STARR-seq, short for self-transcribing active regulatory region sequencing, is a genetic screening method designed to identify and quantify the activity of gene enhancers on a genome-wide scale. Enhancers are specific DNA sequences that function like dimmer switches for genes, dictating when and where they are turned on or off within a cell. These elements control gene expression, which in turn determines cell identity and function. The technique provides a direct and quantitative assessment of the regulatory potential of millions of candidate DNA fragments simultaneously.
The Core Mechanism of STARR-seq
The process of STARR-seq begins with preparing a collection of potential enhancer sequences. Genomic DNA from a cell type of interest is fragmented into small pieces. This collection of fragments represents a library of all the potential regulatory elements within the genome.
These DNA fragments are then inserted into a circular DNA molecule called a reporter plasmid. Each plasmid contains a minimal promoter and a reporter gene. If an inserted DNA fragment functions as an active enhancer, it drives the transcription of the reporter gene and also transcribes itself. This means the enhancer sequence becomes part of the resulting messenger RNA (mRNA) transcript.
The next step involves introducing this library of plasmids into a population of cells, a process known as transfection. The cells then test each potential enhancer’s activity. Total RNA is extracted from these cells.
The amount of RNA produced from a specific fragment directly reflects its enhancer activity. This RNA is converted back into DNA using reverse transcription, and these DNA copies are then sequenced using high-throughput sequencing technologies. The sequencing data provides a direct readout of which genomic fragments were actively transcribed, indicating their enhancer function.
Interpreting STARR-seq Data
The output of a STARR-seq experiment yields quantitative information about enhancer activity across the genome. Results are commonly visualized as peaks along a chromosome map, where each peak corresponds to a specific DNA region tested. The presence of a peak signifies that the DNA fragment at that genomic location exhibited enhancer activity.
The height or intensity of a peak is directly proportional to the number of sequenced RNA reads originating from that particular DNA fragment. A higher peak indicates that the corresponding DNA sequence is a stronger enhancer. Conversely, a lower peak suggests weaker enhancer activity.
This quantitative measurement allows researchers to not only identify where enhancers are located but also to rank them based on their transcriptional strength. Scientists can generate a list of the most active regulatory elements within a specific cell type, under particular experimental conditions. This provides a functional map of the genome’s regulatory landscape.
Applications in Genomic Research
STARR-seq provides insights into how genes are regulated, with applications in genomic research. One primary use is mapping regulatory landscapes across different cell types. By applying STARR-seq to various cell lines, such as neurons versus liver cells, researchers can identify cell-type-specific enhancers that contribute to the unique gene expression patterns defining each cell’s identity and function. This helps to understand the regulatory networks that support cellular diversity and development.
The technique also plays a role in understanding human diseases. Many genetic variations linked to diseases, including cancers or autoimmune disorders, are found in the non-coding regions of the genome, often within or near enhancers. STARR-seq can test if a specific genetic mutation alters the activity of an enhancer, either by creating a new one, disrupting an existing one, or changing its strength. This direct functional assessment helps establish a clear link between a genetic variant and its biological consequence, offering clues into disease mechanisms. For instance, it has been used to assess enhancer activities of DNA fragments containing single nucleotide polymorphisms (SNPs) to determine gene regulatory differences in populations.
STARR-seq in Context with Other Assays
STARR-seq offers an advantage compared to other genomic techniques by directly measuring enhancer activity. For example, ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) identifies regions of “open” or accessible chromatin across the genome. While open chromatin is a prerequisite for enhancer activity, ATAC-seq does not directly measure whether those accessible regions are functionally active enhancers. It simply indicates where the DNA is physically available for regulatory proteins to bind.
ChIP-seq (Chromatin Immunoprecipitation sequencing) identifies genomic locations where particular proteins, such as transcription factors or histone modifications, bind to DNA. Many of these binding events occur at enhancers, but ChIP-seq does not directly quantify the transcriptional output or the activity of the enhancer itself. It shows where certain proteins are, which can suggest regulatory potential, but not how much transcription an enhancer drives. STARR-seq, on the other hand, directly assesses the functional capacity of a DNA sequence to activate transcription, providing a direct measure of enhancer strength irrespective of specific protein binding events or chromatin accessibility.