Mapping of Novel Candidate Functional Elements with Bru-Seq Technology

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Abstract The ENCODE project has provided a tremendous resource for scientists with a treasure trove of data from a large set of different cell types describing RNA expression (RNA-seq), epigenetic signatures (ChIP-seq), chromatin structure (DNase-seq, ATAC-seq, Hi-C, ChIA-PET) and binding patterns of specific proteins to both DNA and RNA (ChIP-seq, CLIP-seq, RIP-seq). While RNA- seq data is very informative in providing signatures of steady-state levels of RNA expressed for particular genes, individual contributions of RNA synthesis and degradation to the steady-state- level of RNA cannot be determined. Furthermore, these expression data sets are static and do not provide dynamic information on specific cell responses. Regarding the ChIP-seq analyses of specific histone modifications, such as high H3K1me1 and H3K27ac and low H3K4me3, they are valuable for the identification of putative enhancer elements in a particular cell type but does not inform on whether such functional elements are functionally active. We have recently developed a set of techniques that are based on the specific labeling of nascent RNA with bromouridine (Bru) followed by lysis, capturing of the Bru-labeled RNA using specific antibodies and deep sequencing. Bru-seq captures the “nascent RNA transcriptome”, a signature of ongoing transcription in the genome where the relative rates of transcription of all genes can be obtained. In BruChase-seq, cells are labeled with Bru and then chased in uridine for different periods of time to allow for the determination of the “RNA stabilome” where the relative RNA degradation rates genome-wide are assessed. Finally, BruUV-seq allows for the capturing of RNA species that normally are rapidly turned over by the RNA exosome and allows for a different view of the nascent transcriptome where active enhancer elements and other candidate functional elements generating unstable RNAs are identified genome-wide. In this UM1 granting period we have used our Bru-seq suite of assays on human cell lines to complement existing data that have previously been generated using various genomic assays. In year 5 of this granting period, we are planning to extend the Bru-seq analysis for 1) COVID-19 cell line experiments, 2) immune/blood cell samples and 3) genetic perturbations.