Defining mechanisms of enhancer-promoter interaction in ZGA with chromatin expansion microscopy

The National Institute of Child Health and Human Development awarded Yale University $100,228 on December 1, 2026, under the Child Health and Human Development Extramural Research program (CFDA 93.865) to define mechanisms of enhancer-promoter interaction in zygotic genome activation using chromatin expansion microscopy.

The project combines chromatin expansion microscopy (ChromEXM) with DNA fluorescence in situ hybridization (FISH) to trace individual gene loci at near-nucleosome resolution in intact zebrafish embryos, with multimodal labeling of proteins, RNA, and histone modifications using antibody labeling, 5-ethynyl-uridine click labeling, and modification-specific intracellular antibodies (MintBodies). The research addresses two primary questions: whether spatial organization of enhancers and promoters reflects RNA polymerase II activity state, and what physical and molecular mechanisms coordinate acetylation at enhancers and promoters with transcriptional activation. The work investigates how dysregulated enhancer-promoter interactions contribute to developmental arrest or disease progression during embryonic development.

Performance is located in New Haven, Connecticut, with a period of performance extending from December 1, 2026, through August 31, 2029. This is a project grant, a standard assistance type under the NICHD extramural research portfolio.

Abstract

Upon fertilization, embryos undergo a dramatic remodeling of chromatin to prepare for zygotic genome activation (ZGA). ZGA requires interactions between enhancers (EN) and promoters (PR) that, when dysregulated, cause developmental arrest or disease progression; yet the physical and molecular mechanisms by which these elements activate transcription in vivo are not fully understood. While recent advances in 3D genomics and live imaging have allowed the capture of EN-PR loops, different studies employing these approaches have found an inconsistent correlation between their proximity and duration and their associated transcription. These findings may be reconciled by a model where transcriptional activation is dynamically related to both contact and the acetylation states of enhancers and promoters. However, understanding how enhancers and promoters converge with epigenetic marks and the transcriptional machinery at individual gene loci requires advanced spatial genomics tools. This study will use a novel approach combining chromatin expansion microscopy (chromexm) with DNA fluorescence in situ hybridization (fish) to trace individual gene loci at near-nucleosome resolution in the nuclei of intact zebrafish embryos, together with multimodal labeling of proteins, RNA and histone modifications by antibody labeling, 5-ethynyl-uridine (EU) click labeling and modification-specific intracellular antibodies (mintbodies). The goals of this project are to ask 1) whether the spatial organization of enhancers and promoters reflects the activity state of RNA polymerase II (POL II), and 2) what physical and molecular mechanisms coordinate acetylation at enhancers and promoters with transcriptional activation. Addressing these two goals, aim 1 describes DNA fish labeling of enhancers and promoters in chromexm in order to relate EN-PR distance with the enrichment and spatial distribution of POL II across paused hubs and active strings. Aim 2 proposes mintbody labeling of the activating mark H3K27AC to compare promoter and enhancer acetylation states during transcription initiation and elongation, and to characterize the enrichment and spatial organization of transcription factors, histone writers and readers, chromatin remodelers and mediator proteins around them. Together, both aims will leverage a novel approach to resolve the 3D structure of EN-PR loops during a key window of transcriptional activation in development, revealing the mechanistic relationships between EN-PR contact, histone acetylation and transcription, and potentially challenging the textbook understanding of transcriptional regulation. The newly expanded chromexm toolkit provides a broadly applicable platform for mapping genome interactions with nanometer precision, not only during ZGA but also in the contexts of cellular differentiation, adult homeostasis and disease.