ZCCHC8 IS REQUIRED FOR THE DEGRADATION OF PERVASIVE TRANSCRIPTS ORIGINATING FROM MULTIPLE GENOMIC REGULATORY REGIONS
Collins, Joshua Wade
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Antibodies are among the most frequently used tools in biomedical research, but they are often subject to many pitfalls including cross-reactivity, production lot-to-lot variability, and loss of activity. Despite the multiple laboratory applications for which antibodies are used, there are few standardized scientific guidelines for validation of antibody usage. Much of the blame for the recent reproducibility crisis in biomedical research has been placed on the failure of antibodies to perform consistently over time. In chapter one, we report a case study of an anti-BTBD7 antibody that displays cross- reactivity, extreme lot-to-lot variability, and loss of activity across multiple applications. We determined the major cross-reacting protein of interest to be ZCCHC8 and provide a general framework for determining the identity of cross-reacting proteins. We also surveyed a panel of anti-ZCCHC8 antibodies that show an array of non-specificity and in some instances apparent cross-reactivity with BTBD7. A mechanistic study of ZCCHC8 and its role in regulating non-coding RNA is detailed in chapter two. The vast majority of mammalian genomes are transcribed as non-coding RNA in what is referred to as “pervasive transcription.” Recent studies have uncovered various families of non-coding RNA transcribed upstream of transcription start sites. In particular, highly unstable promoter upstream transcripts known as PROMPTs have been shown to be targeted for exosomal degradation by the nuclear exosome targeting complex (NEXT) consisting of the RNA helicase MTR4, the zinc- knuckle scaffold ZCCHC8, and the RNA binding protein RBM7. Here, we report that in addition to its known RNA substrates, ZCCHC8 and/or the NEXT complex are responsible for the targeted degradation of pervasive transcripts produced at CTCF binding sites, open chromatin regions, promoters, promoter flanking regions, and transcription factor binding sites. Additionally, we report that a significant number of RIKEN cDNAs and predicted genes display the hallmarks of PROMPTs and are also substrates for ZCCHC8 and/or NEXT complex regulation suggesting these are unlikely to be functional genes. Our results suggest that ZCCHC8 and/or the NEXT complex may play a larger role in the global regulation of pervasive transcription than previously reported.