SPA in planta, and focuses on the phenotypical and molecular characterization of CONSTANS-LIKE 12 (COL12/ BBX10). A domain mapping analysis indicated that the CO, CO-Like, TOC1 (CCT) domain of COL12 is important for binding to COP1 whereas the B-boxes are important for binding to SPA1, indicating that COP1 and SPAs might reinforce substrate binding. In agreement with the protein interactions, the COL12 protein undergoes proteolysis at the 26S proteasome in the darkness in a COP1-dependent manner. Both monochromatic red and blue light enhance COL12 protein stability, which contrasts with the destabilizing effects of red light on CO. Therefore, we conclude that COL12 is a novel COP1/SPA substrate. The phenotypical analysis reveals that overexpression of COL12 delays the plant flowering time and enhances branching. Importantly, COL12 repression of flowering is photoperiod-dependent indicating that the protein takes part in the photoperiodic pathway that controls flowering. At the center of this pathway, the CO-FT accumulation is restricted to the LD. The visible light has a prominent role as a positive regulator of CO protein levels. Here, biochemical analyses reveal that COL12 physically interacts with the master regulator of flowering CO, possibly antagonizing its function. In addition, FT mRNA accumulation is reduced in COL12 ox plants compared to WT. Altogether our experiments suggest that COL12 might constitute a fine-tuning mechanism thereby the visible light can enhance CO function by stabilization of the COL12 repressor. In the second part of this thesis, we isolated the quintuple cop1-5 spaQn mutant seedlings, demonstrating that COP1/SPA do not play a role in embryogenesis. We also isolated the cop1-4 spaQn mutant that is completely devoid in the conserved WD-repeats. These quintuple mutant plants resemble the null cop1-5 mutant. This result indicates that the WD- repeat domains provided by the SPA proteins can partially substitute for the lack of the COP1 WD-repeat domain in the COP1-4 protein. Therefore, the WD-repeats are essential for the signaling activity of COP1/SPA. Timing of flowering and seedling growth. At the molecular level, COP1/SPA acts as an E3 ubiquitin ligase that promotes the selective degradation of its substrates, which mainly consist of transcription factors, i.e. CONSTANS (CO/BBX1). Since the spanQ and cop1 mutants exhibit severe growth and developmental compromises that cannot be explained by the targets described so far, this study aims to identify and characterize a novel COP1/SPA substrate. In addition, COP1 and SPAs have been shown to act in concert to regulate post-embryonic development. However, the degree at which COP1 and SPAs act independently of each other was not addressed so far. Furthermore, it was unknown whether COP1/SPA plays a role in the regulation of embryogenesis. These novel aspects of the COP1/SPAs co-action are addressed in part II of this thesis. Previously, a screening for interactors of COP1/SPA in vitro suggested that several B-Box transcription factors (BBX), that share similarity with CO, interact with COP1 and SPA1. This study confirms that several BBX proteins bind to COP1/SPA in planta, and focuses on the phenotypical and molecular characterization of CONSTANS-LIKE 12 (COL12/ BBX10). A domain mapping analysis indicated that the CO, CO-Like, TOC1 (CCT) domain of COL12 is important for binding to COP1 whereas the B-boxes are important for binding to SPA1, indicating that COP1 and SPAs might reinforce substrate binding. In agreement with the protein interactions, the COL12 protein undergoes proteolysis at the 26S proteasome in the darkness in a COP1-dependent manner. Both monochromatic red and blue light enhance COL12 protein stability, which contrasts with the destabilizing effects of red light on CO. Therefore, we conclude that COL12 is a novel COP1/SPA substrate. The phenotypical analysis reveals that overexpression of COL12 delays the plant flowering time and enhances branching. Importantly, COL12 repression of flowering is photoperiod-dependent indicating that the protein takes part in the photoperiodic pathway that controls flowering. At the center of this pathway, the CO-FT accumulation is restricted to the LD. The visible light has a prominent role as a positive regulator of CO protein levels. Here, biochemical analyses reveal that COL12 physically interacts with the master regulator of flowering CO, possibly antagonizing its function. In addition, FT mRNA accumulation is reduced in COL12 ox plants compared to WT. Altogether our experiments suggest that COL12 might constitute a fine-tuning mechanism thereby the visible light can enhance CO function by stabilization of the COL12 repressor. In the second part of this thesis, we isolated the quintuple cop1-5 spaQn mutant seedlings, demonstrating that COP1/SPA do not play a role in embryogenesis. We also isolated the cop1-4 spaQn mutant that is completely devoid in the conserved WD-repeats. These quintuple mutant plants resemble the null cop1-5 mutant. This result indicates that the WD- repeat domains provided by the SPA proteins can partially substitute for the lack of the COP1 WD-repeat domain in the COP1-4 protein. Therefore, the WD-repeats are essential for the signaling activity of COP1/SPA

Biallelic ZNFX1 variants are associated with a spectrum of immuno-hematological abnormalities

Biallelic changes in the ZNFX1 gene have been recently reported to cause severe familial immunodeficiency. Through a search of our bio/databank with information from genetic testing of >55 000 individuals, we identified nine additional patients from seven families with six novel homozygous ZNFX1 variants. Consistent with the previously described phenotype, our patients suffered from monocytosis, thrombocytopenia, hepatosplenomegaly, recurrent infections, and lymphadenopathy. The two most severely affected probands also had renal involvement and clinical presentations compatible with hemophagocytic lymphohistiocytosis. The disease was less lethal among our patients than previously reported. We identified two missense changes, two variants predicted to result in complete protein loss through nonsense-mediated decay, and two frameshift changes that likely introduce a truncation. Our findings (i) independently confirm the role of ZNFX1 in primary genetic immunodeficiency, (ii) expand the genetic and clinical spectrum of ZNFX1-related disease, and (iii) illustrate the utility of large, well-curated, and continually updated genotype-phenotype databases in resolving molecular diagnoses of patients with initially negative genetic testing findings

The Transcription Factor COL12 Is a Substrate of the COP1/SPA E3 Ligase and Regulates Flowering Time and Plant Architecture

The ambient light environment controls many aspects of plant development throughout a plant's life cycle. Such complex control is achieved because a key repressor of light signaling, the Arabidopsis (Arabidopsis thaliana) COP1/SPA E3 ubiquitin ligase causes the degradation of multiple regulators of endogenous developmental pathways. This includes the CONSTANS (CO) transcription factor that is responsible for photoperiodic control of flowering time. There are 16 CO-like proteins whose functions are only partly understood. Here, we show that 14 CO-like (COL) proteins bind CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and SUPPRESSOR OF PHYTOCHROME A-105 (SPA) 1 in vitro. We subsequently focused on COL12 and show that COL12 binds COP1 and SPA proteins in vivo. The COL12 protein is degraded in darkness in a COP1-dependent fashion, indicating that COL12 is a substrate of the COP1/SPA ubiquitin ligase. Overexpression of COL12 causes late flowering specifically in long day conditions by decreasing the expression of FLOWERING LOCUS T. This phenotype is genetically dependent on CO. Consistent with this finding, COL12 physically interacts with CO in vivo, suggesting that COL12 represses flowering by inhibiting CO protein function. We show that COL12 overexpression did not alter CO protein stability. It is therefore likely that COL12 represses the activity of CO rather than CO levels. Overexpression of COL12 also affects plant architecture by increasing the number of rosette branches and reducing inflorescence height. These phenotypes are CO independent. Hence, we suggest that COL12 affects plant development through CO-dependent and CO-independent mechanisms

Biallelic ZNFX1 variants are associated with a spectrum of immuno-hematological abnormalities

Biallelic changes in the ZNFX1 gene have been recently reported to cause severe familial immunodeficiency. Through a search of our bio/databank with information from genetic testing of >55 000 individuals, we identified nine additional patients from seven families with six novel homozygous ZNFX1 variants. Consistent with the previously described phenotype, our patients suffered from monocytosis, thrombocytopenia, hepatosplenomegaly, recurrent infections, and lymphadenopathy. The two most severely affected probands also had renal involvement and clinical presentations compatible with hemophagocytic lymphohistiocytosis. The disease was less lethal among our patients than previously reported. We identified two missense changes, two variants predicted to result in complete protein loss through nonsense-mediated decay, and two frameshift changes that likely introduce a truncation. Our findings (i) independently confirm the role of ZNFX1 in primary genetic immunodeficiency, (ii) expand the genetic and clinical spectrum of ZNFX1-related disease, and (iii) illustrate the utility of large, well-curated, and continually updated genotype–phenotype databases in resolving molecular diagnoses of patients with initially negative genetic testing findings