Project description:The retinoblastoma tumour suppressor, Rb, has two major functions. First, it represses genes whose products are required for S-phase entry and progression, thus stabilizing cells in G1. Second, Rb synergizes with factors that induce cell cycle exit and terminal differentiation. Dictyostelium lacks a G1 phase in its cell cycle but it has a retinoblastoma orthologue, rblA. Using mRNA-Seq transcriptional profiling, we show that rblA strongly represses hundreds of genes whose products are involved in S-phase and mitosis. Both S-phase and mitotic genes are expressed at a single point in late G2 and again in mid-development, near the time when cell cycling is reactivated. RblA also activates a set of genes unique to slime moulds that function in terminal differentiation. Like its mammalian counterpart, Dictyostelium RblA plays a dual role, regulating cell cycle progression and transcriptional events leading to terminal differentiation. In the absence of a G1 phase, however, RblA functions in late G2 controlling the expression of both S-phase and mitotic genes.
Project description:The retinoblastoma tumour suppressor, Rb, has two major functions. First, it represses genes whose products are required for S-phase entry and progression, thus stabilizing cells in G1. Second, Rb synergizes with factors that induce cell cycle exit and terminal differentiation. Dictyostelium lacks a G1 phase in its cell cycle but it has a retinoblastoma orthologue, rblA. Using mRNA-Seq transcriptional profiling, we show that rblA strongly represses hundreds of genes whose products are involved in S-phase and mitosis. Both S-phase and mitotic genes are expressed at a single point in late G2 and again in mid-development, near the time when cell cycling is reactivated. RblA also activates a set of genes unique to slime moulds that function in terminal differentiation. Like its mammalian counterpart, Dictyostelium RblA plays a dual role, regulating cell cycle progression and transcriptional events leading to terminal differentiation. In the absence of a G1 phase, however, RblA functions in late G2 controlling the expression of both S-phase and mitotic genes. RNA was isolated from an rblA-disruptant and the parental strain when the cells were vegetatively growing or while the cells were developing (at the early culminant stage). Total RNA was also isolated from wild type cells synchronized using the cold-shock protocol (Maeda, J Gen Microbiol 132:1189-1196 (1986)). T0 to T13 represent the hours after the shift up from 9.5C to 22C, a regime that synchronizes the cells in the cell cycle.
Project description:In metazoans, the retinoblastoma tumor suppressor has two major functions. In the first, it represses genes whose products are required for S-phase entry and S-phase progression, and thus stabilizes the G1 phase. In a second function, Rb synergizes with factors that induce cell cycle exit and terminal differentiation. Using mRNAseq and microarray transcriptional profiling, we show that rblA, the Rb ortholog of the Amebozoan Dictyostelium, has both of these functions. We characterize the action of rblA in the Dictyostelium cell cycle in detail, and in particular show that it represses not only S-phase genes but also almost all genes whose products are involved in mitosis. We compare rblA null mutants with their parent strain Ax2 at two developmental stages, vegetative growth and the preculminant stage of fruiting body formation; cell cycle genes show misregulation in the mutant at both stages.
