Research Interests

 

Overview

Experimental Digital Biology

Our lab focuses on developing methods to probe the genome and epigenome at the single-cell and single-molecule levels. Our efforts are split between building new tools to leverage the power of high-throughput sequencing technologies and cutting-edge optical microscopies, and bringing these new technologies to bear against basic biological questions of genomic and epigenomic inheritance and variation in populations.

Motivation: Moving beyond ensemble genomic analysis

Ensemble methods of measuring the genomic characteristics of a population “average out” large-scale cell-to-cell heterogeneities, and "drown out" rare variation within the population. As a simple analogy of this problem, let us consider all the shipping routes from the East Coast to the West Coast of the US (see figure to the left). Some of these routes maneuver through the Panama Canal (red), while another fraction navigate the Straits of Magellan (green). However the ensemble average of these paths (yellow) is a path not taken by any boat -- in fact this path is impossible! The observation of this single yellow path hides the true richness of the situation and supplies misleading information to boot! Similarly, almost all of our understanding of large-scale cellular genomics and epigenomics comes from looking at such ensemble measurements. We hope to move beyond the ensemble by applying single-cell and single molecule methods to biological questions at the systems level.

Beyond the ensemble: Information rich imaging to partition populations

Live cell fluorescence imaging is a powerful means of splitting a complex population of cells into phenotypically relevant sub-populations for downstream genomic analysis. We have interests in extending the capabilities of FACS sorting to address these problems, but are are also interested in single-cell digital FISH (fluorescence in situ hybridization), which allows the digital counting of individual mRNA molecules in intact cells. With this adaptable, information-rich filter as a front end, we hope to leverage the power of revolutionary high throughput sequencing technology to investigate the relationship between expression states and epigenetic marks in small, well defined subpopulations of cells.

Beyond sub-populations: Single cell chromatin positioning and composition

The cell faces a spectacular topological challenge in packing meters of chromosomal DNA inside a ~5 micron nucleus. The cell's solution to this challenge is the hierarchical folding of genomic DNA into regulated structures, the most basic and important of which is the nucleosome. In addition, the positioning and composition of nucleosomes provides an epigenetic layer of information to the genome itself, creating a molecular memory that can help to perpetuate cell-specific gene expression patterns through generations. It is becoming apparent that specific positioning and modifications of histone particles encode a molecular "state machine" of the cell. Chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-Seq) has become an incredibly powerful means to explore these epigenetic markers, however all previous investigations of this "second genetic code" have been ensemble-based measurements, and suffer from the "average out" and "drown out" effects mentioned above. The capacity for single-cell, genome-wide measurements would lift the veil on the cellular nature of the complex, variable characteristics such as epigenetic state and histone positioning.

Recent technical advances demonstrating the detection of individual proteins from eukaryotic cells, as well as ultra-sensitive protocols for chromatin immunoprecipitation (ChIP), have made the investigation of the epigenetic code at the single-cell level an enticing possibility. To investigate the chromatin of single-cells at the level of histone positioning and composition, we will adapt microfluidic cell sorting and laser-based manipulation methods to isolate individual cells within a microfluidic reaction chamber. Following the on-chip generation of nucleic acid fragments associated with specific modified histone particles, these fragments will be sequenced using high-throughput sequencing to map both the position and composition of histone particles within individual cells. This analysis will help lift the veil on cell-to-cell epigenetic variability and the modifications that likely determine cell fate and begin to cast light on the mechanism of heritability of this component of the epigenetic code.

Extracting rare variants: genomic mismatch enrichment

The detection of somatic mutations in cancer and the cataloging of novel and rare SNPs in a population are both difficult tasks for high throughput sequencing, given that these applications squander most sequencing capacity on regions of the genome that are identical to the reference genome. Methods that can specifically enrich for genomic loci that differ from a reference genome would directly enhance the capacity of sequencing technologies to detect rare genomic variation by eliminating this enormous background of normal sequence. We are developing a method of enriching specific locations in the genome that have variability with respect to a “standard” sequence, then sequence only these variable positions significantly improving the depth of coverage of these potentially diagnostically relevant loci. Successful implementation of such and enrichment strategy promises to further amplify the incredible power of high throughput sequencing methods by multiple factors of ten.

Microfluidics and "femtofluidics"

As a natural extension of our interest in individual cells and individual molecules, we are motivated to adapt and develop methods that allow manipulation, observation, and quantification of very small amounts of biomaterials through the use of microfluidic devices. We have also developed a methods of generating minuscule resealable reactions chambers in polydimethylsiloxane. Standard microfluidic devices are constructed on a length scale of 10s or 100s of microns and involve on the order of nanoliters or picoliters. We have developed methods to generate reversibly sealable microreactors that are approximately 10,000 times smaller than these volumes, creating the possibility of generating tens of millions of isolated reactions chambers from a single microliter of liquid. We can image molecules as they freely diffuse confined within the reactor, opening the door to high-throughput single molecule fluorescence correlation spectroscopies, and analysis of DNA binding proteins and transcription factors in a highly parallel, yet single molecule, manner. These resealable femtoreactors also have application to digital PCR and next-generation sample preparation, possibly allowing for efficient and simple amplification of exomes.

Force-based investigations of chromatin structure.

The topological structure of the metaphase chromosome, one of the most recognizable and interesting cellular structures, remains obscure. Because chromatin is not crystalline, the topological structure of condensed chromatin cannot be solved with standard crystallographic techniques -- instead, single molecule methods are uniquely suitable. We hope to help understand this structure by developing innovative new single molecule manipulation methods based on "trappable" magnetic microparticles. These magnetic methods, in combination with optical trapping methods, will allow for both intracellular and extracellular mechanical manipulation of chromatin. These tools, along with ensemble high-throughput sequencing data, will help shed light on the enduring mystery of higher-order chromatin structure.

The chemomechanics of chromatin remodeling

The dynamic mechanical processes whereby nucleosomes are assembled, ejected, exchanged, and moved by molecular motors to make genetic information available to the machinery of expression is at the heart of gene regulation. Recent technical advances in the areas of single molecule biophysics create a tremendous opportunity to explore the mechanics of chromatin dynamics. We are interested in a mechanical characterization of histone-DNA interactions and chemomechanical investigation of the molecular motors required to slide nucleosomes, with the aim of providing a detailed, dynamic physical picture of histone assembly and remodeling, complementing traditional biochemical techniques. We plan to use high-resolution optical trapping methods to directly measure the miniscule molecular motions that accompany the active rearrangements of nucleosomes.

Sound interesting?

We are actively recruiting students and postdoctoral fellows to work on these and other projects in the lab.

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