Dataset Information

Evaluating different DNA binding domains to modulate L1 ORF2p-driven site-specific retrotransposition events in human cells.

ABSTRACT: DNA binding domains (DBDs) have been used with great success to impart targeting capabilities to a variety of proteins creating highly useful genomic tools. We evaluated the ability of five types of DBDs and strategies (AAV Rep proteins, Cre, TAL effectors, zinc finger proteins, and Cas9/gRNA system) to target the L1 ORF2 protein to drive retrotransposition of Alu inserts to specific sequences in the human genome. First, we find that the L1 ORF2 protein tolerates the addition of protein domains both at the amino- and carboxy-terminus. Although in some instances retrotransposition efficiencies slightly diminished, all fusion proteins containing an intact ORF2 were capable of driving retrotransposition. Second, the stability of individual ORF2 fusion proteins varies and difficult to predict. Third, DBDs that require the formation of multimers for target recognition are unlikely to modify targeting of ORF2p-driven insertions. Fourth, the more components needed to assemble into a complex to drive targeted retrotransposition, the less likely the strategy will increase targeted insertions. Fifth, abundance of target sequences present in the genome will likely dictate the effectiveness and efficiency of targeted insertions. Lastly, the cleavage capabilities of Cas9 (or a Cas9 nickase variant) are unable to substitute for the L1 ORF2 endonuclease domain functions, suggestive that the endonuclease domain has alternate functions needed for retrotransposition. From these studies, we conclude that the most critical component for the modification of the human L1 ORF2 protein to drive targeted insertions is the selection of the DBD due to the varying functional requirements and impacts on protein stability.

SUBMITTER: Ade CM

PROVIDER: S-EPMC5734997 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Publications

Evaluating different DNA binding domains to modulate L1 ORF2p-driven site-specific retrotransposition events in human cells.

Ade Catherine M CM Derbes Rebecca S RS Wagstaff Bradley J BJ Linker Sara B SB White Travis B TB Deharo Dawn D Belancio Victoria P VP Ivics Zoltán Z Roy-Engel Astrid M AM

Gene 20171114

DNA binding domains (DBDs) have been used with great success to impart targeting capabilities to a variety of proteins creating highly useful genomic tools. We evaluated the ability of five types of DBDs and strategies (AAV Rep proteins, Cre, TAL effectors, zinc finger proteins, and Cas9/gRNA system) to target the L1 ORF2 protein to drive retrotransposition of Alu inserts to specific sequences in the human genome. First, we find that the L1 ORF2 protein tolerates the addition of protein domains ...[more]

PMID: 29154869

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Project description:Long interspersed nuclear elements-1 (LINE-1 or L1s) are abundant retrotransposons that comprise approximately 20% of mammalian genomes. Active L1 retrotransposons can impact the genome in a variety of ways, creating insertions, deletions, new splice sites or gene expression fine-tuning. We have shown previously that L1 retrotransposons are capable of mobilization in neuronal progenitor cells from rodents and humans and evidence of massive L1 insertions was observed in adult brain tissues but not in other somatic tissues. In addition, L1 mobility in the adult hippocampus can be influenced by the environment. The neuronal specificity of somatic L1 retrotransposition in neural progenitors is partially due to the transition of a Sox2/HDAC1 repressor complex to a Wnt-mediated T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activator. The transcriptional switch accompanies chromatin remodelling during neuronal differentiation, allowing a transient stimulation of L1 transcription. The activity of L1 retrotransposons during brain development can have an impact on gene expression and neuronal function, thereby increasing brain-specific genetic mosaicism. Further understanding of the molecular mechanisms that regulate L1 expression should provide new insights into the role of L1 retrotransposition during brain development. Here we show that L1 neuronal transcription and retrotransposition in rodents are increased in the absence of methyl-CpG-binding protein 2 (MeCP2), a protein involved in global DNA methylation and human neurodevelopmental diseases. Using neuronal progenitor cells derived from human induced pluripotent stem cells and human tissues, we revealed that patients with Rett syndrome (RTT), carrying MeCP2 mutations, have increased susceptibility for L1 retrotransposition. Our data demonstrate that L1 retrotransposition can be controlled in a tissue-specific manner and that disease-related genetic mutations can influence the frequency of neuronal L1 retrotransposition. Our findings add a new level of complexity to the molecular events that can lead to neurological disorders.

Dataset Information

Evaluating different DNA binding domains to modulate L1 ORF2p-driven site-specific retrotransposition events in human cells.

Publications

Evaluating different DNA binding domains to modulate L1 ORF2p-driven site-specific retrotransposition events in human cells.

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