Programmable Readers, Writers, and Erasers of the Epigenetic Cytosine Code

Project Information

EPICODE

Grant agreement ID: 723863

DOI

10.3030/723863

Project closed

EC signature date 22 April 2017

Start date 1 November 2017

End date 30 April 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 979 679,00

EU contribution

€ 1 979 679,00

Coordinated by

TECHNISCHE UNIVERSITAT DORTMUND
Germany

Periodic Reporting for period 4 - EPICODE (Programmable Readers, Writers, and Erasers of the Epigenetic Cytosine Code)

Reporting period: 2022-05-01 to 2023-04-30

Summary of the context and overall objectives of the project

Human DNA contains two types of biologically instructive information: the canonical nucleobases A, G, T, C, and the epigenetic nucleobases mC, hmC, fC, and caC. Canonical nucleobases encode the molecular identity (the sequence) of all RNAs and proteins that are synthesized by a cell, whereas epigenetic nucleobases dynamically regulate this synthesis.
This regulation shapes the phenotype of cells during differentiation and development, and its perturbation is a key trigger of cancer.
Canonical nucleobases can be decoded in a programmable manner by nucleic acids and their analogs via Watson-Crick-base pairing, and the simplicity of this recognition has enabled revolutionary developments in the biological sciences.
In contrast, molecular scaffolds with programmable recognition of epigenetic nucleobases do not exist, which represents a roadblock for developments in epigenetics basic research and cancer diagnostics.
Likewise, the reversible writing, reading and erasing of epigenetic nucleobases (DNA modifications) during cell differentiation, embryonic development and cancer development is highly dynamic, but studying this dynamics is limited by the lack of methodology to write, read, and erase the nucleobases with high spatiotemporal resolution.

Cancer represents a leading cause of death in the human population accounting for ~10 Mio. deaths per year (2020), and the increasing life-expectation further intensifies this problem. Epigenetic nucleobases play key roles in cancer development and serve as important biomarkers for cancer research and diagnostics.
A striking feature of aberrant, cancer-causing nucelobase patterns are their early occurrence during malignant transformation, making them highly valuable biomarkers for early-stage detection. Early detection in turn drastically increases the treatment success rate, and has been identified by the WHO as key factor for reducing cancer deaths. Novel approaches that reveal the actual chemical signals at play thus bear a high potential for biomarker discovery and the development of diagnostics assays. EPICODE dealed with the design and application of novel designer proteins that address fundamental problems in the field of DNA recognition.
These represent basic research tools with potential to be applied in different study settings, including in vitro DNA analytics/Diagnostics, imaging approaches and functional studies.

Specifically, we developed probes that can be programmed to recognize epigenetic DNA nucleobases in user-defined DNA sequences, as well as recognize specific combinations of two epigenetic DNA nucleobases in the two strands of DNA duplexes. Such molecules can serve as versatile probes in vitro and in vivo, i.e. to enrich DNA molecules from complex, genomic DNA backgrounds for targeted and genome-wide analyses, and for direct cellular imaging. We further designed optochemical tools that allow to precisely control the writer and eraser enzymes as well as the reader proteins of epigenetic DNA nucleobases with spatiotemporal resolution by light directly in cells. This allowed us to precisely study the kinetics of nucleobase editing and the triggered downstream chromatin changes in states associated to normal and malignant physiology, and to edit the local epigenetic nucleobase landscape of user-defined genomic loci.
Given the central role of epigenetic nucleobases in cancer and the universality of this approach, EPICODE thus provided enabling and broadly applicable methodology for cancer epigenetics research and diagnosis.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We designed and characterized novel TALE modules and MBD proteins, and developed methodology to apply them for epigenetic nucleobase analyses via affinity enrichment and cellular imaging. We have entered collaborations for mapping studies with the goal to use our new protein probes for cancer diagnostics (the ERC PoC grant COMBICODE has been granted for developments towards commercialization). We filed a patent covering our new MBD mutants and applications. We also started efforts to go beyond our currently employed proteins scaffolds by introducing methods that allow for the identification of novel natural protein-based reader and/or antireader scaffolds of cytosine modifications based on 1.) the enrichment of specific chromatin segments, 2.) protocols for the directed evolution of cDNA libraries (based on a bacterial surface selection protocols established during EPICODE), as well as 3.) pulldown/proteomics assays. Such scaffolds may serve as completely new starting points for probe design.
EPICODE further lead to translational activities, including the ERC PoC-funded project COMBICODE involving an undisclosed European diagnostics company and a collaboration with an undisclosed US-based diagnostics start-up company.

In the second main area of research, we designed optochemical tools that allow to precisely control the writing, reading, and erasing of epigenetic DNA nucleobases with spatiotemporal resolution by light pulses directly in cells.
This allows to precisely study the kinetics of nucleobase editing and of the triggered downstream chromatin changes in normal and malignant cellular states, and to edit the local epigenetic nucleobase landscape of user-defined genomic loci.
Specifically, using a universal strategy for the creation of light-activatable proteins by genetic code expansion, we designed the first eraser enzymes of mammalian DNA methylation that allow for the editing of methylation with spatiotemporal resolution directly in cells. We also developed the first light-activatable writer enzymes – DNA-methyltransferases (DNMT) – and employed them to study the effects of cancer-related DNMT mutations by in vivo kinetic studies, to edit a particular DNA element (SATIII) in a local and temporally controlled fashion, and to study system-wide changes in RNA expression levels. We further developed light-activatable MBD reader proteins to study their recruitment kinetics to pericentromer DNA in dependence on additional domain functions. We already used these tools for biological studies and set out to study the interplay of TET1 and MBD proteins on the kinetic level using light-activated TETs, and discovered that human MBD1 influences the kinetics of human TET1 CD in a dual way, e.g. via an inhibition involving the MBD domain and an activation via the CXXC3 domain.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Since our protein design achievements for the first time address fundamental problems in the field of DNA recognition (Recognition of mammalian DNA modifications in a programmable fashion and in a combined, Duplex-specific way), they represent basic research tools with potential to be applied in many different settings of biological studies, including in vitro DNA analytics/Diagnostics, imaging approaches and functional studies. They thus go significantly beyond the state of the art.

On the other hand, light offers an extremely high spatiotemporal control of biological processes and thus offers a unique and noninvasive way to study the dynamics of chromatin processes, something that is particularly difficult to access. Our light-activated tools offer several of such studies for the first time and provide a particularly tight and selective control of the processes under study, since our genetic code expansion approach offers to directly control the catalytic center of the writer/eraser enzymes, rather than allowing a light-controlled recruitment or mere small molecule inhibition.

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