Institute of Molecular Biology

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Research Interests

Natural and Synthetic Targeting of Chromatin Proteins

The McKnight Lab is interested in the targeting and regulation of chromatin modifying proteins and ATP-dependent chromatin remodeling factors in vivo. Because nucleosomes, the fundamental units of chromatin, are intrinsically inhibitory to DNA-dependent processes like transcription, replication, and DNA repair, proper control of their positions and modification state is critical for cellular processes in all eukaryotic organisms. Importantly, aberrant chromatin dynamics is often associated with developmental malfunctions and human cancers. While much has been learned about mechanisms of chromatin modifying and remodeling proteins in vitro, there has been significantly less mechanistic investigation into how their regulation establishes proper nucleosome positions and modification state under changing environmental conditions in vivo (Figure 1). Additionally, relatively little high resolution work has been done to investigate global targeting of chromatin proteins in three dimensions or to determine how dynamic targeting can lead to reproducible changes in chromatin states. Our lab uses interdisciplinary approaches in biochemistry and genomics and is developing new technologies to probe and manipulate chromatin structure using the budding yeast S. cerevisiae as a model system.

mcknight research Figure 1. Nucleosome positions can control accessibility to DNA elements required for processes like transcription. If a nucleosome is occluding regulatory sequences, the underlying DNA is less likely to be accessed by transcriptional machinery. The positions of nucleosomes are well-defined in specific conditions, but can change in unique environments to support multiple transcriptional states.

“Natural” Targeting of Chromatin Proteins

We are investigating the natural processes leading to proper establishment of chromatin structure. One interesting feature in eukaryotic organisms like budding yeast is that nucleosome positions are stably maintained after disruptive processes like DNA replication, but allow for differential response to changing environments. We are interested in understanding how chromatin proteins can use the same DNA sequence to provide high fidelity inheritance of nucleosome structure while also supporting plasticity. Work in budding yeast suggests that DNA sequence can simultaneously encode multiple distinct nucleosome positioning programs that can be accessed by bridging condition-specific transcription factors to chromatin remodeling proteins. We are interrogating this mechanism using a combination of in vitro biochemical assays and genome-wide analyses to understand regulatory interactions between the Isw2 chromatin remodeling protein and condition-specific Isw2-interacting transcription factors (Figure 2). This work will elucidate conserved mechanisms that are likely involved in environmental responses, cellular differentiation, and the etiology of some cancers.

mcknight research Figure 2. Nucleosome placement by chromatin remodeling proteins can be encoded by transcription factor (TF) binding sites. (left) The interaction of Isw2 with condition-specific transcription factors leads to highly precise nucleosome positioning with respect to the recruitment site. (right) Deletion of Isw2 leads to loss of nucleosome positioning adjacent to dozens of transcription factor binding motifs. We are trying to understand the underlying mechanisms for this precise placement, and how cycling of different transcription factors can dictate sequence-encoded nucleosome positioning changes under different environmental conditions or during developmental processes.

Engineering Chromatin Rearrangements

Because chromatin structure is so vital to the regulation of DNA dependent processes and often perturbed in human diseases and cancers, one major research goal of our lab is to create new ways to directly manipulate chromatin structure in living systems. Earlier work led to the appreciation that artificial targeting of a chimeric Chd1 chromatin remodeling protein using sequence-specific DNA binding domains results in directional repositioning of mononucleosomal substrates. More recently, the feasibility of directly manipulating nucleosome positions in budding yeast was demonstrated, where the targeting of an engineered Chd1 hybrid protein allowed for specific nucleosome rearrangements at recruitment sites (Figure 3). The McKnight Lab is building on this new technology to increase the versatility of targeted chromatin remodeling to manipulate chromatin structure in budding yeast and other organisms. Through engineered chromatin remodeling, we hope to gain insight into principles of transcriptional repression, three dimensional nucleosome folding, interplay between nucleosome positions and histone modifications, and other important biological phenomena. Future efforts will involve targeting other chromatin modifying enzymes (such as histone deacetylases) or structural proteins to establish a tool kit for specifically altering the epigenome for control of DNA dependent processes.

mcknight research Figure 3. General strategy for synthetic targeting of chromatin remodeling proteins in vivo. (left) The catalytic core of the Chd1 chromatin remodeling protein is fused to a localization domain (X) of choice (eg DNA binding domain, histone modification reader) resulting in an engineered enzyme that catalyzes precise movement of specific target nucleosomes in vivo. (right) Meta-analysis of precise, engineered positioning by Chd1[Ume6] at genomic instances of Ume6 binding sites. These factors will be used to test direct effects of nucleosome positions on transcription, chromatin folding, and other chromatin based processes.

Three Dimensional Chromatin Folding

It is becoming increasingly clear that the three-dimensional organization of genetic material has a profound impact on cellular processes, and transitions in three-dimensional genome folding are associated with normal development and cancer. We are taking advantage of Micro-C, a breakthrough technology developed by Oliver Rando that allows for ultra-high resolution insight into the three dimensional folding of chromatin. Our goal is to use this technology to understand how chromatin proteins can orchestrate specific 3-D folding rearrangements in a reproducible manner under changing environmental conditions, identify factors or molecules that can drive these rearrangements, and determine how different chromatin structures can regulate DNA-dependent processes like transcription and DNA repair. In the future we will integrate chromatin engineering with chromatin folding studies to understand how changing nucleosome positions can affect distal chromatin interactions, and we will also develop strategies to engineer designer chromatin folding interactions through synthetic biology approaches.

Recent publications

(pulled from pubmed)