Dissecting functional master-enhancer regimes driving inflammatory stimulation

• Applicants: Professor Dr. Michael Kracht; Professor Dr. Argyris Papantonis
• Subject Area: Pharmacology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 554390251

Inflammation is an evolutionarily conserved process in which inflammatory cellular responses are coordinated and regulated at multiple checkpoints in the body. Failure of these mechanisms leads to excessive acute or chronic inflammation, which is causally associated with organ damage, tissue aging and cancer. Therapeutic successes over the last twenty years highlight the (clinical) importance of pharmacological antagonization of cytokines such as IL-1α/β and TNFα, however inflammatory diseases remain incurable. In this project, molecular mechanisms of cytokine-dependent three-dimensional genome organization and function are being analyzed with the goal of capturing and modulating the nuclear coordination of inflammatory gene expression cascades with the greatest possible precision. In a collaborative effort, the applicants were able to decipher a new level of regulation of the nuclear IL-1α response, whereby IL-1α induces acute changes in chromatin accessibility in regions enriched in single nucleotide polymorphisms (SNPs) relevant to inflammatory diseases. Using CRISPR-Cas9-based microdeletions, they identified two IL-1α-regulated enhancers in the chemokine locus on Chr. 4 and a dominant role of the IL8 "master enhancer" in the regulation of further inflammatory genes.The proposed project will test the hypothesis that the highly coordinated cellular inflammatory response requires specialized genomic "hubs" in which multiple enhancers interact and regulate more than one gene with different strengths. To this end, further inflammatory enhancers of this type and factors that enable these enhancers to achieve a specific cytokine-responsive functional state in the human genome will be characterized as comprehensively as possible. In Work Package (WP) 1, the applicants will comprehensively identify the genome-wide repertoire of dynamic inflammatory enhancers using a combination of micro-C, combined single cell ATAC-seq and RNA-seq, (nascent) RNA-seq, and novel bioinformatics tools and map them based on enhancer strength. In WP2, the molecular composition of enhancer hubs will be revealed by isolating locus-specific protein complexes in the context of transcription factories using proteomic approaches. In WP3, the function of newly discovered "master" enhancers will be elucidated using inactive Cas9 and CRISPR-directed interference screens, molecular imaging of transcription sites, and native 4C assays of perturbed genomic loci and nuclear factors. If successful, the planned approaches will generate new insights into the function of hierarchically organized enhancers in their endogenous context and into enhancer selection and choice in cytokine-responsive systems, bringing us closer to the goal of manipulating genomic enhancers in (chronic) inflammatory diseases.

• DFG Programme: Research Grants