Development of tools for capturing transcriptomes and associated processes.
In previous work, we have developed nuclear run-on based assays that map where actively transcribing polymerase are in the genome by tracking the nascent RNA as it is being transcribed. These approaches include:
- GRO-seq: Maps polymerase location within 30-50bp.
- PRO-seq: Maps polymerase active site at near nucleotide resolution.
- GRO/PRO-cap: The methods above modified to map the sites of transcription initiation (Transcription Start Sites; TSS)
Combined, these approaches allow one to:
- Map all transcripts regardless of the stability, including primary mRNAs, primary miRNAs, enhancer RNAs (eRNAs), RNAs that are upstream and antisense to promoters (uaRNAs), and transcription that continues beyond the 3’-ends of genes.
- Map TSSs of any transcript with high sensitivity.
- Determine immediate and directs transcriptional responses to various stimuli.
- Determine transcription rates from time-course experiments.
- Map transcription from all polymerases, including Pol I, Pol II, and Pol III.
Current projects in this area involve:
- Improving the sensitivity and throughput of these techniques.
- Modification the assays to capture other co-transcriptional processes
Mechanism and function of promoter – and enhancer- associated non-coding RNA transcription.
Transcriptome mapping has revealed that there are many RNAs emanating at or very near to promoters and enhancers. These RNAs are typically, short, non-coding, and rapidly degraded. Nuclear run-on based approaches are ideal for detecting these transcripts since they can be captured before RNA degradation mechanisms take place. We are combining these approaches with other genomic assays to determine the role of protein factors or DNA/RNA sequence elements in various types of non-coding transcription. In addition, we are examining the function of the short transcripts or act of transcription itself in gene regulation.
Mapping transcriptomes and important regulatory DNA sequence elements that are associated with specific developmental or disease states.
Since GRO-seq based approaches can map the entire transcriptome in a single assay, one can obtain a comprehensive gene expression signature of any cell type. In addition, the bidirectional pattern of transcription that emanates from promoters and enhancers allows for identification of these hubs that control gene expression. We are using these transcription signatures to identify and quantify the activity of important regulatory elements associated with development and disease states.