Evaluation of Novel DNA Adenine Methyltransferase Fusion Construct Designs for Enhancer Mapping in Human Cells

Abstract

Enhancers are central regulators of cell-type-specific gene expression, yet direct functional mapping of active enhancer-promoter interactions in single cells remains technically challenging. DNA adenine methyltransferase (Dam)-based chromatin recording offers a strategy to mark genomic regions proximal to proteins associated with active transcription sites in vivo, including enhancer regions, and such adaption requires stringent control of enzymatic Dam-activity to avoid background methylation. In this study, I evaluated whether controlled expression of Dam fused to components of nascent transcript machinery – NCBP2 (cap-binding complex) and Ramac (RNA m7G-methyltransferase-activating subunit) – could enable recording of regulatory chromatin activity associated with active enhancers. Six Dam fusion contracts representing three plasmid architecture strategies – constitutive expression, transcriptional inducibility, and dually combined transcriptional and post-translational regulation – were screened in HEK293T cells across five independent transfection experiments. Dam-mediated methylation was detected for chosen fusion orientation, demonstrating preserved enzymatic activity and fusion compatibility with NCBP2/Ramac. However, basal Dam activity was sufficient to generate substantial background methylation across conditions. Neither transcriptional induction nor dual regulation produced decisive on/off methylation pattern, and endpoint PCR readouts equalized differences in methylation input, limiting quantitative interpretation. These findings establish that Dam fusions to NCBP2/Ramac are functionally viable but highlight critical constraint imposed by Dam processivity and expression leakiness. Achieving tighter temporal control of Dam activity and implementing alternative quantitative readouts will be essential before this approach can be applied to enhancer mapping with single-cell resolution.

Popular Abstract

Developing a method to record gene regulators in living human cells

How does a heart cell know to beat, while a skin cell forms a protective barrier – even though both contain exactly the same DNA? The secret is gene regulation – the molecular switches that control gene activity. This project explores a new strategy to record those switches directly inside living human cells.

All cells share the same genetic blueprint, but they use different parts of it. Small DNA regions called enhancers act like remote controls: they boost expression of specific genes at the right time and in the right cell type. For example, during brain development, certain enhancers activate genes that are needed for neuron formation, while others remain silent. When enhancers are disrupted – by mutations or misregulation – they can contribute to diseases such as cancer or neurodevelopmental disorders.

The challenge is that enhancers are difficult to detect while they are actively working, especially in individual cells.

To address this challenge, the project investigated whether a bacterial protein, DNA adenine methyltransferase (Dam), could serve as a molecular “recorder”. Dam leaves small chemical marks on DNA. The idea was to genetically fuse, i.e. attach, Dam to proteins that are involved in actively expressed DNA regions. If successful, Dam would piggyback its way to active enhancer sites and leave behind traceable marks. Later, by analyzing these marks, a reconstruction of which regions had been active would be possible.
In this project, six different genetic constructs with Dam fused to proteins were designed and tested in human cells. Different fusion orders (“Dam-Protein” or “Protein-Dam”) were tested, as well as different proteins (“Dam-Protein1” or “Dam-Protein2”). All Dam-protein fusions worked – they were capable of marking DNA! However, they also marked many regions that were not actively involved in gene regulations. Even small amounts of uncontrolled Dam-marking activity created background “noise”, making it difficult to separate true signal from accidental marking.

To conclude, the key insight from this work is that while Dam-based recording of active regulatory regions is technically feasible, achieving precise control of Dam-activity is critical. If refined, this approach could help scientists map gene regulation in individual cells – improving our understanding of development, tissue specialization and disease at a very detailed level.

• author: Jirström, Lykke
• supervisor: Christina Sakellariou; Robert Krautz
• organization: Department of Immunotechnology
• course: KIMM05 20252
• year: 2026
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• language: English
• id: 9223438
• date added to LUP: 2026-03-02 17:37:06
• date last changed: 2026-03-02 17:37:06