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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 4 - Enhancer ID (Identification and functional characterization of mammalian enhancers and transcriptional co-factors during cellular signaling and cell fate transitions)

Reporting period: 2020-03-01 to 2022-01-31

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 activities, activity changes and the underlying DNA sequence features have largely remained elusive. Furthermore, fundamental questions about transcriptional cofactors have remained unanswered even though they regulate enhancer activities and have become attractive therapeutic targets, e.g. for cancer treatment.

Our project 'Enhancer ID' undertook a functional genomics approach to identify transcriptional enhancers using the recently developed quantitative enhancer activity assay STARR-seq. We adapted STARR-seq to mammalian cells, identified enhancers and measured their activities and activity changes. We also determined chromatin and sequence properties of the identified enhancers, particularly the DNA sequence motifs that underly enhancer activity and strength. Finally, we systematically dissected the functional relationship between enhancers and transcriptional cofactors using rapid cofactor depletion with the recently developed auxin-inducible-degron (AID) technology.

This proposal addressed fundamental questions in enhancer biology and complemented the genome-wide profiling of gene expression and chromatin states (e.g. by ENCODE). We gained insights into the genomic organization of enhancers and revealed their chromatin and sequence features. Finally, we defined cofactor requirements for enhancer function and revealed that different types of enhancers exist that differentially depend on different cofactors, an important advance towards the understanding of enhancer biology and gene regulation.
We have optimized the STARR-seq enhancer screening methodology for genome-wide application in human cells (Muerdter & Boryn et al., 2018). We have further optimized the quantitativeness of the method and enabled the use of defined candidate fragments (Neumayr et al., 2019). We have performed screens in the presence and absence of innate immune (cGAS/STING & interferon) signaling and thereby created the first genome-wide set of interferon-responsive enhancers in mammalian cells (Muerdter & Boryn et al., 2018). Moreover, we dissected the DNA sequences of fly and human enhancers and modeled enhancer activity based on DNA sequences and motif content, revealing important conceptual insights into how enhancer activities are encoded by the presence of certain transcription factor motif types and the number and relative arrangement of the corresponding motif instances (Almeida et al., Nature Genetics, in press).

We have implemented technologies to tag and rapidly deplete cofactor proteins and performed genome-wide enhancer-activity screens using STARR-seq under conditions in which cofactors are present or depleted. This revealed the first genome-wide map of enhancers and their functional dependencies on different cofactor proteins. Importantly, our results show that different enhancers depend on different cofactors and these distinct dependencies can be used to define enhancer types. These different enhancer types differ in other properties, including chromatin features and transcription factor motif content and factor binding. In particular, we found that P53-target enhancers are insensitive to Mediator depletion and Brd4-independent enhancers are specifically compatible with TATA-box promoters (Neumayr & Haberle et al., in revision).
The project has moved beyond the state of the art in several important ways: we developed a new and improved STARR-seq protocol that enables quantitative enhancer activity screens across entire mammalian genomes or with defined candidate fragments. This protocol identifies and overcomes several problems that exist for widely used enhancer-activity assays. The project furthermore achieved an unprecedented understanding of the DNA sequence basis for enhancer activities in flies and human cells, including the highly accurate prediction of enhancers, transcription-factor-motif importance and the de novo design of enhancers. Finally, the project is the first to systematically map cofactor-requirements of all enhancers in a human model cell line and to define distinct enhancer types that differ by their cofactor dependencies, including enhancers independent of Brd4 or insensitive to Mediator depletion. The project therefore moved beyond the state of the art for both, our basic understanding of transcriptional regulation but by informing novel therapeutic strategies that are based on the inhibition of different transcriptional cofactors.
Graphical Abstract Enhancer-COF Dependencies