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Regulation of microtubule assembly and organization in mitosis by the AAA+ ATPase Pontin.

ABSTRACT: To identify novel proteins important for microtubule assembly in mitosis, we have used a centrosome-based complementation assay to enrich for proteins with mitotic functions. An RNA interference (RNAi)-based screen of these proteins allowed us to uncover 13 novel mitotic regulators. We carried out in-depth analyses of one of these proteins, Pontin, which is known to have several functions in interphase, including chromatin remodeling, DNA repair, and transcription. We show that reduction of Pontin by RNAi resulted in defects in spindle assembly in Drosophila S2 cells and in several mammalian tissue culture cell lines. Further characterization of Pontin in Xenopus egg extracts demonstrates that Pontin interacts with the gamma tubulin ring complex (gamma-TuRC). Because depletion of Pontin leads to defects in the assembly and organization of microtubule arrays in egg extracts, our studies suggest that Pontin has a mitosis-specific function in regulating microtubule assembly.

SUBMITTER: Ducat D

PROVIDER: S-EPMC2441676 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Regulation of microtubule assembly and organization in mitosis by the AAA+ ATPase Pontin.

Ducat Daniel D Kawaguchi Shin-Ichi S Liu Hongbin H Yates John R JR Zheng Yixian Y

Molecular biology of the cell 20080507 7

To identify novel proteins important for microtubule assembly in mitosis, we have used a centrosome-based complementation assay to enrich for proteins with mitotic functions. An RNA interference (RNAi)-based screen of these proteins allowed us to uncover 13 novel mitotic regulators. We carried out in-depth analyses of one of these proteins, Pontin, which is known to have several functions in interphase, including chromatin remodeling, DNA repair, and transcription. We show that reduction of Pont ...[more]

PMID: 18463163

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Project description:Maintenance of the correct level and organization of nucleosomes is crucial for genome function. Here we uncover a role for a conserved bromodomain AAA-ATPase, Abo1, in maintenance of nucleosome architecture in fission yeast. Cells lacking abo1+ experience both a reduction and mis-positioning of nucleosomes at transcribed sequences in addition to increased intragenic transcription, phenotypes that are hallmarks of defective chromatin re-establishment behind RNA polymerase II. Abo1 is recruited to gene sequences and associates with histone H3 and the histone chaperone FACT. Furthermore, the distribution of Abo1 on chromatin is disturbed by impaired FACT function. The role of Abo1 extends to some promoters and also to silent heterochromatin. Abo1 is recruited to pericentromeric heterochromatin independently of the HP1 ortholog, Swi6, where it enforces proper nucleosome occupancy. Consequently, loss of Abo1 alleviates silencing and causes elevated chromosome mis-segregation. We suggest that Abo1 provides a histone chaperone function that maintains nucleosome architecture genome-wide. A chromatin-seq/MNase-seq approach called Chromatin Particle Spectrum Analysis (Kent et al., (2011) Nucleic Acids Res. 39:e26) was used to map and compare nucleosome position in wild-type (strain 972) and isogenic abo1 knock-out (strain HM463) fission yeast cells. For each strain, three independent in vivo MNase digest bio-reps were performed and the purified DNA pooled. CPSA was performed using paired-end mode Illumina technology with pooled samples multiplexed over two HiSeq2000 lanes. Each CPSA paired read describes a microcococcal nuclease (MNase) resistant DNA species from chromatin, with the insert-size equivalent to the size of DNA protection. For each strain type, two analysed data sets are provided here: one listing the genomic distribution of MNase-protected DNAs of 150bp (â150bp CPSA size classâ) and corresponding to mono-nucleosomes; the other listing the genomic distribution of MNase-protected DNAs of 300bp (â300bp CPSA size classâ) and corresponding to di-nucleosomes.

Dataset Information

Regulation of microtubule assembly and organization in mitosis by the AAA+ ATPase Pontin.

Publications

Regulation of microtubule assembly and organization in mitosis by the AAA+ ATPase Pontin.

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