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2. Evolutionary conserved relocation of chromatin remodeling complexes to the mitotic apparatus

Author

Messina, G, Prozzillo, Y, Monache, F, Santopietro, M, Dimitri, P, Messina, Giovanni, Prozzillo, Yuri, Monache, Francesca Delle, Santopietro, Maria Virginia, Dimitri, Patrizio, Messina, G, Prozzillo, Y, Monache, F, Santopietro, M, Dimitri, P, Messina, Giovanni, Prozzillo, Yuri, Monache, Francesca Delle, Santopietro, Maria Virginia, and Dimitri, Patrizio

Abstract

Background ATP-dependent chromatin remodeling complexes are multi-protein machines highly conserved across eukaryotic genomes. They control sliding and displacing of the nucleosomes, modulating histone-DNA interactions and making nucleosomal DNA more accessible to specific binding proteins during replication, transcription, and DNA repair, which are processes involved in cell division. The SRCAP and p400/Tip60 chromatin remodeling complexes in humans and the related Drosophila Tip60 complex belong to the evolutionary conserved INO80 family, whose main function is promoting the exchange of canonical histone H2A with the histone variant H2A in different eukaryotic species. Some subunits of these complexes were additionally shown to relocate to the mitotic apparatus and proposed to play direct roles in cell division in human cells. However, whether this phenomenon reflects a more general function of remodeling complex components and its evolutionary conservation remains unexplored. Results We have combined cell biology, reverse genetics, and biochemical approaches to study the subcellular distribution of a number of subunits belonging to the SRCAP and p400/Tip60 complexes and assess their involvement during cell division progression in HeLa cells. Interestingly, beyond their canonical chromatin localization, the subunits under investigation accumulate at different sites of the mitotic apparatus (centrosomes, spindle, and midbody), with their depletion yielding an array of aberrant outcomes of mitosis and cytokinesis, thus causing genomic instability. Importantly, this behavior was conserved by the Drosophila melanogaster orthologs tested, despite the evolutionary divergence between fly and humans has been estimated at approximately 780 million years ago. Conclusions Overall, our results support the existence of evolutionarily conserved diverse roles of chromatin remodeling complexes, whereby subunits of the SRCAP and p400/Tip60 complexes relocate from the interphase ch

Published
2022

8. Additional file 4: Figure S2. of eEF1Bγ binds the Che-1 and TP53 gene promoters and their transcripts

Author

Pisani, Cinzia, Onori, Annalisa, Gabanella, Francesca, Monache, Francesca Delle, Borreca, Antonella, Ammassari-Teule, Martine, Fanciulli, Maurizio, Certo, Maria Di, Passananti, Claudio, and Corbi, Nicoletta

Abstract

A. Chromatin immunoprecipitation was performed in hSH-SY5Y cells using anti-eEF1Bγ rabbit polyclonal antibodies or no-Ab as a control. Immunoprecipitates from each sample were analyzed by PCR performed with primers specific for the human Che-1 promoter and for the human TP53 promoter. The thymidine kinase human promoter was amplified as a negative control. A sample representing linear amplification of the total input chromatin (input) was included in the PCR as a control. B. Co-localization of endogenous Che-1, performed with the anti-Che-1 rat polyclonal antibody (green), and the mitochondrial marker Tom20 (red), in hSH-SY5Y cells. Extensive co-localization (yellow) between Che-1 and Tom20 is visualized by the merged-color image. The boxed area represents a high magnification image of co-localization. Nuclei were labeled with DAPI (blue). Scale bars: 10 μm. C. Western blot analysis of hSH-SY5Y whole-cell lysate and mitochondrial enriched fraction. The quality of mitochondrial-enriched fractions was monitored using anti-HSP60 monoclonal antibodies and anti-Tom20 rabbit polyclonal antibodies. D. Quantitative real time PCR (qPCR) analysis of the eEF1Bγ and Che-1 mRNAs in hSH-SY5Y cells (siRNA-Control and siRNA-eEF1Bγ). The gene expression ratio between eEF1Bγ and GAPDH and between Che-1 and GAPDH are shown as the mean ± SD from three independent experiments performed in triplicate. E. Representative Western blot of hSH-SY5Y whole-cell lysates treated or un-treated with siRNA as shown. The antibodies that were used are indicated. (PDF 199 kb)

Published
2016
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10. Additional file 3: Figure S1. of eEF1Bγ binds the Che-1 and TP53 gene promoters and their transcripts

Author

Pisani, Cinzia, Onori, Annalisa, Gabanella, Francesca, Monache, Francesca Delle, Borreca, Antonella, Ammassari-Teule, Martine, Fanciulli, Maurizio, Certo, Maria Di, Passananti, Claudio, and Corbi, Nicoletta

Abstract

A. RIP assay output was analyzed by semi-quantitative RT-PCR with specific primers to validate some of mRNAs co-immunoprecipitated with eEF1Bγ and they are listed in Table S2. B. The myc-eEF1Bγ and MS2-GFP fusion proteins were expressed in HeLa cells with the report mRNA carrying both the MS2 binding site and the indicated 3′ UTR. C. RIP assay-eEF1Bγ mRNAs (Additional file 2: Table S2) analyzed by quantitative real time PCR (qPCR) in HeLa whole-cell lysates treated with siRNA as shown. The gene expression ratio between mRNAs and GAPDH are shown as the mean ± SD from three independent experiments performed in triplicate. D. Representative western blot of HeLa whole-cell lysates treated or un-treated with siRNA as shown. The antibodies that were used are indicated. Densitometric analysis represents the mean ± S.D. of 3 independent experiments (right panel). (PDF 208 kb)

Published
2016
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11. eEF1Bγ binds the Che-1 and TP53 gene promoters and their transcripts.

Author

Pisani, Cinzia, Onori, Annalisa, Gabanella, Francesca, Monache, Francesca Delle, Borreca, Antonella, Ammassari-Teule, Martine, Fanciulli, Maurizio, Di Certo, Maria Grazia, Passananti, Claudio, and Corbi, Nicoletta

Subjects
ELONGATION factors (Biochemistry), RNA-binding proteins, MITOCHONDRIA, DNA damage, RNA polymerases
Abstract

Background: We have previously shown that the eukaryotic elongation factor subunit 1B gamma (eEF1Bγ) interacts with the RNA polymerase II (pol II) alpha-like subunit "C" (POLR2C), alone or complexed, in the pol II enzyme. Moreover, we demonstrated that eEF1Bγ binds the promoter region and the 3' UTR mRNA of the vimentin gene. These events contribute to localize the vimentin transcript and consequentially its translation, promoting a proper mitochondrial network. Methods: With the intent of identifying additional transcripts that complex with the eEF1Bγ protein, we performed a series of ribonucleoprotein immunoprecipitation (RIP) assays using a mitochondria-enriched heavy membrane (HM) fraction. Results: Among the eEF1Bγ complexed transcripts, we found the mRNA encoding the Che-1/AATF multifunctional protein. As reported by other research groups, we found the tumor suppressor p53 transcript complexed with the eEF1Bγ protein. Here, we show for the first time that eEF1Bγ binds not only Che-1 and p53 transcripts but also their promoters. Remarkably, we demonstrate that both the Che-1 transcript and its translated product localize also to the mitochondria and that eEF1Bγ depletion strongly perturbs the mitochondrial network and the correct localization of Che-1. In a doxorubicin (Dox)-induced DNA damage assay we show that eEF1Bγ depletion significantly decreases p53 protein accumulation and slightly impacts on Che-1 accumulation. Importantly, Che-1 and p53 proteins are components of the DNA damage response machinery that maintains genome integrity and prevents tumorigenesis. Conclusions: Our data support the notion that eEF1Bγ, besides its canonical role in translation, is an RNA-binding protein and a key player in cellular stress responses. We suggest for eEF1Bγ a role as primordial transcription/translation factor that links fundamental steps from transcription control to local translation. [ABSTRACT FROM AUTHOR]

Published
2016
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