NIH grant: Reconstituting gene regulatory landscapes to identify mechanisms of transcriptional control and dysregulation (1R35GM165813-01)

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NIH grant 1R35GM165813-01: Reconstituting gene regulatory landscapes to identify mechanisms of transcriptional control and dysregulation
Institute: {'code': 'GM', 'abbreviation': 'NIGMS', 'name': 'National Institute of General Medical Sciences', 'admin_org_id': '12500024', 'admin_funding_url': ''}. PI: Judith  Kribelbauer-Swietek. Award: $426145.
Start: 2026-08-01T00:00:00.
PROJECT SUMMARY:
Mammalian gene expression is governed by a ‘separation of function’ principle, by which gene regulatory activity
is split between promoters (P) and enhancers (E). Conceptually, enhancers work by recruiting a combination of
transcription factors (TFs), which bind DNA in a sequence-specific manner. Once bound, TFs interact with
coregulators to induce epigenetic changes. While many examples indicate a sequence-dependent E-P
compatibility code, little is known about the exact molecular interactions that mediate communication. Shedding
light on this process is critical to advance our understanding of how enhancer-centric mutations fuel
transcriptional dysregulation and disease – from cancer to rare genetic disorders.
The goal of this research program is to investigate the molecular mechanisms that control mammalian E-P
compatibility. Our focus is on the development of scalable experimental tools that can break down regulatory
complexity to a human-interpretable level. Such insight is needed to design therapies that directly target
‘transcriptional dysregulation’. We and others have shown that TFs cooperate to boost enhancer activity and
communication. However, all these observations are based on average effects across unique genomic contexts,
precluding insight into the contributions of individual TFs and TF pairs. Our first goal is therefore to develop a
reduced-complexity, genome-integrated reporter assay that can quantify how distinct TF binding site
combinations, their affinities and copy number, modulate enhancer activity in different cellular contexts. This work
will provide a catalogue of TF combinatorial function, which will aid the interpretation and identification of disease-
linked non-coding mutations. Our second goal is to identify how a TF combinatorial code establishes E-P and E-
E communication. Specifically, we will probe how enhancer sequence alters epigenetic landscapes and how it is
interpreted by different promoter architectures. We will optimize the EXTRA-seq tool I previously developed to
reconstitute thousands of kilobase-scale E-P and E-E-P landscapes in a single experiment. We will track the
transcriptional and epigenetic output for each de novo created genotype in different cellular contexts to identify
the minimally required set of TF and promoter motifs that mediate E-P and E-E communication. While perturbing
DNA is an important first step to decode regulatory grammar, it only provides correlative insight into the protein
interactions that mediate E-P communication. Our third goal is therefore to directly map the protein domains
involved in this process.
This research will shed light on the molecular layers involved in translating enhancer sequence to transcriptional
output, insight we lack, and which is necessary to advance personalized therapies whose aim it is to disrupt
transcriptional dysregulation at the source – perturbing regulatory interactions that drive disease states without
affecting ‘normal’ gene regulation.

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