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Single molecule microscopy

Our lab specializes in using a novel single molecule approach to investigate how interactions between proteins and nucleic acids give rise to biological outcomes. We employ a combination of materials engineering and biochemistry to isolate and arrange individual strands of DNA or chromatin fibers. We then use fluorescence microscopy to directly observe how proteins interrogate and process this genetic material.

Chromatin

Barriers

DNA

Pedestals

Lipid bilayer

Chromatin Curtains

H2B Atto488

DNA Curtains

DNA-YOYO1

Cas9-3XFlag

Questions

What are the biological consequences and requirements of genome organization?

We study the fundamental physics of how DNA is packaged into nuclei. Our goal is to understand the role of genome organization in cellular decision making. One of the few broadly applicable rules of genome organization is that inactive genes and repetitive DNA elements are often collected in separate nuclear compartments from those that contain active genes. We are engaged in determining the driving forces behind establishing these domains and their physical characteristics. We use our single molecule assay to probe how the actions of individuals give rise to the emergent phenomena of large networks of biomolecules observed in the cell. An example of this approach can be seen below where Heterochromatin protein 1 (HP1) rapidly assembles on DNA and compacts it into a focused volume. In the cell, HP1 is involved in sequestering heterochromatic sequences into compact structures. Our experiments isolate the early steps of this process, when the HP1-mediated heterochromatin domains are just forming, providing a unique lens into the dynamics of genome packaging.

Watch the video!

How influential are dynamics on epigenetic states?

All DNA is not treated equally in the cell. Some sequences are transcribed into RNA, while others are not; some sequences act as physical landmarks, while others have no described function. In order to specify the locations at which certain activities will occur, cells add small chemical modifications to their chromosomes. Some of these modifications are to the DNA itself; others are to nucleosomal proteins which nearly coat the genome from end to end. These modifications and the order in which they appear are critical informational layers that help the cell respond to developmental and external cues.

methylated DNA

MBD1-Atto488

unmethylated DNA

MBD1-Atto488

We want to know the rules by which the cell establishes, maintains, and interprets these additional inputs and the consequences of that framework on cellular function. To tackle these questions, our lab is developing methods to visualize the proteins which deposit and edit epigenetic modifications,

as well as strategies to visualize the chemical modifications themselves. One of these modifications, cytosine methylation, is written to the DNA at millions of sites in the genome. At a subset of those sites, an eraser protein removes these marks to varying degrees. As seen above, we can detect the presence or absence of methylated cytosines with high precision. From data like these, we can develop maps of the chemical activity of chromatin modifying enzymes and then couple that information with direct visualization of the enzymes. Through these types of experiments and the data they provide, we can build a deeper understanding of how dynamic processes that alter the context of our DNA can give rise to stable cellular decisions.