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Identification and functional characterization of mammalian enhancers and transcriptional co-factors during cellular signaling and cell fate transitions

Periodic Reporting for period 3 - Enhancer ID (Identification and functional characterization of mammalian enhancers and transcriptional co-factors during cellular signaling and cell fate transitions)

Reporting period: 2018-09-01 to 2020-02-29

A major goal in biology is to understand how gene regulatory information is encoded by the human genome and how it defines different gene expression programs and cell types. This is an important goal for both basic science and medical research, as mis-regulation of genes underlies many diseases such as cancer. Enhancers are genomic elements that control transcription, yet despite their importance, only a minority of enhancers are known and functionally characterized. In particular, their activity changes during cellular signalling or cell type transitions are largely elusive. Furthermore, fundamental questions about transcriptional co-factors have remained unanswered even though they regulate enhancer activities and have become attractive therapeutic targets, e.g. for cancer treatment.

Our project 'Enhancer ID' undertakes a functional genomics approach in mammalian cells with three specific objectives: First, we aim to identify and functionally characterize transcriptional enhancers in selected human and mouse cells using the recently developed quantitative enhancer activity assay STARR-seq. Second, we will determine enhancer activity changes quantitatively in the presence and absence of cellular signaling cues. Third, we will systematically dissect the functional relationship of enhancers and transcriptional co-factors using small molecule inhibitors and recently developed CRISPR/Cas9-based technologies.

This proposal addresses fundamental questions in enhancer biology and complement the genome-wide profiling of gene expression and chromatin states (e.g. by ENCODE). We will gain insights into the genomic organization of active enhancers and reveal chromatin or sequence features associated with dynamic activity changes. I also expect that we will be able to define co-factor requirements for enhancer function and reveal if different types of enhancers exist, an important advance towards the understanding of gene regulation in mammals.
In the first funding period, we have optimized the STARR-seq enhancer screening methodology for genome-wide application in human cells. We have further performed pilot screens in both fly and human cell lines to assess the cells’ suitability and optimal screening conditions for different signaling conditions. Finally, we have implemented technologies to tag and rapidly deplete cofactor proteins and performed pilot screens under conditions in which cofactors are inhibited or depleted.

Our main results from this period are a new and improved STARR-seq protocol and optimized parameters and protocols for screening under signaling conditions. The pilot experiments using cofactor inhibition further revealed the existence of enhancers with different cofactor dependencies, which we will now follow up.
The new and improved STARR-seq protocol makes this high-throughput enhancer activity assay applicable to entire mammalian genomes in mammalian cell lines and overcomes several problems that exist for widely used enhancer-activity assays. Applied systematically, it should help identify human enhancers of different strengths - with potential implications for biotechnological transgene-expression - as well as enhancers that are important during development and/or associated with different diseases, including cancer.

The systematic characterization of the cofactor-requirements of different enhancers should not only be important for our basic understanding of transcriptional regulation but might also inform novel therapeutic strategies that are based on the inhibition of different transcriptional cofactors.