Megan King - US grants

[unreadable] DESCRIPTION (provided by applicant): The nuclear envelope (NE) has long been appreciated as a gatekeeper to the genome of eukaryotic cells, restricting genomic access only to those molecules capable of traversing the NE via nuclear pore complexes (NPCs). It is increasingly evident, however, that the NE performs active and essential roles in genomic organization beyond providing simple compartmentalization of DMA. NE-associated complexes are important for maintenance of chromatin structure, gene regulation, and DMA replication. Disruption of the NE structure can lead to disregulation of these processes. This proposal focuses on the role of NE membrane proteins in maintaining the structural integrity provided by the NE, with a particular focus on the role of the transmembrane (TM) and lumenal domains of NE membrane proteins. [unreadable] [unreadable] [unreadable]

DESCRIPTION (Provided by the applicant) Abstract: The ability of an organism to thrive pits two fundamental processes against each other: the desire to maintain genomic integrity with the necessity to explore different genetic states in order to adapt to a changing environment. In this proposal, I suggest that the genomic context can influence the likelihood for genetic change at a given locus, providing a mechanism to ""target"" alterations to specific genes. Further, I suggest a model in which tethering of change-vulnerable loci to elements of the nuclear architecture restrains genetic change under supportive growth conditions, whereas stress releases such constraints, leading to an increase in genetic change. I propose to study genetic changes in loci encoding cell surface proteins in fission yeast as a model for antigenic variation employed by many pathogens. Primarily, we will utilize assays for intra- and intergenic recombination within and between copies of genes encoding adhesin family members. We will examine the effect of environmental stress on the probability of changes in the adhesin loci. Based on our preliminary data, we will pursue the hypothesis that adhesin loci are associated with the nuclear periphery, which we believe represses recombination. In addition to investigating how exposure to stress affects the nuclear position of adhesin loci, we will also identify the molecules that serve as nuclear envelope tethers. This model predicts that loss of these peripheral tethers will recapitulate the effects of stress on recombination, which we will test. Lastly, we will carry out genetic screens to reveal how stress triggers release of adhesin loci from the nuclear periphery. The ability to intervene during this signaling cascade would provide new avenues to combat the pathogenecity of a wide array of microbes. Public Health Relevance: In order to cope with an environmental insult, organisms respond by adaptive change. Although a substantial response can be achieved through regulation of the genome, pathogenic organisms utilize genetic change to increase the potential for expressing novel proteins that support virulence and allow them to evade the host immune system. The goal of this proposal is to discover how pathogens mobilize the adaptive response so that interventions might be devised to more effectively treat infection.

The research objective of this award is to examine how individual nuclear components contribute to the mechanical properties of the cell nucleus using a novel optical-tweezers assay in conjunction with nuclei from the genetically-facile fission yeast, S. pombe. Studies conducted under this award will test how altering the size of the nucleus, the compaction of the genome, the fluidity of the nuclear membrane, and association of chromatin with the nuclear envelope affect nuclear mechanical properties. Lamins, which are key in determining the mechanical properties of mammalian nuclei, but do not occur natively in S. pombe, will be introduced heterologously to dissect their mechanical properties from their roles in mammalian signaling and gene regulation. Fluorescence imaging of specific chromosome locations during force application to the S. pombe LINC complex, which spans the nuclear membrane, will elucidate how force is communicated into the nuclear mechanical network.

If successful, these studies will elucidate how the mechanics of the nucleus emerge from its constituents, and delineate how the mechanical environment, which is essential for the differentiation programs that give rise to specific cell types, is communicated to the nucleus, where cell fate is largely determined. They will also establish S. pombe as a model system in which the mechanical role of lamins can be decoupled from their regulatory functions, thus providing a platform for future studies of lamin mutants that cause such diseases as lipodystrophy, muscular dystrophy, and progeria. Because embryonic stem (ES) cells and yeast express very low levels or no lamins, respectively, these studies will also lead to new insights into how extracellular mechanical cues drive ES cell differentiation. On the educational side, the students involved in these studies will become a new generation of teacher-scientists, who excel at quantitative approaches and possess the biological sophistication to identify and tackle cutting-edge biological problems.

DESCRIPTION (provided by applicant): For over one hundred years, scientists have observed spatial preferences for the organization of the chromosomes within the nucleus. While we have now deciphered the entire genome sequences of eukaryotic species from yeasts to humans, how those genomes are physically organized to support genomic functions remains an open question. Within the last few years, several studies in yeast and mammalian models have suggested that association of genes with the nuclear periphery, likely with inner nuclear membrane proteins, acts to regulate transcription. The importance of these studies is underscored by the discovery that several human diseases are associated with mutations within integral inner nuclear membrane proteins including emerin, which causes Emery-Dreifuss Muscular Dystrophy (EDMD). Treatment strategies are extremely challenging to devise, however, since there remains substantial confusion over two key issues of how the nuclear periphery influences transcription: 1) in yeast, association with the nuclear periphery appears to activate some genes while repressing others, and 2) it remains controversial whether heterologously driving a reporter gene to the periphery is sufficient to induce transcriptional repression in mammalian cells. In this proposal, our strategy is to derive genome-wide maps of chromatin-inner nuclear membrane interactions in two yeast models, S. cerevisiae and S. pombe (Aim 1). These organisms display correspondence of 80% of their protein coding genes while lacking significant synteny; we can leverage these properties to identify genes (and the regulatory pathways they contribute to) with conserved sites for inner nuclear membrane association, which we predict will reflect functional importance. In Aim 2, we will examine how regulatory stimuli alter the association of genes with the nuclear periphery, assess the requirement for specific inner nuclear membrane proteins in the regulation of these genes, and test the ability of engineered and reversible tethers to recapitulate gene regulation at the nuclear periphery. Our long-term goal is to understand how we might ameliorate the changes in the transcriptome that result from mutations in inner nuclear membrane proteins, and establish paradigms to allow us to devise strategies to tackle a growing number of genetic diseases including EDMD, which are collectively called nuclear envelopathies.

Cells are subject to numerous types of mechanical stress, from forces exerted on the skin to fluid shear within blood vessels. Because these forces can be transmitted to the cell nucleus, which houses the genome, mechanisms to adapt to and dissipate mechanical stress are necessary for cell survival, particularly within the nucleus itself. Importantly, defects in the mechanical properties of the nucleus can compromise cell survival during normal cellular processes, like cell migration, sometimes leading to disease. In addition, a cell's mechanical environment is defined by the tissue in which it resides, and is a key determinant of cell and tissue development. Therefore, it is an essential challenge to understand how a cell's external mechanical environment is communicated to the nucleus, where cell fate is largely determined. Moreover, the mechanical properties of the nucleus must be tuned to its tissue environment, a process that is poorly understood. This project will address these challenging questions through an interdisciplinary approach combining a genetic model organism, live-cell imaging, and biophysical tools. In addition, the supported graduate students will be trained to become the next generation of researchers and educators, who both excel at quantitative approaches and possess the biological sophistication to tackle cutting-edge biological problems. Supported graduate students will participate in Yale's Integrated Graduate Program in Physical and Engineering Biology (IGPPEB), for which the PIs serve on the executive committee, mentor students, and teach program courses. IGPPEB provides training in communication skills, outreach activities, and teaching. Summer research opportunities will also be provided to high school students.


This project will test and further develop a physical model for cell nuclear mechanics by combining novel live-cell imaging and force-spectroscopy tools capable of probing the mechanical properties of nuclei at biologically-relevant temporal, spatial and force scales, taking advantage of the genetic model system fission yeast (Schizosaccharomyces pombe). First, it will elucidate how changing the heterochromatin-euchromatin balance can alter the mechanics of the nucleus. Second, it will implement biosensors that directly measure the tension on the chromatin-inner nuclear membrane protein interface in living yeast cells in wild-type cells as well as cells with perturbed nuclear mechanics and/or chromatin states. Third, it will exploit a novel optical tweezers assay capable of applying calibrated force to nuclei in living yeast cells, thereby enabling measurements of nuclear viscoelasticity and chromatin flow in vivo. Throughout these experiments, the new information gleaned will be fed back in to a developing mathematical model of nuclear mechanics, ultimately leading to a comprehensive picture of the mechanisms that define the mechanics of nuclei. Finally, this project will test the ability of the models developed to explain nuclear blebbing, which can drive loss of nuclear integrity in transformed mammalian cells.

The ability to design genomes towards new functions has the potential to exert a broad, positive influence on society. Built from deceptively simple four bases (A, T, C and G), DNA is packaged by association with proteins into chromatin, which helps to condense the DNA into the nucleus and to regulate the DNA output. Previous work has established the importance of several factors, including chromatin compaction, the identity and modifications of the chromatin-associated proteins, as contributors to DNA output. However, we still lack understanding of the mechanisms that will allow us to effectively design DNA and chromatin architectures towards new functions. This project will take on the challenges of discovery and synthesis to meet this need by engaging teams of scientists bridging engineering and biology. For the graduate students who participate in this project, Yale's Integrated Graduate Program in Physical and Engineering Biology will serve as a framework for training a new generation of powerful practitioners of interdisciplinary science. This project will also recruit undergraduates studying biology, engineering and physics from under-represented groups to this interdisciplinary team to participate in well-defined, yet independent, projects. Engaging these students in mentoring and leadership roles for outreach programs at the high school and middle school levels will enhance their growth and confidence as scientists, engineers and scholars, while also bringing the fundamental concepts of bio-inspired design to the broader community.

The scientific goals of this project are to define the design principles that underlie chromatin organization and to leverage the genome as a device to measure and record dynamic chromatin states. Historically, a major barrier to the quantitative and comprehensive understanding of chromatin organization is the lack of versatile, tractable systems in which to probe and interpret chromatin dynamics. This project leverages methods to combine high-resolution observations of the dynamic behavior of specific chromatin loci in individual living cells with a systems-level image and data analysis pipeline that sorts single-particle-tracking data from a population of cells into discrete diffusive states. Powerful genetic tools will be employed, in combination with modular engineering strategies, to alter key factors that influence chromatin structure, including the SMC protein complexes, cohesin and condensin, to create changes in the local epigenetic landscape. Simulations will be employed to test emerging models for the origin of topologically-associating domains, such as loop extrusion; these models will be further enhanced by accounting for both the dynamics and excluded volume of the chromatin polymer in three dimensions as well as the processes that drive loop formation. These insights will be leveraged to develop an entirely novel method to record transient chromatin conformations as "memories" in the genome itself through recombinase-based state machine designs, extending biological computing to the dynamic sampling of a cell biological state.

This award is co-funded by the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences in the Biological Sciences Directorate, by the Physics of Living Systems Program in the Division of Physics in the Mathematical and Physical Sciences Directorate, and by the Emerging Frontiers in Research and Innovation Program in the Division of Emerging Frontiers and Multidisciplinary Activities in the Engineering Directorate.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

SUMMARY The nuclear lamina is a compositionally complex structure that serves functions in chromatin organization, transcriptional regulation, genome protection and mechanotransduction. In cells and tissues, the nuclear lamina is mechanically integrated into the actin, microtubule, and intermediate filament cytoskeletons via nuclear envelope-spanning Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes. Through these cytoskeletal connections, forces exerted on plasma membrane adhesions from either the extracellular matrix or from adjacent cells can be transmitted to the nuclear interior. In cells in which LINC complex function has been altered, investigators have observed correlative changes in gene regulation. However, to date it has been extremely challenging to decipher whether mechanical forces transmitted by the LINC complex, potentially through interactions at the nuclear lamina, directly influence specific genetic programs. Our published work and preliminary studies demonstrate that the LINC complex enables a critical crosstalk between cellular adhesions, the cytoskeleton, and components of the nuclear periphery. Most important for this proposal, we used a mouse model to reveal that A-type lamins and the LINC complex drive opposite effects on pro-fibrotic signaling, which is classically driven by SMAD-dependent signaling downstream of TGF?. Taking these insights together with our global transcriptome studies, we suggest that tension exerted on the nuclear lamina by the LINC complex influences nuclear events necessary for SMAD gene targets to be properly regulated by TGF? inputs (despite normal cytoplasmic events necessary to drive this signaling pathway). Building on this, here we propose three complementary Aims that will address both molecular mechanisms as well as physiological contexts in which these mechanisms play critical roles. First, we will take an unbiased approach to define how the LINC complex, in combination with substrate inputs from the extracellular matrix, influences the nuclear lamina interactome in situ, ultimately employing a cross-linking mass spectrometry approach. Second, we will investigate the mechanisms by which LINC complex ablation influences SMAD function in the nucleus, including the analysis of SMAD target binding, nuclear position of SMAD target genes, and the influence of integral inner nuclear membrane proteins on SMAD-dependent gene output. Lastly, we will test if (and how) LINC complex function intersects with that of A-type lamins in this TGF??SMAD-fibrotic axis using both in vitro and in vivo approaches, including mouse models of interstitial fibrosis of the myocardium and lung injury models of pulmonary fibrosis. Taken together, the Aims of this proposal will reveal both molecular mechanisms of mechanotransduction through the LINC complex while also placing this detailed understanding into its physiological and disease contexts.

Summary Altered nuclear shape and appearance has long been known to be pathognomonic for cellular transformation; as a consequence, it is a critical parameter used in cancer diagnosis and tumor grading. Despite an increasingly mechanistic understanding of oncogenic and tumor suppressor pathways, as well as burgeoning genomic data that heralds the possibility of personalized treatments, we still lack a firm understanding of the relationship between nuclear architecture and cancer. In particular, it has yet to be defined if changes in the nucleus are causal or simply a consequence of transformation. Here, we sidestep this question, and instead ask: can the changes in nuclear architecture typical of cancer cells be exploited as a liability? Altered nuclear shape is intimately tied to mechanical defects of the nuclear envelope; recently, such defects have been linked to either transient or catastrophic losses of nuclear integrity, which can lead to cell death through two potential mechanisms. First, permanent losses of nuclear integrity are incompatible with cellular viability. Second, even transient losses of the nuclear barrier expose the DNA to cytoplasmic DNA sensors such as cGAS, which can drive a STING-dependent innate immune response that, at least in some cases, is sufficient to drive cell- autonomous death. In the latter case, loss of nuclear integrity also boosts the immune response to the tumor. Importantly, pathways that recognize and ?heal? ruptures of the nuclear envelope have also been recently defined; perhaps not surprisingly, these repair mechanisms become critical for cell viability in contexts where nuclear integrity is compromised. Taken together, these new insights make a strong case that further weakening nuclear integrity in tumor cells can be exploited to drive cell death and immune system recognition. Here, in Aim 1, we propose to leverage an unbiased, genome-wide CRISPR dropout screen to identify synthetic lethal interactions of 1) normal cells with either weakened nuclear integrity or defective nuclear repair mechanisms or 2) cancer cell lines, with and without further compromise of their nuclear integrity pathways. In Aim 2, we will apply systems level approaches to organize the resulting context-dependent fitness genes into functional nodes. Beyond the strength of the genetic interaction, targets for in depth analysis will be further prioritized based on the availability of chemical inhibitors and representation in The Cancer Genome Atlas. Mechanistic experiments will explicitly examine these high priority synthetic genetic relationships in the context of nuclear shape, nuclear ruptures, and innate immune pathway activation. Completion of these two Aims will lead to the development of novel targets that exploit a key pathognomonic structure for cancer.