Browsing by Subject "Metnase"

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    The Chimeric Fusion Protein SETMAR Functions as a Chromatin Organizing Factor
    (2020-08) Bates, Alison Melissa; Georgiadis, Millie M.; Mosley, Amber L.; Quilliam, Lawrence A.; Fehrenbacher, Jill C.
    About 50 million years ago, an Hsmar1 transposon invaded an early primate genome and inserted itself downstream of a SET methyltransferase gene, leading to the birth of a new chimeric protein now called SETMAR. While all other Hsmar1 sequences in the human genome have suffered inactivating mutational damage, the transposase domain of SETMAR has remained remarkably intact, suggesting that it has gained a novel, evolutionarily advantageous function. While SETMAR can no longer transpose itself throughout the genome, it has retained its ancestral sequence-specific DNA binding activity, the importance of which is currently unknown. To investigate this, we performed ChIP-seq to examine SETMAR binding in the human genome. We also utilized RNA-sequencing to assess SETMAR overexpression as well as SETMAR deletion on the human transcriptome. Additionally, we explored SETMAR’s transposase-derived chromatin-looping ability using chromosome-conformation-capture-on-ChIP (4C) in the presence of SETMAR overexpression and performed genome-wide Hi-C to assess the impact of complete SETMAR silencing on global chromatin interactions. ChIP-seq revealed that SETMAR amassed 7,332 unique binding sites, 69% of which included a TIR motif. RNA-sequencing in cells overexpressing SETMAR indicated 177 differentially regulated transcripts, including repression of 17 histone transcripts, suggesting a possible role in chromatin dynamics. RNA-sequencing of parental and SETMAR knockout clones highlighted an average of 5,000 altered transcripts in each cell line, with 343 transcripts significantly differentially expressed in all three knockout clones, many of which participate in embryonic development pathways. 4C analysis in the presence of SETMAR overexpression discovered multiple intrachromosomal looping interactions, and Hi-C analysis of SETMAR knockout cell lines uncovered genome-wide loss of chromatin interactions and disruption of TAD boundaries. The prevalence of SETMAR binding in the human genome combined with its chromatin looping capability and its dramatic effects on the transcriptome suggest a previously undiscovered role for SETMAR as a novel chromatin organizing factor.
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    Functional Analysis of Two Novel DNA Repair Factors, Metnase and Pso4
    (2008-10-13T18:49:36Z) Beck, Brian Douglas; Lee, Suk-Hee
    Metnase is a novel bifunctional protein that contains a SET domain and a transposase domain. Metnase contains sequence-specific DNA binding activity and sequence non-specific DNA cleavage activity, as well as enhances genomic integration of exogenous DNA. Although Metnase can bind specifically to DNA sequences containing a core Terminal Inverted Repeat sequence, this does not explain how the protein could function at sites of DNA damage. Through immunoprecipitation and gel shift assays, I have identified the Pso4 protein as a binding partner of Metnase both in vitro and in vivo. Pso4 is essential for cell survival in yeast, and cells containing a mutation in Pso4 show increased sensitivity to DNA cross-linking agents. In addition, the protein has sequence-independent DNA binding activity, favoring double-stranded DNA over single-stranded DNA. I demonstrated that the two proteins form a 1:1 stochiometric complex, and once formed, Metnase can localize to DNA damage foci as shown by knockdown of Pso4 protein using in vivo immunofluorescence. In conclusion, this shows that Metnase plays an indispensable role in DNA end joining, possibly through its cleavage activity and association with DNA Ligase IV.
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    Investigation of Protein – Protein Interactors of Setmar Using Tandem Mass Tag Mass Spectrometry
    (2022-03) Segizbayeva, Lana; Georgiadis, Millie M.; Mosley, Amber L.; Wells, Clark D.
    The nuclear protein SETMAR has been reported to be involved in many processes such as non-homologous end joining (NHEJ), di-methylation (arguably) of K36 of histone H3, restart of stalled replication forks, chromosome decatenation, enhancing of TOPII inhibitors which results in resistance to chemotherapeutics in cancer patients, etc. All these purported functions are impossible to execute without interaction with other proteins. It is established that SETMAR binds specifically to DNA at terminal inverted repeat sequences and can loop DNA. This DNA sequence specific pull-down exploits this attribute to identify possible protein interactors of SETMAR. As a result of this experiment several proteins have been identified for further research: BAG2, c12orf45, PPIA, XRCC5/6, and ZBTB43, all of which are found in higher statistical abundances in full length SETMAR samples.
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    Metnase promotes restart and repair of stalled and collapsed replication forks
    (Oxford University Press, 2010-05-10) De Haro, Leyma P.; Wray, Justin; Williamson, Elizabeth A.; Durant, Stephen T.; Corwin, Lori; Gentry, Amanda C.; Osheroff, Neil; Lee, Suk-Hee; Hromas, Robert; Nickoloff, Jac A.; Biochemistry and Molecular Biology, School of Medicine
    Metnase is a human protein with methylase (SET) and nuclease domains that is widely expressed, especially in proliferating tissues. Metnase promotes non-homologous end-joining (NHEJ), and knockdown causes mild hypersensitivity to ionizing radiation. Metnase also promotes plasmid and viral DNA integration, and topoisomerase IIα (TopoIIα)-dependent chromosome decatenation. NHEJ factors have been implicated in the replication stress response, and TopoIIα has been proposed to relax positive supercoils in front of replication forks. Here we show that Metnase promotes cell proliferation, but it does not alter cell cycle distributions, or replication fork progression. However, Metnase knockdown sensitizes cells to replication stress and confers a marked defect in restart of stalled replication forks. Metnase promotes resolution of phosphorylated histone H2AX, a marker of DNA double-strand breaks at collapsed forks, and it co-immunoprecipitates with PCNA and RAD9, a member of the PCNA-like RAD9–HUS1–RAD1 intra-S checkpoint complex. Metnase also promotes TopoIIα-mediated relaxation of positively supercoiled DNA. Metnase is not required for RAD51 focus formation after replication stress, but Metnase knockdown cells show increased RAD51 foci in the presence or absence of replication stress. These results establish Metnase as a key factor that promotes restart of stalled replication forks, and implicate Metnase in the repair of collapsed forks.