Simply Put
The development and normal aging of every cell in the body is guided by a precise “choreography” of gene expression, dictated by regulatory elements in our DNA known as enhancers. These enhancers have traditionally been viewed as independent switches that are turned on by binding to sequence-specific proteins known as transcription factors. However, we recently uncovered an unprecedented interaction between different enhancers, in which the activity at one enhancer directly controls the physical ability of a separate enhancer on the same chromosome to bind its specific transcription factors. Such inter-enhancer communication increases the complexity and flexibility in the logic of transcriptional circuitry controlling development and aging, but the underlying mechanisms are currently unknown.
This project investigates the fundamental rules of this inter-enhancer communication. By using a specific gene system – the IRF8 superenhancer locus – as a high-resolution model, we are mapping how these sequential “handoffs” establish cell identity of the type 1 conventional dendritic cell. We believe this represents a universal principle of genomic organization. Understanding how this coordination is maintained, and how it might break down, is critical for deciphering the predictable decline of cell function during aging. Our goal is to move beyond viewing DNA as a static blueprint and instead reveal the dynamic, interdependent network that governs life across the human lifespan.
Description
Lineage specification is often governed by the sequential activation of stage-specific enhancers. In our model, three enhancers located at +56kb, +41kb, and +32kb downstream from the Irf8 promoter are activated by distinct transcription factors: C/EBPa initiates expression in early progenitors, followed by E proteins in the common dendritic cell progenitor (CDP), and finally BATF3, which maintains expression throughout the cDC1’s lifespan.
Our recent findings revealed a previously unappreciated cis-dependent interaction between these elements. In compound genetic models, we found that the deletion of an earlier enhancer renders a subsequent, physically intact enhancer to be functionally inaccessible on the same chromosome, unable to bind its transcription factors, causing a failure in cDC1 development. This occurs even when the necessary transcription factors are present at normal levels, suggesting that the activity of a prior enhancer is a mechanistic requirement for the chromatin opening of subsequent regulatory sites.
The project will employ a multi-layered approach to dissect this mechanism:
Single-Cell Multiomics: We will utilize paired scRNA-seq and scATAC-seq to map the chromatin and transcriptional landscapes of bone marrow progenitors across multiple genetic variants.
eRNA Characterization: We will develop targeted RNA-capture sequencing to map low-abundance enhancer RNAs (eRNAs) at high resolution to test their role in inter-enhancer communication.
Functional Perturbation: Using CRISPR interference (CRISPRi), we will selectively block eRNA transcription to determine if the process of transcription itself is necessary for the recruitment of lineage-determining factors.
3D Chromatin Architecture: We will apply Hi-ChIP and modified Hi-C techniques to visualize the physical looping interactions between enhancers and the promoter as they evolve during development.
By integrating these data, we will generate a 4D model of gene regulation. This work reframes our understanding of distal regulatory elements not as independent switches, but as a coordinated, interdependent network. This discovery has broad implications for understanding how subtle epigenetic shifts can lead to the loss of cell populations, providing a roadmap for future interventions in aging and regenerative medicine.
Year 1 Objectives: Mapping the Epigenetic Landscape and Enhancer RNA Dynamics.
The first year of the project establishes a high-resolution molecular atlas of the regulatory transitions at the Irf8 locus. This foundational work is essential for moving from observation to functional interrogation of the sequential “handoff” mechanism.
Single-Cell Multiomic Profiling of Progenitors: We will utilize paired scRNA-seq and scATAC-seq to simultaneously measure chromatin accessibility and gene expression within precise bone marrow progenitor populations, including MDPs, CDPs, and pre-cDCs.
Genetic Variant Comparison: This analysis will compare wild-type controls with a suite of genetic variants, including individual enhancer mutants D32, D41, and D56 and F1 intercrosses (D32/D41, D41/D56, and D32/D56) to observe how specific deletions disrupt the developmental trajectory.
eRNA Targeted Capture & Mapping: We will initiate the development of a highly sensitive RNA-capture sequencing method using biotinylated probes to detect and map low-abundance, potentially non-polyadenylated enhancer RNAs (eRNAs) at the +56kb, +41kb, and +32kb sites.