Abstract
Biochemical analysis of molecular interactions in specific genomic regions requires their isolation while retaining molecular interactions in vivo. Here, we report isolation of telomeres by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using a transcription activator-like (TAL) protein recognizing telomere repeats. Telomeres recognized by the tagged TAL protein were immunoprecipitated with an antibody against the tag and subjected to identification of telomere-binding molecules. enChIP-mass spectrometry (enChIP-MS) targeting telomeres identified known and novel telomere-binding proteins. The data have been deposited to the ProteomeXchange with identifier PXD000461. In addition, we showed that RNA associated with telomeres could be isolated by enChIP. Identified telomere-binding molecules may play important roles in telomere biology. enChIP using TAL proteins would be a useful tool for biochemical analysis of specific genomic regions of interest.
Highlights
Biochemical analysis of molecular interactions in specific genomic regions requires their isolation while retaining molecular interactions in vivo
EnChIP using transcription activator-like (TAL) proteins would be a useful tool for biochemical analysis of specific genomic regions of interest
Scheme of engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). enChIP consists of the following steps[5] (Fig. 1): (i) A DNA-binding molecule/complex (DB) recognizing a target DNA sequence in a genomic region of interest is engineered (Fig. 1a, b)
Summary
Biochemical analysis of molecular interactions in specific genomic regions requires their isolation while retaining molecular interactions in vivo. We report isolation of telomeres by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using a transcription activator-like (TAL) protein recognizing telomere repeats. We recently developed the insertional chromatin immunoprecipitation (iChIP) technology to isolate specific genomic regions retaining molecular interactions in vivo[2,3]. In iChIP, recognition sequences of an exogenous DNA-binding molecule such as LexA are inserted into a target genomic region. IChIP enables us to isolate specific genomic regions of interest and dissect chromatin components, insertion of recognition sequences of LexA or other DNA-binding molecules is a time-consuming step. To eliminate the step of insertion of recognition sequences of an exogenous DNA-binding molecule from iChIP and make the procedure more straightforward, we recently developed a novel method, engineered DNAbinding molecule-mediated chromatin immunoprecipitation (enChIP), for purification of specific genomic regions[5]. EnChIP using TAL proteins would be a useful tool for biochemical analysis of specific genomic regions of interest
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