DNA elimination analysis of Tetrahymena cells defective in heterochromatin body formation

In the ciliated protozoan Tetrahymena, de novo heterochromatin body formation is accompanied by programmed DNA elimination. Here, we show that the novel heterochromatin body component Jub1p promotes heterochromatin body formation and dephosphorylation of the Heterochromatin Protein 1 (HP1)-like protein Pdd1p. Through the identification and mutagenesis of the phosphorylated residues of Pdd1p, we demonstrate that Pdd1p dephosphorylation promotes the electrostatic interaction between Pdd1p and RNA in vitro and heterochromatin body formation in vivo. We therefore suggest that heterochromatin bodies are assembled by the Pdd1p-RNA interaction. Jub1p and Pdd1p dephosphorylation are required for heterochromatin body formation and DNA elimination but not for local heterochromatin assembly, indicating that heterochromatin body of itself plays an essential role in DNA elimination. Overall design: Micronuclei (MICs) and new macronuclei (MACs) of exconjugants were isolated from different mutants at 36 hpm, and the genomic DNA was analyzed by high-throughput sequencing.

在纤毛原生动物四膜虫（Tetrahymena）中，从头异染色质体形成伴随程序性DNA消除过程。本研究发现，新型异染色质体组分Jub1p可促进异染色质体形成，以及异染色质蛋白1（HP1）同源蛋白Pdd1p的去磷酸化。通过鉴定并诱变Pdd1p的磷酸化残基，我们证实Pdd1p去磷酸化可在体外增强Pdd1p与RNA的静电相互作用，并在体内促进异染色质体形成。据此我们提出，异染色质体通过Pdd1p与RNA的相互作用组装而成。Jub1p及Pdd1p的去磷酸化是异染色质体形成与DNA消除所必需的，但并不参与局部异染色质组装，这表明异染色质体本身在DNA消除过程中发挥关键作用。总体实验设计：于接合后36小时（hpm）从不同突变体中分离接合后体的微核（MICs）与新形成的大核（MACs），提取基因组DNA并通过高通量测序进行分析。