Browsing by Subject "SETMAR"

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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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    Crystallization of and selenomethionine phasing strategy for a SETMAR–DNA complex
    (International Union of Crystallography, 2016-08-26) Chen, Qiujia; Georgiadis, Millie; Biochemistry and Molecular Biology, School of Medicine
    The DNA-binding domain of SETMAR was successfully crystallized in a complex with its ancestral terminal inverted repeat and a variant of this sequence through a systematic approach, and initial Se SAD phasing was achieved through the judicious addition of Met residues., Transposable elements have played a critical role in the creation of new genes in all higher eukaryotes, including humans. Although the chimeric fusion protein SETMAR is no longer active as a transposase, it contains both the DNA-binding domain (DBD) and catalytic domain of the Hsmar1 transposase. The amino-acid sequence of the DBD has been virtually unchanged in 50 million years and, as a consequence, SETMAR retains its sequence-specific binding to the ancestral Hsmar1 terminal inverted repeat (TIR) sequence. Thus, the DNA-binding activity of SETMAR is likely to have an important biological function. To determine the structural basis for the recognition of TIR DNA by SETMAR, the design of TIR-containing oligonucleotides and SETMAR DBD variants, crystallization of DBD–DNA complexes, phasing strategies and initial phasing experiments are reported here. An unexpected finding was that oligonucleotides containing two BrdUs in place of thymidines produced better quality crystals in complex with SETMAR than their natural counterparts.
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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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    A role for SETMAR in gene regulation: insights from structural analysis of the dna-binding domain in complex with dna
    (2016-08) Chen, Qiujia; Georgiadis, Millie M.; Hurley, Thomas D.; Wek, Ronald C.; Turchi, John J.; Kelley, Mark R.
    SETMAR is a chimeric protein that originates from the fusion of a SET domain to the mariner Hsmar1 transposase. This fusion event occurred approximately 50 million years ago, after the split of an anthropoid primate ancestor from the prosimians. Thus, SETMAR is only expressed in anthropoid primates, such as humans, apes, and New World monkeys. Evolutionary sequence analyses have revealed that the DNA-binding domain, one of the two functional domains in the Hsmar1 transposase, has been subjected to a strong purifying selection. Consistent with these analyses, SETMAR retains robust binding specificity to its ancestral terminal inverted repeat (TIR) DNA. In the human genome, this TIR sequence is dispersed in over 1500 perfect or nearly perfect sites. Given that many DNA-binding domains of transcriptional regulators are derived from transposases, we hypothesized that SETMAR may play a role in gene regulation. In this thesis, we determined the crystal structures of the DNA-binding domain bound to both its ancestral TIR DNA and a variant TIR DNA sequence at 2.37 and 3.07 Å, respectively. Overall, the DNA-binding domain contains two helix-turn-helix (HTH) motifs linked by two AT-hook motifs and dimerizes through its HTH1 motif. In both complexes, minor groove interactions with the AT-hook motifs are similar, and major groove interactions with HTH1 involve a single residue. However, four residues from HTH2 participate in nucleobase-specific interactions with the TIR and only two with the variant DNA sequence. Despite these differences in nucleobase-specific interactions, the DNA-binding affinities of SETMAR to TIR or variant TIR differ by less than two-fold. From cell-based studies, we found that SETMAR represses firefly luciferase gene expression while the DNA-binding deficient mutant does not. A chromatin immunoprecipitation assay further confirms that SETMAR binds the TIR sequence in cells. Collectively, our studies suggest that SETMAR functions in gene regulation.