Student Research

Nano-environmental Engineering

Measuring, modeling, & modulating the chromatin nano-environment for transcriptional regulation

Nano-environmental EngineeringAdvances in molecular biology have made it easier than ever to explore the functions of the human genome. We tend to now think of the functional properties of the genome with respect to the activities each gene carries out and the overarching molecular regulators of that behavior. Comparatively underexplored is the physical organization of the genome and its effects on the functions of genes. In much the same way that the organization of a city influences our behavior (e.g. driving in Boston vs. Chicago, public transit in Los Angeles vs. NYC), the structure of the genome has a major impact on molecular behavior. To study the effects of physical organization on genomic function requires the capability to measure the relevant structures in real-time, develop models of how this organization alters behavior, and test them experimentally. My research has therefore focused on all of these aspects – helping to develop microscopic techniques that are nanoscale sensitive to image the physical structure of the genome within seconds, developing mathematical models that account for the role of the measured physical structure on processes such as gene expression, and testing these predictions using molecular techniques.

While the understanding of the role physical forces play on genomic function is at its earliest stages, we have been able to apply this approach to develop compounds that control the physical structure of the genome in order to manipulate global patterns in gene expression, so called “Macrogenomic Engineering.” My goal is to develop this physico-chemical Macrogenomic Engineering as a complement to existing gene editing tools: while gene editing techniques work at the level of the linear genetic code and thus individual genes, the regulation of the genomic structure affects the global patterns of gene expression. As most diseases are inherently genomic and multifactorial in nature, the combination of these techniques may eventually allow us to address diseases where the global reprogramming of gene expression plays a role, such as cancer, inflammation, and neurodegeneration. For further details, see Almassalha et al., The Global Relationship between Chromatin Physical Topology, Fractal Structure, and Gene Expression. – Luay Almassalha

Forming Brain Tissue

Uncovering how integrin is needed for cells to come together and assemble organs in regeneration

Forming Brain TissueThe use of stem cells to regenerate damaged tissue has the potential for far-reaching medical applications. Certain animals have evolved the ability to harness their stem cells for complete regeneration after injury and are models for understanding natural mechanisms of tissue repair. The flatworm planarian Schmidtea mediterranea is famous for its ability to regenerate after removal of any body part because of robust mechanisms to control the activity of its potent adult stem cells. My research uses planarians as a model system to study how stem cells differentiate into specialized cell types and how their progeny assemble to form functional organs, such as the brain, during regeneration. We found that β1-Integrin, a cell surface molecule involved in adhesion to the extracellular matrix, is required for the proper organization of brain tissue in planarian regeneration. Inhibition of the β1-Integrin gene caused regenerating planarians to generate disorganized brains in which neurons aggregated with each other randomly rather than forming distinct and symmetric brain lobes. Intriguingly, these aggregates resemble organoids, a type of synthetic tissue that human stem cells form in petri dishes in the absence of a natural environment for growth. Planarians depleted of integrin activity also produced excess brain neurons that were placed at abnormal locations, suggesting that integrin signaling is additionally required to determine appropriate numbers and positions of mature brain neurons formed through regeneration. Together these results showed that integrin signalling regulates planarian stem cell activity to control the organization and scale of tissues formed during regeneration. For further details, see Bonar & Petersen, Integrin suppresses neurogenesis and regulates brain tissue assembly in planarian regeneration. – Nicolle Bonar

Genomics in C. elegans

Leveraging genomic and computational approaches to understand biology

cook reflectionI have a long-standing interest in the application of novel computational tools to answer important biological questions. During my PhD studies in the IBiS program at Northwestern, I focused on understanding the genomes of a tiny translucent nematode called Caenorhabditis elegans. Specifically, I studied the role genetic differences play in making each individual in a population distinct from one another. My projects have focused on three areas. First, I characterized natural differences in the genomes of wild strains by developing bioinformatic pipelines to identify single nucleotide variants (SNVs) and other genomic features. Second, our lab measures correlations of these genetic differences with physical differences (using a technique known as genome-wide association) to identify which specific genetic differences make individuals distinct from one another. For example, we identified a specific genetic difference that contributes to the regulation of the ends of chromosomes (telomeres) across the natural population. Finally, I developed tools that make the study of natural variation across the species easier for the scientific community. Towards this end, I created an interactive web portal - The C. elegans Natural Diversity Resource - available at elegansvariation.org, and developed a command-line utility for processing genome sequence data known as VCF-kit. For further details, see Cook et al., CeNDR, the Caenorhabditis elegans natural diversity resource. – Daniel Cook

It's about Time!

A superior algorithm to infer gene regulatory networks from time-series expression data

Finkle & WuMany genetic components are involved in diseases such as cancer, and genome-scale experiments now allow us to uncover information about them faster than ever. Untangling how genes interact is essential for developing more effective treatments for disease. An emerging approach to this problem is to infer gene regulatory networks (GRNs) from microarray or RNA-seq experiments. GRNs condense thousands of changes in gene expression into a map that depicts the flow of information through genes using directed edges. Although the computational tools for doing this have consistently improved, many do not incorporate temporal information from time-series measurements, leading them to make false predictions.

We developed a novel algorithm that improves network inference from time-series gene expression data, which we call Sliding Window Inference for Network Generation (SWING). The key idea behind SWING is to account for time delays between regulatory events that exist due to the many steps required for genes to respond to stimuli. SWING partitions experimental data into many small "windows," which we found improves algorithm accuracy for revealing interactions that occur within short periods of time. It also pairs this with the concept of Granger causality—a statistical idea from economics that states that an event can be forecasted by another causal event in the past—to improve the inference of GRNs from time-series gene expression data. Using simulated GRNs, where we know the true interactions, we show that SWING can reconstruct network maps more accurately by accounting for time delays. We identify many time-delayed interactions from simulated systems and in vitro E. coli and S. cerevisiae data, and show that SWING can improve network inference in these contexts. Overall, we demonstrate the importance and utility of accounting for temporal ordering to understand biological systems. In other words, it’s about time!  For further details, see Finkle, Wu, and Bagheri, Windowed Granger causal inference strategy improves discovery of gene regulatory networks. – Justin Finkle & Jia Wu 

Understanding Stem Cells

Exploring how cell-cell communication mediates cell fate decisions in the early embryo

GearyStem cells of the early embryo, termed embryonic stem cells, will give rise to all the cell types of the mature organism. Cells are eventually partitioned into one of three different tissue layers, called germ layers, with each preparing cells for a very different function. Neural crest stem cells, which are unique to vertebrate organisms, are part of a germ layer where cells are destined to become either skin cells or neural cells, yet they are not instructed to become either of these specialized cell types. Instead, these cells exhibit a stem cell-like state until later stages of development to make contributions to mature structures like muscle, cartilage and bone, and the valves of the heart. These cells resist the push to become specialized and provide a great system to answer the enigmatic question, how do stem cells maintain their remarkable potential? My work uses Xenopus as a model organism to explore how cell-cell communication is involved in regulating stem cell potential, providing novel insights into the cell signals that are essential for both stem cell pluripotency and for neural crest formation. I found that fibroblast growth factor (FGF) signals regulate the gene expression of pluripotent stem cells, and that there is a striking switch in the signaling cascades activated by FGF signals as cells exit the stem cell state and commence lineage restriction. Specifically, pluripotent stem cells display and require Map Kinase signaling, whereas PI3 Kinase/Akt signals increase as cells undergo lineage restriction. Importantly, retaining a high Map Kinase/low Akt signaling profile is essential for establishing Neural Crest stem cells. Thus, my work sheds important light on the signal-mediated control of pluripotency and the molecular mechanisms governing the genesis of the Neural Crest.  For further details, see Geary and LaBonne, FGF mediated MAPK and PI3K/Akt Signals make distinct contributions to pluripotency and the establishment of Neural Crest. – Lauren Geary

What Are Methanobactins?

The biosynthesis, regulation, and transport of an unusual family of metal-binding natural products

What Are Methanobactins?Methanotrophic bacteria – bacteria for which the only source of carbon is methane – are a major area of research for the Rosenzweig group. The enzymes responsible for the oxidation of methane to methanol are metalloenzymes, and metal homeostasis plays a major role in governing methane oxidation. My project focuses on the natural product family known as methanobactins. These compounds are secreted from the cells of methanotrophic bacteria, and once outside of the cell, they bind copper with high affinity. Copper-bound methanobactins are then re-admitted to the cell and the internalized copper is ultimately incorporated into some of the metalloenzymes responsible for methane oxidation. Over my time in the Rosenzweig group, my work has provided insight into how these unusual natural products are biosynthesized, how they are transported out and into the cell, and how their production is regulated by the availability of copper. For further details, see Kenney et al., Characterization of Methanobactin from Methylosinus sp. LW4Grace Kenney

Positioning Organelles

Uncovering a role for mitochondrial anchors in nuclear migration

Inter-organelle contacts facilitate communication between organelles and impact fundamental cellular functions. Investigations into the molecular mechanisms of inter-organelle tethering are still in the early stages, and we are just beginning to appreciate the number and variety of inter-organelle tethers that exist. My work is focused on understanding how cells establish, maintain, and alter contacts between organelles at the right time and place to impact a wide array of cellular functions. I am studying an organelle contact site in the model organism budding yeast. Using live-cell microscopy to visualize organelles and contact site proteins, I found that the assembly of a mitochondria-ER-cortex anchor, called MECA, requires mitochondria. Once assembled, MECA persistently anchors mitochondria to the cell cortex. MECA also serves as a cortical anchor for dynein during mitotic spindle positioning. I found that disrupting the mitochondria-dependent assembly of MECA leads to defects in dynein-mediated spindle positioning. Therefore, MECA-mediated mitochondria-plasma membrane tethering not only impacts the spatial distribution of mitochondria within cells but also the position of the spindle. This work helps us to understand how the regulated assembly of a contact site integrates the positioning and inheritance of two essential organelles and expands our knowledge of organelle contact site function. For further details, see Kraft & Lackner, Mitochondria-driven assembly of a cortical anchor for mitochondria and dynein– Lauren Kraft

Memories of Transcription

Mechanism and evolution of GAL gene epigenetic transcriptional memory

Varun SoodHow do cells remember previous experiences?  One way is through epigenetic processes in which a stimulus can induce heritable (although not necessarily permanent) changes in behavior or phenotype.  Transcriptional memory is such a phenomenon: certain inducible genes show a much faster reactivation for several generations after they were previously expressed.  This type of memory has been described in budding yeast, fruit flies and humans.  For my thesis project, I performed a genetic screen for mutants in budding yeast that were disrupted for memory of previous growth in galactose, which activates the GAL genes.  This type of transcriptional memory lasts for eight generations and provides a huge adaptive advantage in galactose, which is a sub-optimal sugar.  I found that a point mutation in the co-activator Gal1 specifically blocked memory by disrupting a physical interaction with its target.  Because Gal1 is made at high levels when cells are grown in galactose, this supported the idea that proteins produced in the first round of activation can serve to promote faster reactivation for several generations through cytoplasmic inheritance.  I also found that Gal1 stimulates an inter-domain interaction within Gal4 transcription factor, leading to stronger expression of GAL genes.  By comparing how different Saccharomyces species adapt to galactose, I found that GAL transcriptional memory evolved from an older, constitutive poising mechanism that is genetically encoded through low level, basal expression of GAL1.  My work suggests that the evolution of GAL transcriptional memory in budding yeast is the product of competing pressures to retain the fitness benefits of previous growth in galactose while maximizing fitness in glucose-galactose mixtures.  For further details see Sood & Brickner, Genetic and Epigenetic Strategies Potentiate Gal4 Activation to Enhance Fitness in Recently Diverged Yeast Species. – Varun Sood

Seeing Nucleosomes Up Close

High-res mapping of nucleosomes in embryonic stem cells with new biochemical and computational tools

What Are Methanobactins?Nucleosomes affect transcription primarily by regulating the accessibility of DNA-binding proteins to the underlying DNA sequence. Understanding how nucleosomes regulate transcription requires knowing precisely where they are positioned. Recent advances in sequencing technologies have made it possible to map the detailed locations of nucleosomes across the genomes of a multitude of organisms and cell types. These genome-wide nucleosome maps have significantly expanded our understanding of the role of nucleosome positioning and dynamics in eukaryotic transcriptional regulation. My research focuses on the development of chemical and computational approaches to charting the nucleosome landscape in mammalian cells. Recently, we implemented a chemical nucleosome mapping strategy in mouse embryonic stem cells and uncovered new insights into the dynamic interplay between nucleosomes, transcription, and splicing. For further details, see Voong et al., Insights into Nucleosome Organization in Mouse Embryonic Stem Cells through Chemical Mapping. – Lilien Voong

These and other student projects have led to many awards and papers.