Aaron F. Straight - US grants

DESCRIPTION (provided by applicant): Accurate genome segregation is essential for the survival and development of all organisms. Mistakes in chromosome segregation result in cellular aneuploidies that give rise to human genetic diseases such as Down syndrome and that characterize most human cancer types. A major question in cell biology is what are the cellular mechanisms that ensure high fidelity chromosome segregation to avoid genetic instability and the resulting aneuploidy. Our studies focus on the formation and function of human centromeres and kinetochores. The functions of the kinetochore are to bind microtubules, to monitor proper chromosome attachment via the mitotic checkpoint and to segregate chromosomes in anaphase. Defects in any of these processes result in chromosome segregation errors. The centromere is sole the assembly site for the mitotic kinetochore on the chromosome. Centromere function is determined by a specialized histone variant called centromere protein A (CENP-A) and mutation or loss of CENP-A causes centromere and kinetochore dysfunction. Our first objective in this proposal is to identify the mechanisms that assemble CENP-A into chromatin. We propose to do this by identifying how two of the key proteins required for CENP-A assembly, HJURP and M18BP1, are targeted to centromeres to assemble CENP-A nucleosomes during telophase and G1. Second, we propose to characterize the mechanisms by which the essential centromere protein CENP-C interacts with arrays of centromeric chromatin. Using insights from biochemical experiments, we will test models for CENP-C function in human cells in organizing and reinforcing centromere and kinetochore structure. Third, we propose to use a novel in vitro centromere and kinetochore assembly system to understand the role of chromatin structure in promoting centromere and kinetochore function. Together our aims should provide new insight into the assembly and function of vertebrate centromeres and how their activities ensure faithful chromosome segregation.

DESCRIPTION (provided by applicant): This application requests funds to purchase a multi-use fluorescence microscope to be shared by three NIH R01 supported Principal Investigators and one NIH K99 supported investigator in the Department of Biochemistry at Stanford University School of Medicine. The laboratories of Jim Spudich, Suzanne Pfeffer, Aaron Straight and Rajat Rohatgi share a common interest in microscopy based dynamic analysis of cellular systems and mechanistic dissection of those systems through biochemical analysis. Central to the research in our laboratories is the use of fluorescence microscopy to study cellular protein and organelle dynamics. In order to analyze complex cell biological processes, our laboratories combine optical, chemical, and genetic perturbation of cells with high-resolution immunofluorescence imaging and live cell timelapse imaging. To understand how individual cellular components participate in cellular functions we apply microscopy-based analysis to reconstituted biochemical systems and cell extract based reactions that recapitulate cellular processes. Specifically, we use methods ranging from single molecule analysis of motor protein movement on cytoskeletal polymers by optical trapping and TIRF microscopy to epifluorescence imaging of chromosome segregation and vesicle trafficking in cell extracts. We are requesting funds to purchase a shared microscope capable of live cell time-lapse fluorescence and brightfield microscopy, TIRF microscopy, patterned fluorescence photobleaching and photoactivation and iterative deconvolution. These combined modalities will significantly enhance the NIH supported research in our laboratories by providing a shared resource to address our microscopy needs.

DESCRIPTION (provided by applicant): Our understanding of fundamental cell biological processes is both driven by and limited by our ability to visualize the organization of structures and molecules within cells and tissues. The development of super-resolution fluorescence imaging methods that circumvent the resolution limits of conventional light microscopy, achieving lateral resolution of d100 nm, are transforming cell biological research. Access to super-resolution fluorescence imaging technology is now essential for scientists wishing to push the frontiers of cell biological research. This proposal requests funds to purchase the OMX BLAZE 2D, 3D-SIM fast super-resolution imaging system (Applied Precision, Inc.). This super-resolution system can achieve both lateral and axial resolution at twice the diffraction limit of conventional light microscopy and is capable of multi- channel super-resolution far beyond the cover glass. The fast imaging capability of OMX BLAZE is also designed to overcome speed limitations for 3D live-cell imaging. This advanced imaging system will be a shared resource, located in a well-established, multi-user microscopy facility at Stanford: the Cell Sciences Imaging Facility. The requested OMX BLAZE 3D-SIM imaging system will support NIH funded projects from 14 researchers. These projects investigate a wide range of topics, including: meiotic chromosome segregation (Villeneuve); genetic recombination (Villeneuve); centrosome structure, function and duplication (Stearns); centromere and kinetochore assembly (Straight); biogenesis and function of the primary cilium (Nachury, Rohatgi, Stearns); architecture of neural circuits (Smith); molecular mechanisms underlying epithelial cell rearrangements during gastrulation (Nelson); actin filament assembly and dynamics (Nelson); molecular mechanisms of secretory vesicle docking and fusion (Pfeffer); development of neural synapses (Shen); mechano-electrical transduction in touch receptor neurons (Goodman); recombination and genetic engineering (Porteus); Hedgehog pathway signaling in development and cancer (Chen, Scott, Beachy, Rohatgi). These studies investigate critical functional and structural questions regarding fundamental cell biological processes and cover NIH research areas with implications for diverse aspects of human health and disease, including cancer, birth defects, obesity, kidney cysts (in the inherited Bardet-Biedl Syndrome) and neurodegenerative disease. All these projects require multi-channel super-resolution imaging and simultaneous multi-channel fast imaging; this combination of capabilities is most effectively provided by the requested OMX BLAZE 3D SIM system.

The organization of the nucleus and the regulated folding of the genome plays essential roles in regulating gene expression, chromosome segregation and chromosome structure. Long range interactions in chromatin are required for activation of gene transcription and repression of genes during the differentiation of eukaryotic cells. Long-range contacts between different chromosomal loci also regulate processes such as antibody diversity and mitotic chromosome condensation. Despite the wide range of processes that involve chromatin loops we know very little about the mechanisms that drive chromatin folding and stabilize long range interactions in the genome. This proposal is focused on developing new methods to measure the formation of looped domains dependent upon the activity of the chromatin protein CTCF. CTCF is known to be required for the stabilization of looped regions in the genome but how it generates or stabilizes looped domains is unknown. We propose to first characterize the dynamics of CTCF dependent looping using defined chromatin substrates in vitro and on chromatinized plasmids in cell extracts. Using mutagenesis and depletion we will alter the binding affinity and dimerization properties of CTCF and its interaction with the loop stabilizing protein cohesin to determine how these activities regulate the frequency of loop generation. Using the insight we gain from these in vitro experiments we will compare the dynamics of loop formation to the statistics of long range interactions at the human globin locus. By depleting CTCF and cohesin we will relate the cellular statistics of loop formation to the in vitro mechanics of loop stabilization. Our studies should provide unique and novel insight into the processes that regulate the formation of long range chromatin interactions and how they relate to essential developmental and cell biological processes.

DESCRIPTION (provided by applicant): The development of an adult multi-cellular organism from a single fertilized egg requires the proliferation and differentiation of a large number of cells. In many species, the early post-fertilization divisions occur rapidly and synchronously without growth phases and cell cycle checkpoints. These early embryos are almost entirely transcriptionally inactive and therefore driven by maternally supplied RNAs. At the Mid-Blastula Transition (MBT), the embryo initiates large-scale transcription from the zygotic genome and cells gain growth phases and checkpoints. Previous work suggested that the MBT is initiated by the increased DNA-to-cytoplasmic ratio resulting from repeated rounds of DNA replication and cell division without cell growth. This led to the hypothesis that the progressive titration of an inhibitory factor present in the embryo allows the initiation of zygotic transcription. To understand the nature of this inhibitory activity we replicated early studies, which had been performed in intact embryos, in Xenopus laevis egg extracts and showed similar DNA concentration dependent transcriptional repression. Using this cell free system we have shown that adding DNA-coated beads to extracts lowers the threshold DNA concentration for transcriptional activation indicating that DNA beads titrate the inhibitory activity. Pre-incubatio of DNA-coated beads in egg extract reduces the beads' ability to induce transcription when the beads are added to a naive extract. Thus we are now able to biochemically assay and quantify the inhibitory activity. Using this assay we propose to biochemically purify the inhibitory factor() from Xenopus egg extract and to understand how the factor(s) contributes to transcriptional induction during development. )

? DESCRIPTION (provided by applicant): The development of an adult multi-cellular organism from a single fertilized egg requires the proliferation and differentiation of a large number of cells. In many species, the early post-fertilization divisions occur rapidly and synchronously without growth phases and cell cycle checkpoints. These early embryos are almost entirely transcriptionally inactive and therefore driven by maternally supplied RNAs. At the Mid-Blastula Transition (MBT), the embryo initiates large-scale transcription from the zygotic genome and cells gain growth phases and checkpoints. Previous work suggested that the MBT is initiated by the increased DNA-to-cytoplasmic ratio resulting from repeated rounds of DNA replication and cell division without cell growth. This led to the hypothesis that the progressive titration of an inhibitory factor present in the embryo allows the initiation of zygotic transcription. Using a cel free system that recapitulates zygotic genome activation in vitro, we purified the transcriptional inhibitory activity present in the Xenopus egg cytoplasm and identified histones H3/H4 as DNA-titrated inhibitors of the MBT. Manipulating histone levels quantitatively shifts the onset of zygotic transcription and cell cycle lengthening in vivo, demonstrating a specific role for chromatin state in MBT initiation. This raises the question as to how histone titration and chromatin state are mechanistically linked to transcription and cell cycle duration. To address this, we will measure zygotic transcription and nucleosome occupancy genome wide at unprecedented temporal resolution through early development in control and histone manipulated embryos. We will determine mechanism linking DNA replication and histone levels. Successful completion of these aims will identify the mechanism through which global histone levels can be used to coordinate transcription and cell division with development. Since activating zygotic transcription is the first major transition after fertilization in human embryos our work determining how chromatin based mechanisms regulate the initiation of transcription will also provide insight global gene regulation and developmental reprogramming in the early vertebrate embryo.

In addition to the transcription of protein coding genes in the genome, a large amount of transcription encodes RNA molecules that do not generate mRNA. These noncoding RNAs play important roles in the cell that include regulating dosage compensation, controlling genomic imprinting and regulating transcription. However, human cells transcribe thousands of noncoding RNAs and we have only ascribed functions to a small number. One of the main challenges to understanding the functions of noncoding RNAs is that technologies to rapidly identify and characterize noncoding RNAs are lacking. In this proposal, we develop a novel method that makes it possible to identify, in any cell type, all of the noncoding RNAs that interact with chromosomes and at the same time map the sites where those RNAs bind chromatin. Our approach involves directly linking noncoding RNAs to the underlying DNA by generating a covalent chimera between a chromosome bound RNA and DNA. Using next generation sequencing, we can identify the RNAs in the cell that are likely to regulate chromosome structure or function and define their sites of action on the chromosome. In our first Aim we use Drosophila cells to develop this approach, taking advantage of the fact that established chromosomal RNAs, roX1 and roX2, are known to coat the X chromosome to accomplish dosage compensation in the fly. We then broaden this approach in Aim 2 and identify the RNAs that bind chromatin throughout the human genome and develop a new analytical infrastructure to classify and functionally assign these RNAs. In Aim 3 develop perturbation experiments to test the functions of noncoding RNAs and RNA motifs for their impact on local chromosome accessibility, histone modification state and transcriptional output. We apply a system to redirect noncoding RNAs to new genomic regions to test their functional impact on chromosomes and to regulate different genomic regions through RNA dependent control. By defining the landscape of chromatin associated RNAs in humans and the sites that they regulate in the cell our proposal how these RNAs function as well as the impacts of defects in RNA dependent control that result in cellular dysfunction.

Abstract Myosin molecular motors play crucial, dynamic roles in most cellular processes, including contraction, movement, and shape change. A variety of diseases owe their origins to defects in the myosin family of molecular motors. A prime example is inherited familial hypertrophic cardiomyopathy (HCM), which leads to hyper-contractility of the heart. HCM results from mutations in various cardiac muscle proteins, with mutations in ?-cardiac myosin and its associated thick filament protein myosin binding protein C (MyBPC) accounting for about 80% of these cases. HCM is not rare, affecting as many as 1 in 200 people.  Current therapeutic interventions for cardiomyopathies are limited to symptomatic relief, in large part because the molecular underpinnings of the disease ? how mutations affect the biomechanical interaction of myosin with its sarcomeric partners, and thus sarcomeric force, velocity, and power output ? are not well understood. Studies using human ?-cardiac myosin have shown that mutation-induced changes in the basic biochemical and biomechanical parameters of the myosin motor do not adequately account for the cardiac hypercontractility that is a clinical hallmark of HCM. Rather, it has recently been shown that HCM-causing mutations in the myosin motor domain disrupt intramolecular interactions that stabilize a folded-back, off state of myosin. This results in an increase in the number of heads functionally accessible to interact with actin, which in turn may lead to hypercontractility. In this proposal, the effects of HCM-causing point mutations in different regions of human ?-cardiac myosin will be explored, including the motor domain and both the proximal and distal portions of the alpha-helical coiled coil tail that allows myosin to form bipolar thick filaments. The interaction of myosin with MyBPC has also been implicated in regulation of the folded-back state of myosin. The effects of potential physiological regulators of this interaction, including phosphorylation and calcium binding, will be assessed using binding and functional assays. The effects of point mutations in different regions of myosin and in different domains of MyBPC will also be determined. Finally, a variety of structural approaches will be employed to determine the structure of the folded-back state of myosin in the absence and presence of MyBPC. Both negative stain and cryo-electron microscopy will be used to study the folded-back form of myosin and the myosin-MyBPC complex, and this work will be supplemented by cross-linking mass spectrometry to define interfacial residues. FRET probes will be placed on the human ?-cardiac myosin to observe its transition between the on-and-off states. This measurement will be critical in the HCM mutant myosins which have been shown to disrupt the off-state of myosin. Transient time-resolved FRET measurements will enable us to measure the nanosecond dynamics of myosin as it undergoes the on-to-off transition. Thus, we will use this more complex reconstituted system and an array of assays to determine the role of ?-cardiac myosin and MyBPC on the sequestration of myosin heads under physiological and pathophysiological conditions.