An international scientific group led by Lawrence Berkeley National Laboratory (Berkeley Lab) reports the development of a novel method for identifying gene enhancers in the genomes of humans and other mammals. Called SIF-seq, for site-specific integration fluorescence-activated cell sorting followed by sequencing, the technique complements existing genomic tools, such as ChIP-seq (chromatin immunoprecipitation followed by sequencing), and offers some additional benefits, according to the researchers.
“While ChIP-seq is very powerful in that it can query an entire genome for characteristics associated with enhancer activity in a single experiment, it can fail to identify some enhancers and identify some sites as being enhancers when they really aren’t,” says Diane Dickel, Ph.D., a geneticist with Berkeley Lab’s genomics division and member of the SIF-seq development team. “SIF-seq is currently capable of testing only hundreds to a few thousand sites for enhancer activity in a single experiment, but can determine enhancer activity more accurately than ChIP-seq and is therefore a very good validation assay for assessing ChIP-seq results.”
Dr. Dickel is the lead author of a paper (“Function-based identification of mammalian enhancers using site-specific integration”) in Nature Methods describing the new methodology.
With the increasing awareness of the important role that gene enhancers play in normal cell development as well as in disease, there is strong scientific interest in identifying and characterizing these enhancers. This is a challenging task because an enhancer does not have to be located directly adjacent to the gene whose expression it regulates, but can instead be located hundreds of thousands of DNA base pairs away. The challenge is made even more difficult because the activity of many enhancers is restricted to specific tissues or cell types.
“For example, brain enhancers will not typically work in heart cells, which means that you must test your enhancer sequence in the correct cell type,” Dr. Dickel says.
Currently, enhancers can be identified through chromatin-based assays, such as ChIP-seq, which predict enhancer elements indirectly based on the enhancer’s association with specific epigenomic marks, such as transcription factors or molecular tags on DNA-associated histone proteins. Dr. Dickel and colleagues developed SIF-seq in response to the need for a higher-throughput functional enhancer assay that can be used in a wide variety of cell types and developmental contexts.
“Targeted single-copy genomic integration into pluripotent cells, reporter assays, and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences,” wrote the investigators.” By functionally interrogating >500 kilobases (kb) of mouse and human sequence in mouse embryonic stem cells for enhancer activity we identified enhancers at pluripotency loci. In in vitro–differentiated cardiomyocytes and neural progenitor cells, we identified cardiac enhancers and neuronal enhancers, respectively. SIF-seq is a powerful and flexible method for de novo functional identification of mammalian enhancers in a potentially wide variety of cell types.”
The ability of SIF-seq to use reporter assays in mouse embryonic stem cells to identify human embryonic stem cell enhancers that are not present in the mouse genome opens the door to intriguing research possibilities, according to Dr. Dickel.
“Human and chimpanzee genes differ very little, so one hypothesis in evolutionary genomics holds that humans and chimpanzees are so phenotypically different because of differences in the way they regulate gene expression. It is very difficult to carry out enhancer identification through ChIP-seq that would be useful in studying this hypothesis,” she pointed out. “However, because SIF-seq only requires DNA sequence from a mammal and can be used in a variety of cell types, it should be possible to compare the neuronal enhancers present in a large genomic region from human to the neuronal enhancers present in the orthologous chimpanzee region. This could potentially tell us interesting things about the genetic differences that differentiate human brain development from that of other primates.”