DNA Bridges Govern Ground State Pluripotency

Unrestricted self-renewal and the ability to form germline cells, sperm, and ova are hallmarks of stem cells with the innate potential to become any cell in the body, a state known as pluripotency. Understanding the physical basis of pluripotency is key in regenerative medicine, which aims to replace, develop, or regenerate diseased or aged cells.

Previous studies have found that a tightly regulated protein called NANOG is found in abundance with increased cellular capacity for reprogramming and renewal, and in minute amounts with increased tendencies for spontaneous cellular differentiation and specialization. Furthermore, human stem cells in culture can be coaxed into the baseline state of pluripotency—a state of naïve, untrained “stemness”—by enhanced NANOG expression. So far, however, it has been unclear how NANOG accomplishes this.

In a new study from Baylor College of Medicine and collaborating institutions, scientists uncover mechanistic insights into Nanog’s unique properties and how it works in activating pluripotency. These results were published in the journal on April 28, 2022 nature cell biologyin an article entitled “NANOG prion-like assembly mediates DNA bridging to relief chromatin reorganization and activation of pluripotency.”

Researchers found that NANOG’s “super stickiness” enabled it to form large clumps at very low concentrations. These clumps interact with chromatin—DNA packaged in tight coils on protein scaffolds—to rearrange its organization in a way that induces pluripotency.

The study’s lead author, Josephine Ferreon, PhD, an assistant professor of pharmacology and chemical biology and a member of the Dan L. Duncan Comprehensive Cancer Center in Baylor, said: “Resetting specialized cells to a pluripotent state requires massive chromatin reorganization and changes in the gene Expression – turning on genes involved in pluripotency and turning off genes that specify specialized cells.”

Josephine Ferreon added: “Coordinated gene activation often requires bringing DNA elements that are far apart closer together to allow for gene expression. We have found that the properties of NANOG — its naturally floppy, flexible 3D shape and a C-terminal tail structurally related to prion-like proteins — allow it to achieve this.”

Prions are a type of infectious misfolded protein that can transfer their misfolded shape to normal variants of the same protein. The researchers found that NANOG’s prion-like tail spontaneously transitions into a gel-like phase.

The stickiness of NANOG is a problem as it forms oligomers even at nanomolar concentrations. Researchers therefore resorted to single-molecule Förster resonance energy transfer and fluorescence cross-correlation techniques to study NANOG and demonstrated its oligomerization-bridge DNA elements in vitro.

Using chromatin immunoprecipitation sequencing and Hi-C 3.0 in cells, the authors validated that the prion-like domain arrangement of NANOG is required to recognize specific regions of DNA and to bridge distant chromatin regions. Hi-C, a benchmark tool for studying genome organization, uses chromosome conformation (3C) sensing to detect interacting pairs of chromatin throughout the genome.

“In this study, we applied single-molecule and fluorescence fluctuation microscopy techniques, which allow us to visualize whether two molecules interact with each other. The experiments were performed at very small concentrations, picomolar to nanomolar, where we can typically avoid aggregation and study proteins prone to aggregation,” said co-author Allan Chris Ferreon, PhD, assistant professor of pharmacology and chemical biology at Baylor.

Allan Chris Ferreon added: “However, with NANOG we still found aggregation even at extremely low concentrations. Nevertheless, we were able to show that NANOG aggregation is essential for its function as a master transcription factor and mediator of DNA bridging. This phenomenon is possibly unique to NANOG.”

“We believe this phenomenon is why NANOG expression is key to establishing pluripotency. When the NANOG level is low, cells tend to differentiate, and when its level is high, the pluripotent ground state or ‘full reset’ is reached and maintained,” Josephine Ferreon said.

The authors theorize that NANOG’s tendency to aggregate acts like a molecular glue, triggering and stabilizing bridged conformations of chromatin required for pluripotency. It also explains its role as a molecular “hub” that interacts with many regulatory proteins that recognize, open, and modify specific regions of chromatin.

Gaining greater clarity on how NANOG, with its prion-like tail, works as part of a team of transcription factors, coactivators and epigenetic modulators that remodel chromatin architecture remains the team’s goal in future investigations.