IMAGING AND SEQUENCING PROTEIN-DNA INTERACTIONS IN SINGLE CELLS USING INTEGRATED MICROFLUIDICS

IMAGING AND SEQUENCING PROTEIN-DNA INTERACTIONS IN SINGLE CELLS USING INTEGRATED MICROFLUIDICS

Researchers at UC Berkeley have developed an integrated microfluidics method, µDamID (microfluidic DamID), capable of co-determining the cellular location and nucleotide sequence of DNA that is contacted by a protein of interest in a single cell.

Genome regulation depends on carefully programmed protein-DNA interactions that maintain or alter gene expression states, often by influencing chromatin organization. Most studies of these interactions have relied on bulk immunoprecipitation methods, which can overlook important biological heterogeneity, especially in single cells. Furthermore, these bulk methods are difficult to pair with imaging data, which can reveal spatial information of protein-DNA interactions within the nuclei of living single cells.

Stage of Research

The inventors have developed an integrated microfluidic platform that enables paired single-cell imaging and sequencing data of protein-DNA interactions. The authors make use of DNA adenine methyltransferase identification (DamID), a technique to measure a protein’s DNA-binding history by assessing methylated adenine bases near each protein-DNA interaction site. When genetically fused to a protein of interest, Dam enzyme deposits methyl groups near the protein-DNA contacts at the N6 positions of adenine bases (^m6A). DamID reads out these chemical recordings of protein-DNA interactions by specifically amplifying and then sequencing fragments of DNA containing the interaction site. Further, these ^m6A methylation marks can be visualized in cells using additional fluorescent proteins that bind to methyladenines, such as ^m6A-Tracer, before cells are selected and prepared for sequencing. The authors validate the utility of µDamID by mapping the sequence and spatial location of nuclear lamina interactions in single cells.

Applications

Paired imaging and sequencing data in single cells for joint analysis of the nuclear localization, sequence identity, and variability of protein-DNA interactions.
Identification of the in vivo target loci of chromatin proteins in a single cell, or in populations of living cells

Advantages

Integrated microfluidics platform allows single cell capture and high-magnification imaging on inverted microscopes in serial, before cell lysis and processing performed “on-chip” in parallel
µDamID can be used to assess direct and indirect protein-DNA interactions, as well as transient DNA association, up to the length of a cell cycle
Reduced input requirement compared to immunoaffinity purification methods, such as ChIP-seq

Stage of Development

Research – in vitro

Publications

Altemose N, Maslan A, Lai A, White JA, Streets A. “μDamID: a microfluidic approach for imaging and sequencing protein-DNA interactions in single cells” Cell Systems 11(4) Oct 2020. DOI: 10.1016/j.cels.2020.08.015