Phosphorylation of an HP1-like protein regulates RNA-bridged heterochromatin body assembly for DNA elimination
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ABSTRACT:
ORGANISM(S): Tetrahymena thermophila
SUBMITTER: Kazufumi Mochizuki
PROVIDER: E-GEOD-70083 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description: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. New macronuclei (MACs) of exconjugants were isolated from wild-type different mutant cells at 12 hpm, shared chromatin was immunoprecipitated and precipitated DNA was analyzed by high-throughput sequencing.
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Project description:In the ciliated protozoan Tetrahymena, de novo heterochromatin body formation is accompanied by programmed DNA elimination. We previously reported that dephosphorylation of the HP1-like protein Pdd1p is required for the formation of heterochromatin bodies during the process of programmed DNA elimination in the ciliated protozoan Tetrahymena. Here, we show that the heterochromatin body component Jub4p is required for Pdd1p phosphorylation, heterochromatin body formation and DNA elimination. Moreover, our analyses of unphosphorylatable Pdd1p mutants demonstrate that Pdd1p phosphorylation is required for heterochromatin body formation and DNA elimination, while it is dispensable for local heterochromatin assembly. Therefore, both phosphorylation and the following dephosphorylation of Pdd1p are necessary to facilitate the formation of heterochromatin bodies. We suggest that Jub4p-mediated phosphorylation of Pdd1p creates a chromatin environment that is a prerequisite for subsequent heterochromatin body assembly and DNA elimination. New macronuclei (MACs) of exconjugants were isolated from wild-type and various mutant cells at 12 hpm (hours post-mixing), sheared chromatin was immunoprecipitated andprecipitated DNA was analyzed by high-throughput sequencing
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2016-07-15 | E-GEOD-83252 | biostudies-arrayexpress
Project description: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. 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.
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Project description:In order to identify epigenetic protein complexes recruited to histone PTMs in the deadly human malaria parasite Plasmodium falciparum, we set out to perform histone peptide pulldowns with various histone peptides. Biotinylated peptides corresponding to P. falciparum histone variant H2A.Z, H2B.Z and H3.3, canonical P. falciparum histone H4 or human histone H4 tail sequences (differing on position 21 from the P. falciparum peptide) bearing multiple histone post-translational modifications (acetylations and/or methylations) as well as the unmodified control peptides were coupled to beads and incubated with native nuclear extract obtained from mixed-stage asexual cultures. Proteins preferentially binding to the modified histone peptide were identified by quantitative proteomics using di-methyl labelling. This yielded 6 out of 7 P. falciparum bromo-domain proteins preferentially binding to the acetylated histone peptides, as well as many putative bromo-domain protein interactors. In addition, a PHD-domain containing protein was found to be recruiting a PfSAGA-like complex to H3K4 di- and tri-methylated peptides. 7 reader-domain containing proteins (BDP1, BDP2, BDP4, TAF1, GCN5, PHD1, PHD2) and 5 putative interactors (PF3D7_0306100, PF3D7_1124300, PF3D7_1128000, PF3D7_1225200, PF3D7_1451200) were endogenously tagged by GFP or HA and used in quantitative GFP-/HA-IP-MS/MS experiments to further characterize epigenetic reader-complex composition. As negative control, GFP- and HA-IPs were performed on native nuclear extract not encoding tagged protein. Most HPP experiment employed a 3-label setup, while few only relied on “light” and “heavy”-label sample pools (labels and conditions listed below). GFP- and HA-IP experiments were using a 2-label “light” and “heavy” di-methyl setup only. For all experiments technical replicates were performed using a label-swap approach (called F (forward) and R (reverse)) and independent biological replicates were performed for each. For GFP- and HA-IP forward reactions are set-up as: GFP-/HA-beads = “heavy” di-methyl label, control-beads = “light” di-methyl label. Reverse reactions are set-up as: GFP-/HA-beads = “light” di-methyl label, control-beads = “heavy” di-methyl label. For GFP-/HA-IP raw files the bait and tag used for fishing are included in the file name.
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