In a new study, researchers at the Institute for the Advanced Study of Human Biology (ASHBi), report a new approach to reveal the temporal regulation of hundreds of gene regulatory motifs involved in neural differentiation.

The findings are published in the journal Nature Communications in a paper titled, “Massively parallel reporter perturbation assays uncover temporal regulatory architecture during neural differentiation,” and led by ASHBi associate professor Fumitaka Inoue, PhD.

The researchers sought to determine the role and function gene regulatory elements play a key role in orchestrating gene expression during cellular differentiation.

“Here, we perform perturbation-based massively parallel reporter assays at seven early time points of neural differentiation to systematically characterize how regulatory elements and motifs within them guide cellular differentiation,” the researchers wrote. “By perturbing over 2,000 putative DNA binding motifs in active regulatory regions, we delineate four categories of functional elements, and observe that activity direction is mostly determined by the sequence itself, while the magnitude of effect depends on the cellular environment.”

“Enhancers have important effects on gene expression and thus determine whether cells will grow into different organs. Despite their consequences, however, we understand relatively little about how enhancers function,” explained Inoue.

A number of biochemical assays are available to study enhancers, but massively parallel reporter assays (MPRAs) allow for the quantitative analysis of thousands of enhancers at one time.

“MPRAs are useful for studying the interplay between enhancers and transcription factors. To extract even more information from the assays, we modified the assays to introduce perturbations in the regulatory motifs where transcription factors bind,” continued Inoue.

“We broke these motifs into four groups. Essential and contributing motifs either were required or contributed to the activation. Likewise, silencing and inhibiting motifs were required or contributed to dampening transcription,” said Inoue.

Further observation revealed that among the genes enhanced or dampened, many are involved in promoting neural differentiation or stem cell pluripotency. Overall, said Inoue, the findings demonstrate an intricate network of regulatory sequences that interact with one another to achieve neural differentiation.

“Overall, our results provide an atlas of motif function across early time points of neural differentiation by directly testing hundreds of regulatory regions for the function of the motifs they harbor,” concluded the researchers.