Background

The interaction between enhancer regions, which regulate gene expression, and promoter regions, which initiate transcription, within the genome has long been a mystery. Enhancers, although located far from genes, form chromatin loops in three-dimensional space to contact promoters and activate genes. The academic community has primarily focused on cohesin or mediator complexes as key connectors. However, the process by which such physical contact leads to the recruitment of RNA polymerase II (Pol II), which transcribes genes, has remained unclear. In particular, the specific structural roles of non-coding RNAs transcribed in these regions have not been elucidated. To uncover the initiation mechanism of gene expression, it was necessary to identify molecular mediators linking loop formation and polymerase recruitment.

Key Findings

Professor Xue Yuanchao's research team at the Chinese Academy of Sciences (CAS) revealed that the RNA-binding protein hnRNPK acts as a molecular bridge that mediates communication between the two regions and recruits Pol II. The research team visualized this mechanism using RIC-seq (RNA in situ conformation sequencing), HiChIP, CUT&Tag, and super-resolution imaging technologies.

When enhancer RNA (eRNA) transcribed from enhancers and upstream antisense RNA (uaRNA) near promoters bind, hnRNPK was found to simultaneously bind to both RNAs. This complex formation contributes to stabilizing the chromatin loop structure. hnRNPK was shown to induce liquid-liquid phase separation (LLPS) on its own, forming aggregates with internal void spaces. These aggregates directly bind to the RPB3 subunit of Pol II and play a role in recruiting the polymerase to the promoter region.

When hnRNPK was rapidly depleted in cells, loop interactions weakened, Pol II recruitment was blocked, and overall transcriptional activity was suppressed. Furthermore, the pathological effects of the hnRNPK gene mutation (c.953+1dupG) associated with Au-Kline syndrome were investigated. In the mutant mouse model, hnRNPK aggregates were observed to transition from a liquid to a gel-like state. This physical change was found to disrupt loop formation and block polymerase recruitment, leading to developmental disorders.

Significance and Prospects

This study clearly elucidates the mechanism by which enhancer-promoter interactions lead to transcription, a central question in molecular biology. It was confirmed that chromatin loop formation and Pol II recruitment are closely linked through hnRNPK and phase separation. This demonstrates that RNA plays a structural role in determining three-dimensional genome architecture and regulating expression, beyond merely acting as an information carrier.

The study also provides new directions for drug development by proving that changes in the state of intracellular aggregates can directly cause disease. Approaches to prevent or revert the gelation of aggregates caused by mutations, such as in Au-Kline syndrome, back to a normal liquid state are now feasible.

However, challenges remain in linking phase separation control technology to pharmaceutical development. Precision is required to selectively modulate specific aggregates among various intracellular protein aggregates at target genes. Ensuring a drug delivery system that avoids systemic transcriptional suppression or side effects is also a challenge.