Nucleosome Fibre Topology Guides Transcription Factor Binding to Enhancers

Academic Background

The establishment of cellular identity relies on the concerted action of multiple transcription factors (TFs) bound to enhancers of cell-type-specific genes. Although TFs recognize specific DNA motifs within accessible chromatin, this information alone is insufficient to explain how TFs select enhancers. This study compares four different TF combinations, analyzing their genome occupancy, chromatin accessibility, nucleosome positioning, and 3D genome organization at nucleosome resolution, revealing how nucleosome fibre topology guides TF binding to enhancers.

Source of the Paper

This paper was co-authored by Michael R. O’Dwyer, Meir Azagury, Katharine Furlong, and others, from institutions including the University of Edinburgh, The Hebrew University-Hadassah Medical School, and Albert Einstein College of Medicine. The paper was published online on November 1, 2024, in the journal Nature.

Research Process

1. Overexpression of TF Combinations and Cell Fate Transformation

The study began by overexpressing four different TF combinations in mouse embryonic fibroblasts (MEFs), inducing four distinct cell fates. These combinations included: - OSKM: Induced pluripotent stem cells (iPS cells) - GETM: Induced trophoblast stem cells (iTS cells) - GETMR: Induced either iPS cells or iTS cells depending on culture conditions - BS9G4M: Did not induce cell reprogramming

2. Mapping TF Genome Occupancy

Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), the researchers mapped the genome occupancy of all TFs 48 hours after induction and after the completion of reprogramming. The results showed limited overlap between early reprogramming binding sites and fully reprogrammed cells, indicating off-target binding in the early stages.

3. Measuring Chromatin Accessibility and Nucleosome Positioning

Using ATAC-seq and MNase-seq, the researchers measured chromatin accessibility and nucleosome positioning in MEFs, iPS cells, and iTS cells. The results revealed that most TFs primarily targeted closed chromatin regions during early reprogramming, while in fully reprogrammed cells, they relocated to cell-type-specific cis-regulatory elements in open chromatin.

4. Topological Analysis of Nucleosome Arrays

Using Micro-C technology, the researchers mapped the 3D chromatin architecture at single-nucleosome resolution. The results showed that TFs bound to nucleosome arrays with specific 3D organization during early reprogramming, guiding their binding to cell-type-specific enhancers.

Key Findings

1. Cooperative Binding of TFs

The study found that cooperative binding of TFs on nucleosome arrays depends on specific motif arrangements. For example, the OSK (Oct4, Sox2, Klf4) combination displayed orientation-specific motif distributions on nucleosome arrays, while the GET (Gata3, Eomes, Tfap2c) combination showed specific motif arrangements at the borders of nucleosome arrays.

2. Nucleosome Fibre Topology Guides TF Binding

The study proposed a “guided search” model, suggesting that TFs recognize motif patterns on nucleosome fibres, guiding their binding to enhancers. The topology of nucleosome fibres acts as “signpost elements,” reducing the dimensionality of the genome that TFs need to explore.

3. Formation and Dissolution of Chromatin Loops

Using Micro-C, the researchers found that TFs initially bind to the borders of chromatin loops during early reprogramming. As reprogramming progresses, these loops dissolve, and TFs relocate to enhancer regions, accompanied by changes in chromatin accessibility and histone modifications.

Conclusion

This study reveals the critical role of nucleosome fibre topology in guiding TF binding to enhancers. By recognizing specific motif patterns on nucleosome arrays, TFs are directed to cell-type-specific enhancers. This discovery not only deepens our understanding of cellular reprogramming mechanisms but also provides new insights for developing more efficient cell reprogramming strategies.

Research Highlights

1. Nucleosome Fibre Topology Guides TF Binding: The study is the first to reveal the critical role of nucleosome fibre topology in guiding TF binding to enhancers.
2. Guided Search Model: A “guided search” model is proposed, suggesting that TFs recognize motif patterns on nucleosome fibres to guide their binding to enhancers.
3. Formation and Dissolution of Chromatin Loops: Using Micro-C, the study uncovers the process of TF binding to chromatin loops and their subsequent dissolution, providing new perspectives on the role of 3D chromatin organization in gene regulation.

Research Significance

This study holds significant scientific value and provides new theoretical foundations for the fields of cellular reprogramming and regenerative medicine. By revealing the role of nucleosome fibre topology in guiding TF binding to enhancers, future strategies for more efficient cell reprogramming can be designed, advancing the field of regenerative medicine.