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). 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 relative stability of all transcripts are assessed. Finally, BruUV-seq allows for the capturing of RNA species that normally are rapidly turned over by the RNA exosome and can be used to map active enhancer elements producing eRNA genome-wide. In this UM1 grant application we are proposing to use these three Bru-seq techniques to obtain critical novel information on the nascent RNA transcriptome, RNA stabilome and active enhancome that will complement and enrich existing ENCODE data. We are proposing to focus our mapping efforts on two specific Aims:
Aim 1 : Obtain nascent RNA transcriptome, RNA stabilome and active enhancome data for human and mouse ENCODE cell lines.
Aim 2 : Obtain dynamic signatures of nascent transcription, RNA stability and activity of enhancer elements genome-wide following cellular exposure to environmental perturbations. The mapping efforts proposed in this UM1 grant application are seeking to identify novel candidate regulatory elements genome-wide that will complement and extend existing ENCODE data. Furthermore, dynamic studies will be initiated to assess the activities of regulatory elements during selected cell transitions and specific cellular responses. GOALS: The main goal of this proposal is to generate comprehensive novel data sets using our newly developed Bru-seq analysis platform that will complement and extend existing ENCODE data. The emphasis will be on mapping candidate functional elements in the human genome.
The experiments proposed in the UM1 grant application are seeking to provide critical new information on transcriptional and post-transcriptional regulation in human cells that will complement and enrich existing ENCODE data. We will use three techniques recently developed in the lab to obtain novel genome-wide maps of nascent transcription and active enhancer elements and signatures of RNA stability. We will also provide comprehensive signatures of dynamic changes in transcription and utilization of enhancer elements directly following exposure of cells to important environmental factors or conditions.
