Cracking the genomic code

During embryonic development, cells start out as pluripotent, or have the potential to become many different cell types through differentiation. Naive-state is the name for the earliest stage of development for pluripotent stem cells. These cells are considered totipotential, with the ability to become every kind of cell in a multicellular organism.

The first step in gene expression is called transcription. During transcription, proteins bind to sequences of DNA, such as promoters and enhancers, inside the cell to directly impact gene expression. 

A research lab at Lewis & Clark College in Portland, Oregon is exploring the characteristics of enhancers that influence the expression of a gene called Klf4, important for establishing and maintaining naive-state pluripotent stem cells.

“Understanding how gene expression is controlled in pluripotent stem cells is important for being able to harness these cells in medical applications, particularly regenerative medicine, where there is potential to produce new tissues for patients,” said Sharon Torigoe, professor of biology at Lewis & Clark and the principal researcher. 

To function, enhancers are interpreted by transcription factors, or proteins, that bind to DNA sequences and then signal to other factors about whether to express a target gene. Several transcription factors are involved in the enhancers that initiate the process of activating Klf4 expression, and the central ones are OCT4 and SOX2. 

Through experiments, Torigoe and her students observed that the binding sites in Klf4 enhancers for the transcription factors OCT4 and SOX2 were “low-affinity.” In other words, the transcription factors did not bind very well to the enhancer sequence. They were initially surprised by this finding, but eventually confirmed that this “low affinity” was crucial for driving the naive-state specific activities of the enhancers for Klf4. In fact, further experimentation demonstrated that if they replaced the binding sites with better, high-affinity versions, the Klf4 enhancers did not function optimally. They have published their findings in PLOS ONE, in an article called: “A suboptimal OCT4-SOX2 binding site facilitates the naïve-state specific function of a Klf4 enhancer.”

“We found that the low affinity of the binding sites for the central transcription factors were crucial for specifying enhancer function,” says Torigoe. “Our work suggests that the affinity of OCT4-SOX2 binding sites could facilitate enhancer functions in specific states of pluripotency.”

When Torigoe and her students dug through the literature, they found that researchers had previously observed similar functions for low-affinity transcription factor binding sites in other enhancers and organisms. To Torigoe’s knowledge, her work is among the first to confirm the importance of low-affinity binding sites for an enhancer in naive-state pluripotent stem cells.

“The genome contains a huge amount of information that makes an organism function, and we

know so little about it, even the parts that we thought we knew,” says Torigoe. “We’ve known about enhancers since the 1980s, and we still have much to understand about them.”

This research advances scientists’ knowledge of the mechanisms of gene expression more broadly.

“We have so much ability to sequence genomes now, but we do not yet understand what all of that information means,” says Torigoe. “Gaining insights into enhancers, from a sequence viewpoint, will help us to interpret any genome, whether for new organisms to understand evolution or for patients to understand the genetic basis of human disease.”