Histone 2B monoubiquitination complex integrates transcript elongation with RNA processing at circadian clock and flowering regulators

Altres ajuts: CERCA Programme/Generalitat de CatalunyaHISTONE 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

Characterization of the circadian clock function in the control of cell cycle progression to modulate growth in Arabidopsis thaliana

La función circadiana es esencial para el crecimiento y adaptación de las plantas a su entorno. La maquinaria molecular responsable de la generación de ritmos circadianos está basada en la expresión rítmica de genes cuyo pico de expresión oscila en diferentes fases durante el día y la noche. Los ritmos de expresión génica se traducen en oscilaciones de procesos fisiológicos y de desarrollo. El crecimiento de las plantas está regulado por una plétora de procesos que en última instancia operan a través del control de la proliferación y diferenciación celular. La proliferación celular depende de la progresión del ciclo mitótico, el cual está dividido en 4 fases: S (Síntesis del ADN), M (Mitosis) y de las interfases G1 y G2 (en inglés Gap 1 y 2) que ocurren antes de las fases S y M respectivamente. El proceso de diferenciación celular coincide con el cambio al endociclo, una variante del ciclo mitótico en la que el ADN genómico se duplica pero sin posterior división, es decir en ausencia de fase M. Aunque la regulación circadiana y el ciclo celular han sido individualmente estudiados en plantas, no se ha demostrado hasta la fecha la posible conexión de ambos ciclos en plantas. El trabajo realizado durante esta Tesis Doctoral se ha centrado en el estudio del papel del reloj circadiano en el control del ciclo celular durante la regulación del crecimiento de la planta. Los resultados obtenidos muestran que plantas con un reloj circadiano de ritmo lento desaceleran la progresión del ciclo celular, mientras que un reloj de ritmo rápido lo acelera. El componente esencial del reloj denominado en inglés TIMING OF CAB EXPRESSION 1 (TOC1) controla la transición de la fase G1 a la fase S, regulando así el ritmo del ciclo mitótico durante los estadios tempranos del desarrollo foliar. Asimismo, TOC1 también controla la ploidía somática característica del endociclo durante estadios tardíos del desarrollo foliar y en las células del hipocotilo. Utilizando técnicas de citometría de flujo y parámetros de cinéticas de crecimiento foliar se pudo determinar que en plantas que sobre-expresan TOC1 la fase S es más corta, lo que se correlaciona con la represión diurna del gen CELL DIVISION CONTROL 6 (CDC6). Este gen codifica un factor esencial en la formación de los complejos de pre-replicación que determinan los orígenes de replicación del ADN. Mediante técnicas de inmunoprecipitación de cromatina encontramos que la represión de CDC6 ocurre a través de la unión directa de TOC1 al promotor de CDC6. Los análisis de interacción genética demostraron que los fenotipos de crecimiento reducido y de ploidía somática alterada observados en plantas que sobre-expresan TOC1, quedaban revertidos al sobre-expresarse también CDC6. Estos resultados confirman que la función de TOC1 en el ciclo celular ocurre en gran medida a través de la represión de CDC6. La desaceleración de la progresión del ciclo celular en plantas que sobre-expresan TOC1 afecta no solo el desarrollo de los órganos de la planta, sino también el desarrollo tumoral en los tallos de las inflorescencias. Por lo tanto, nuestros estudios demuestran que la función de TOC1 es importante en la regulación rítmica de la maquinaria pre-replicativa del ADN para controlar el crecimiento de las plantas en resonancia con el medio ambiente.The circadian function is essential for plant growth and its adaptation to the environment. The molecular machinery responsible for the establishment of the circadian rhythmicity relies on the rhythmic oscillation of differentially expressed genes with different peaks of expression along the day and night. The rhythms in gene expression are translated into oscillations of physiological and developmental processes. Plant growth is controlled by a plethora of different processes that ultimately work through the control of cell proliferation and differentiation. Cell proliferation relies on the proper progression of the mitotic cycle, which is divided in 4 phases: S (DNA synthesis), M (Mitosis) and two gap phases G1 and G2, that take place before S and M phases, respectively. Cell differentiation coincides with the entry into the endocycle, a variant of the mitotic cycle in which genomic DNA duplicates without further division or mitosis. Even though the circadian clock and cell cycle as separate pathways have been well documented in plants, the possible direct interplay between these two cyclic processes has not been previously addressed. The work performed during this Thesis has focused on the characterization of the role of the circadian clock in the control of the cell cycle during plant growth. We found that plants with slower than Wild-Type circadian clocks slow down the progression of the cell cycle, while plants with faster clocks speed it up. The core clock component TIMING OF CAB EXPRESSION 1 (TOC1) controls the G1 to S-phase transition, thereby regulating the rhythm of the mitotic cycle during the early stages of leaf development. Likewise, TOC1 controls somatic ploidy during later stages of leaf development and of hypocotyl cell elongation. The use of flow cytometry analyses and of leaf growth kinetics showed that in plants over-expressing TOC1, the S-phase is shorter, which correlates with the diurnal repression of the CELL DIVISION CONTROL 6 (CDC6) gene. This gene encodes an essential component of the pre-replication complex, which is responsible for the specification of DNA origins of replication. Chromatin immunoprecipitation assays showed that the diurnal repression of CDC6 most likely relies on the direct binding of TOC1 to the CDC6 promoter. Genetic interaction analyses showeed that the reduced growth and altered somatic ploidy phenotypes observed in plants over-expressing TOC1 were reverted when CDC6 was over-expressed. Thus, our results confirm that TOC1 regulation of the cell cycle occurs through CDC6 repression. The slow cell cycle progression in plants over-expressing TOC1 has an impact not only in organ development but also on tumor growth in stems and inflorescences. Thus, TOC1 sets the time of the DNA pre-replicative machinery to control plant growth in resonance with the environment

Characterization of the circadian clock function in the control of cell cycle progression to modulate growth in Arabidopsis thaliana /

La función circadiana es esencial para el crecimiento y adaptación de las plantas a su entorno. La maquinaria molecular responsable de la generación de ritmos circadianos está basada en la expresión rítmica de genes cuyo pico de expresión oscila en diferentes fases durante el día y la noche. Los ritmos de expresión génica se traducen en oscilaciones de procesos fisiológicos y de desarrollo. El crecimiento de las plantas está regulado por una plétora de procesos que en última instancia operan a través del control de la proliferación y diferenciación celular. La proliferación celular depende de la progresión del ciclo mitótico, el cual está dividido en 4 fases: S (Síntesis del ADN), M (Mitosis) y de las interfases G1 y G2 (en inglés Gap 1 y 2) que ocurren antes de las fases S y M respectivamente. El proceso de diferenciación celular coincide con el cambio al endociclo, una variante del ciclo mitótico en la que el ADN genómico se duplica pero sin posterior división, es decir en ausencia de fase M. Aunque la regulación circadiana y el ciclo celular han sido individualmente estudiados en plantas, no se ha demostrado hasta la fecha la posible conexión de ambos ciclos en plantas. El trabajo realizado durante esta Tesis Doctoral se ha centrado en el estudio del papel del reloj circadiano en el control del ciclo celular durante la regulación del crecimiento de la planta. Los resultados obtenidos muestran que plantas con un reloj circadiano de ritmo lento desaceleran la progresión del ciclo celular, mientras que un reloj de ritmo rápido lo acelera. El componente esencial del reloj denominado en inglés TIMING OF CAB EXPRESSION 1 (TOC1) controla la transición de la fase G1 a la fase S, regulando así el ritmo del ciclo mitótico durante los estadios tempranos del desarrollo foliar. Asimismo, TOC1 también controla la ploidía somática característica del endociclo durante estadios tardíos del desarrollo foliar y en las células del hipocotilo. Utilizando técnicas de citometría de flujo y parámetros de cinéticas de crecimiento foliar se pudo determinar que en plantas que sobre-expresan TOC1 la fase S es más corta, lo que se correlaciona con la represión diurna del gen CELL DIVISION CONTROL 6 (CDC6). Este gen codifica un factor esencial en la formación de los complejos de pre-replicación que determinan los orígenes de replicación del ADN. Mediante técnicas de inmunoprecipitación de cromatina encontramos que la represión de CDC6 ocurre a través de la unión directa de TOC1 al promotor de CDC6. Los análisis de interacción genética demostraron que los fenotipos de crecimiento reducido y de ploidía somática alterada observados en plantas que sobre-expresan TOC1, quedaban revertidos al sobre-expresarse también CDC6. Estos resultados confirman que la función de TOC1 en el ciclo celular ocurre en gran medida a través de la represión de CDC6. La desaceleración de la progresión del ciclo celular en plantas que sobre-expresan TOC1 afecta no solo el desarrollo de los órganos de la planta, sino también el desarrollo tumoral en los tallos de las inflorescencias. Por lo tanto, nuestros estudios demuestran que la función de TOC1 es importante en la regulación rítmica de la maquinaria pre-replicativa del ADN para controlar el crecimiento de las plantas en resonancia con el medio ambiente.The circadian function is essential for plant growth and its adaptation to the environment. The molecular machinery responsible for the establishment of the circadian rhythmicity relies on the rhythmic oscillation of differentially expressed genes with different peaks of expression along the day and night. The rhythms in gene expression are translated into oscillations of physiological and developmental processes. Plant growth is controlled by a plethora of different processes that ultimately work through the control of cell proliferation and differentiation. Cell proliferation relies on the proper progression of the mitotic cycle, which is divided in 4 phases: S (DNA synthesis), M (Mitosis) and two gap phases G1 and G2, that take place before S and M phases, respectively. Cell differentiation coincides with the entry into the endocycle, a variant of the mitotic cycle in which genomic DNA duplicates without further division or mitosis. Even though the circadian clock and cell cycle as separate pathways have been well documented in plants, the possible direct interplay between these two cyclic processes has not been previously addressed. The work performed during this Thesis has focused on the characterization of the role of the circadian clock in the control of the cell cycle during plant growth. We found that plants with slower than Wild-Type circadian clocks slow down the progression of the cell cycle, while plants with faster clocks speed it up. The core clock component TIMING OF CAB EXPRESSION 1 (TOC1) controls the G1 to S-phase transition, thereby regulating the rhythm of the mitotic cycle during the early stages of leaf development. Likewise, TOC1 controls somatic ploidy during later stages of leaf development and of hypocotyl cell elongation. The use of flow cytometry analyses and of leaf growth kinetics showed that in plants over-expressing TOC1, the S-phase is shorter, which correlates with the diurnal repression of the CELL DIVISION CONTROL 6 (CDC6) gene. This gene encodes an essential component of the pre-replication complex, which is responsible for the specification of DNA origins of replication. Chromatin immunoprecipitation assays showed that the diurnal repression of CDC6 most likely relies on the direct binding of TOC1 to the CDC6 promoter. Genetic interaction analyses showeed that the reduced growth and altered somatic ploidy phenotypes observed in plants over-expressing TOC1 were reverted when CDC6 was over-expressed. Thus, our results confirm that TOC1 regulation of the cell cycle occurs through CDC6 repression. The slow cell cycle progression in plants over-expressing TOC1 has an impact not only in organ development but also on tumor growth in stems and inflorescences. Thus, TOC1 sets the time of the DNA pre-replicative machinery to control plant growth in resonance with the environment

The circadian clock sets the time of DNA replication licensing to regulate growth in Arabidopsis

The circadian clock and cell cycle as separate pathways have been well documented in plants. Elucidating whether these two oscillators are connected is critical for understanding plant growth. We found that a slow-running circadian clock decelerates the cell cycle and, conversely, a fast clock speeds it up. The clock component TOC1 safeguards the G(1)-to-S transition and controls the timing of the mitotic cycle at early stages of leaf development. TOC1 also regulates somatic ploidy at later stages of leaf development and in hypocotyl cells. The S-phase is shorter and delayed in TOC1 overexpressing plants, which correlates with the diurnal repression of the DNA replication licensing gene CDC6 through binding of TOC1 to the CDC6 promoter. The slow cell-cycle pace in TOC1-ox also results in delayed tumor progression in inflorescence stalks. Thus, TOC1 sets the time of the DNA pre-replicative machinery to control plant growth in resonance with the environment

The circadian clock sets the time of DNA replication licensing to regulate growth in Arabidopsis

The circadian clock and cell cycle as separate pathways have been well documented in plants. Elucidating whether these two oscillators are connected is critical for understanding plant growth. We found that a slow-running circadian clock decelerates the cell cycle and, conversely, a fast clock speeds it up. The clock component TOC1 safeguards the G1-to-S transition and controls the timing of the mitotic cycle at early stages of leaf development. TOC1 also regulates somatic ploidy at later stages of leaf development and in hypocotyl cells. The S-phase is shorter and delayed in TOC1 overexpressing plants, which correlates with the diurnal repression of the DNA replication licensing gene CDC6 through binding of TOC1 to the CDC6 promoter. The slow cell-cycle pace in TOC1-ox also results in delayed tumor progression in inflorescence stalks. Thus, TOC1 sets the time of the DNA pre-replicative machinery to control plant growth in resonance with the environment

The elongator complex regulates hypocotyl growth in darkness and during photomorphogenesis

Elongator promotes RNA polymerase II-mediated transcript elongation through epigenetic activities such as histone acetylation. Elongator regulates growth, development, immune response, sensitivity to drought and abscisic acid. We demonstrate that elo mutants exhibit defective hypocotyl elongation but have a normal apical hook in darkness and are hyposensitive to light during photomorphogenesis. These elo phenotypes are supported by transcriptome changes, i.e. downregulation of circadian clock components, positive regulators of skoto- or photomorphogenesis, hormonal pathways and cell wall biogenesis-related factors. The downregulated genes LHY, HFR1 and HYH are selectively targeted by Elongator for histone H3K14 acetylation in darkness. The role of Elongator in early seedling development in darkness and light is supported by hypocotyl phenotypes of mutants defective in components of the gene network regulated by Elongator, and double mutants between elo and mutants in light or darkness signaling components. A model is proposed in which Elongator represses the plant immune response and promotes hypocotyl elongation and photomorphogenesis via transcriptional control of positive photomorphogenesis regulators and a growth-regulatory network that converges on genes involved in cell wall biogenesis and hormone signaling