Lin He - US grants

This award by the Chemistry Division supports a Research Experiences for Undergraduates (REU) site at North Carolina State University for the summers of 2006-2008. The program director is Lin He who is assisted by co-PI Paul Maggard. Nine students each year will do chemical research in the fields of materials science and life science. Special efforts will be made to assist students in career and academic decisions by broadening their perspectives on chemical research careers and by providing research opportunities for rising juniors/seniors of all backgrounds with demonstrated academic excellence. The program goals will be accomplished by developing a program that focuses on independent research projects as the central foundation, with incorporation of facility tours, a research presentation and conference participation that complements the research experience. Participants will have an exposure to government and industrial research experiences through invited seminars and on-site laboratory visits. Students will also have the opportunity to present their work at a National ACS Meeting in the spring following their summer internship. Recruiting efforts will focus on the inclusion of underrepresented minority groups, particular consideration will be given to rising juniors/seniors from HBCUs in the southeast region.

The Analytical and Surface Chemistry (ASC) program of the Division of Chemistry will support the CAREER development plan of Prof. Lin He of North Carolina State University. Prof. He's integrated research and education program focuses on the development of a simple yet sensitive molecular amplification method to detect DNA hybridization. Prof. He and her research group will employ a controlled polymerization process known as atom transfer radical polymerization (ATRP) to grow polymer brushes at DNA hybridization sites. The visible polymer growth will be indicative of the presence of specific DNA targets. The research of Prof. He will provide excellent training opportunities for graduate and undergraduate students, and postdoctoral research associates in a highly multidisciplinary area at the forefront of scientific research. Prof. He will integrate her research project with an on-going educational effort to increase involvement of undergraduate students from small local colleges and historically black colleges and universities (HBCUs), and high school students and teachers in bioanalytical and material science and technology.

[unreadable] DESCRIPTION (provided by applicant): Malignant transformation represents the phenotypic endpoint of successive genetic lesions that confer uncontrolled proliferation and survival, unlimited replicative potential, and invasive growth. Recent evidence has suggested that non-coding RNAs, in particular, microRNAs (miRNAs), are subjected to changes in gene structure and expression regulation in tumors. I identified a polycistronic miRNA cluster, mir17-92, as a target of chromosome 13q31 amplicon found in human B-cell lymphomas. In a mouse model for B-cell lymphoma, enforced mir17-92 expression cooperates with c-myc and accelerates tumor growth by repressing cell death. These findings provided some of the first functional evidence that changes in miRNAs could contribute to oncogenesis. The work described in this application continues my studies on the oncogenic effects of mir17-92 using both cell culture systems and animal tumor models. First, I propose to identify the oncogenic miRNA components within the mir17-92 cluster, and to dissect the molecular basis for the tumorigenic effects of mir17-92. Second, the effects of mir17-92 in tumor maintenance and therapy response will be investigated. Finally, combined expression studies, copy number studies and functional characterization will be applied to examine more broadly the miRNA pathways in the oncogenic and tumor suppressor network. These studies, if successful, will produce fundamental insights into the functions of miRNAs during tumor development and tumor maintenance, which can be applied for discovery of both diagnostic markers and therapeutic targets. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): p53, the most frequently mutated tumor suppressor, is regarded as the guardian of the genome. Intensive studies over the past three decades have placed p53 in the center of a complex molecular network regulating diverse physiological responses to cancer- related stress signals. In previous studies, the components of the p53 network solely consist of protein coding genes, including those acting upstream to regulate p53 activity, those functioning downstream to mediate p53 effects, and those forming a regulatory feedback loop regulating the dynamics of the p53 activity. Recent studies from our group and others have identified a family microRNAs (miRNAs), mir-34a, b and c, as bona fide transcriptional targets of p53. These findings, for the first time, revealed the interplay between proteins and non-coding RNAs in this most important tumor suppressor pathway. miRNAs are a novel class of small, regulatory non-coding RNAs that mediate post-transcriptional gene silencing of a large number of target mRNAs. The ectopic expression of mir-34 miRNAs mimics the biological effects of p53 in growth arrest and apoptosis, possibly through their ability to dampen the expression of pro-proliferation and pro-survival genes. Given these preliminary findings, we hypothesize that the endogenous mir-34 miRNAs are key effectors to mediate many of the p53 downstream effects, and that the functions of mir-34 have profound impacts on tumorigenesis and tumor maintenance. Using cell culture studies and mouse genetic studies, we propose to determine whether mir-34 miRNAs mediate p53 induced growth arrest and apoptosis, and to what extent mir-34 miRNAs contribute to p53 mediated tumor suppression and tumor regression.

1015026
Tirrell

Intellectual Merit:

This interdisciplinary program aims to bring new developments in self-assembled materials to bear on frontier problems in bioactive nucleic acid (NA) delivery and gene expression. Materials comprising nucleic acids and lipids, assembled based on a balance of electrostatic, hydrophobic and hydration forces, form stable, layered films, alternating nucleic acid and lipid layers. Recently published work from our group has shown that this structure can be manipulated in various ways, for example, by changing the temperature or state of hydration, or by varying the molecular weights of the nucleic acids included. This provides a versatile platform of potentially enabling technology to advance and develop new capabilities in gene delivery and the regulation of gene expression. The first aim of the proposed work is to optimize these constructs for the stated delivery applications. Preliminary work included in the proposal demonstrates that nucleic acid delivery, leading to transfection of stem cells, is possible with these constructs. Maximization of this capability will be explored by varying lipid choices (to have the best possible disassembly characteristics and to minimize any toxicity), by including multiple nucleic acids to have the desired structural features, and more importantly, to be able to have simultaneous, multiple transfection ability. The structures and disassembly profiles of all of these constructs will be thoroughly characterized. A second aim of this work, which will be conducted in parallel with the first, is to use these constructs to examine transfection efficiency for mouse embryonic stem cells in culture, using expression of green fluorescent protein as an indicator of efficiency. Stem cells are notably difficult to transfect; our aim is to exploit the high concentration of nucleic acids in our constructs, and the direct physical contact of the cells with the nucleic acid-lipid layers to increase transfection efficiency. A third aim of the work will be to apply our new material delivery vehicles to the delivery of microRNAs. MicroRNAs (miRNAs) are a novel class of small, regulatory non-coding RNA, serving as potent regulators for gene expression at posttranscriptional level. Contact-mediated delivery will be explored to accomplish this goal. The further goal of the third phase of this work is to integrate plasmid DNA and miRNA delivery to reprogram adult cells into induced, pluripotent stem cells. iPS cells have numerous profound scientific and biomedical implications in personalized therapies and platforms for high-throughput screening of pharmaceuticals. The technical challenge with microRNA delivery for reprogramming is not delivery efficiency, per se, but rather sustained delivery to achieve sustained expression over a span of approximately ten days.

Interdisciplinary Nature of the Proposed Research:

The team assembled spans several different disciplines from chemical engineering and materials science, to stem cell and tissue engineering, to the molecular and cellular biology of gene regulation. The proposed work will take a discovery in materials science quite far toward new enabling technology in engineering nucleic acid delivery and gene expression. The connection this team embodies, among chemical and materials engineering, biology and biological engineering, is essential to realize the potential of this new delivery system, both to have the insight into how to optimize the materials involved, and to assure that the biological engineering objectives are achieved in a meaningful way.


Broader Impacts:

An exciting, interdisciplinary development project such as this is an ideal opportunity to bring undergraduate engineering students to the forefront of an important research area. The very nature of this project, spanning three laboratories in chemical, materials and biological engineering, as well as molecular and cellular biology, gives capacity to bring undergraduates with varied interests into participation in this work. The specific plan is to engage two undergraduate students per year, in the summer (six total over the project lifetime), from underrepresented groups as participants in this research. These students will be admitted to the Amgen Scholars Summer Research Program at UC Berkeley. The Amgen Scholars Program is a national program attracting approximately 25 participants each year. Joining this group of 25, the two undergraduate participants will benefit significantly in numerous ways as members of the summer research cohort. They will participate in all program activities including weekly meetings and the poster session and oral presentations at the end of the summer. As a result of these collaborative activities, the undergraduate participants in this project will be fully involved in a broad and comprehensive summer experience.

DESCRIPTION (provided by applicant): Malignant transformation represents the phenotypic endpoint of successive genetic lesions that alter gene function and gene regulation in oncogene and tumor suppressor networks. While extensive investigations have characterized protein-coding gene functions in these molecular networks, the functional importance of non-coding RNAs (ncRNAs) is just beginning to be revealed. microRNAs (miRNAs) encode a class of small regulatory ncRNAs with great capacity for post-transcriptional gene regulation by targeting a broad range of mRNAs. Our previous studies identified an important miRNA oncogene, mir17-92, that promotes the development of B-cell lymphomas. mir17-92 encodes a polycistronic miRNA transcript that yields six individual miRNA components, which have distinct biological functions and differential gene regulation during tumorigenesis. This context-dependent differential regulation of polycistronic miRNA components could ultimately determine the oncogenic activity of mir17-92. Aberrant regulation of miRNA biogenesis, stability and function has been frequently observed in human cancer, yet the underlying molecular basis still remains unclear. This is largely due to the lack of an effective methodology to study cancer-related miRNA ribonucleoprotein (miRNP) complexes. Here, we propose to develop a powerful biochemical strategy to isolate and characterize specific miRNA ribonucleoprotein (miRNP) complexes with important functions during tumorigenesis. We engineered a bacterial endonuclease, Csy4, to achieve high affinity binding and inducible cleavage of a specific 16-nucleotide (nt) RNA sequence. Using this 16-nt RNA as a tag, we aim to isolate specific miRNP complexes both in vitro and in vivo. These proposed studies will allow us to identify important miRNP complex components that differentially regulate components of a polycistronic miRNA oncogene in cancer cells. More importantly, our technology can be easily adapted to study cancer related RNP complexes containing other ncRNA species. With the increasing number of functionally important ncRNAs in cancer, our proposed studies will bring fundamental insights into an area of cancer research that has been largely unexplored.

The PI has begun the development of an intriguing amplification alternative to PCR that could potentially be much simpler and more cost-effective. The PI's previous work on a free-radical "amplification?by-polymerization" approach demonstrated that it was possible to detect fewer than 2000 copies of DNA, with false positive and false negative rates comparable to PCR. This proposal seeks to extend this novel approach by using Ring-Opening Metathesis Polymerization (ROMP), as a way to avoid the problems oxygen plays with free-radical polymerization and to combine ROMP with lateral flow assay as the basis of a new fieldable biosensor. The proposed research is transformative and has the potential and has the potential to become the basis of a field assay and have an impact in several biomedical sensing areas. These results will be used to improve the efficiency of using ROMP for amplifying the presence of target analytes and could serve as a first-line screening for, e.g., cytomegalovirus (CMV). The PI has an excellent record for training underrepresented minorities at all levels and this proposed work will add to that outreach effort. Her productivity under the previous CAREER is quite good with 16 publications and 21 conference talks.

? DESCRIPTION (provided by applicant): Motile cilia are tubulin-based cell protrusions that beat in a coordinated fashion to mediate movement of extracellular fluids to generate directional flow. The functional importance of motile cilia are evident in a wide variety of developmental and physiological processes in complex multicellular animals. Structural and functional defects in motile cilia are associated with a variety of human conditions, including impaired mucociliary clearance in the airway, male and female infertility, cerebrospinal fluid flow, and left-right axis pattern formation. Although it has become increasingly clear that non-coding RNAs are integral components of the molecular network for development and disease, most studies on motile ciliogenesis to date have focused on the functional characterization of protein-coding genes. Our preliminary studies identified miR-34/449 miRNAs as the first non-coding RNAs that play an essential role in regulating motile ciliogenesis. The miR-34/449 miRNA family consists of six highly homologous and evolutionarily conserved miRNAs that collectively exhibit a high-level expression in all tissues containing motile cilia, and particularly, in multiciliated cells of the respiratory epithelia. The redundancy of the miR-34/449 family in the mammalian genome, combined with their dominant expression patterns in multiciliated cells, confer a robust functional regulation on motile ciliogenesis. mice deficient for all miR-34/449 miRNAs exhibited frequent postnatal mortality, strong respiratory dysfunction, and infertility. In particular, the postnatal mortality due to miR-34/449 deficiency was mostly caused by defective mucociliary clearance in the airway, which is largely due to ciliation defects in the respiratory such as mucociliary clearance in respiratory track, fertility, cerebrospinal fluid flow, and left-right axi pattern formation. Using mouse and frog genetics, cell biology and molecular biology approaches, we proposed to carefully characterize the ciliation defects in miR-34/449 deficient MCCs and animals both in development and in their physiological response to smoke exposure. In addition, we propose to elucidate the transcriptional regulation of miR-34/449 miRNAs during motile ciliogenesis, and to investigate the molecular and cellular mechanisms underlying the miR-34/449 functions during motile ciliogenesis. Taken together, these proposed studies will not only deepen our understanding on the molecular basis of motile ciliogenesis, but also provide important insights into the development of new diagnostic markers and therapeutical agents for treating respiratory conditions.

Project summary The choroid plexus is a highly vascularized, secretory tissue that protrudes into the brain ventricles, whose primary function is to produce cerebrospinal fluid (CSF). CSF provides nutritional and metabolic support for brain development and mediates efficient waste removal. Choroid plexus consists of an apical monolayer of ciliated epithelial cells that surround a stromal core of capillaries and connective tissues. Choroid plexus epithelial cells (CPECs) each forms one to two dozen cilia, which are microtubule-based organelles that project from the apical membrane. Emerging evidence has suggested the importance of cilia in choroid plexus development and function, as a connection has been established between a defective ciliogenesis in choroid plexus and the excessive CSF production in mice. Our preliminary studies identified a highly redundant miRNA family, miR-34/449 miRNAs, as the key regulators for CPEC ciliogenesis. The miR-34/449 family comprises six evolutionarily conserved, homologous miRNAs that are highly enriched in the ciliated CPECs. miR-34/449-deficient mice are characterized by reduced brain ventricle size, aberrant CPEC morphology and impaired brain development, suggesting a reduced CSF production due to defective choroid plexus functions. Interestingly, the earliest defect we can detect in miR-34/449-deficient choroid plexus is the aberrant ciliogenesis in CPECs, characterized by a significant increase of the cilium number per cell and a greater length of the axonemes. Given the functional importance of CPEC cilia in negatively regulating CSF production, we hypothesize that the miR-34/449 deficiency leads to excessive CPEC ciliogenesis, which represses CSF production and impairs brain development. Here, we propose to functionally characterize miR-34/449 miRNAs in CPEC ciliogenesis and CSF production, and to elucidate the underlying cellular and molecular mechanisms. Using genetic mouse models, choroid plexus in vitro culture, cell biology and molecular biology approaches, we will characterize the cellular and molecular defects in miR-34/449-TKO choroid plexus, and investigate the functional connection between CPEC cilia and choroid plexus function. We will also identify the key miR-34/449 targets that mediate CPEC ciliogenesis and CSF production. Our studies will not only reveal a highly robust regulatory mechanism for CPEC ciliogenesis, but also provide important insights into the functional importance of CPEC cilia in choroid plexus function.

In respiratory epithelia, goblet cells secrete mucus to trap foreign particles and invading pathogens; multiciliated cells (MCCs) provide synchronized beating of motile cilia, driving the extruded mucus out of the respiratory tract. The coordinated functions of MCCs and goblet cells constitute the basis for defense mechanism against respiratory infections. A single MCC contains hundreds of motile cilia that beat coordinately to generate continuous and directional movement of extracellular fluid for pulmonary clearance. Hence the motile ciliogenesis in MCCs is particularly important for the pulmonary defense in respiratory epithelia. Although it has become increasingly clear that non-coding RNAs are integral components of the molecular network for development and disease, most studies on motile ciliogenesis and MCC bioology have focused on protein- coding genes. Using mouse models, our preliminary studies identified miR-200 miRNAs with an essential role in respiratory epithelia. The miR-200 family consists of five highly homologous and evolutionarily conserved miRNAs that collectively exhibit a high-level expression in respiratory epithelia, and particularly, in multiciliated cells. The redundancy of the miR-200 family in the mammalian genome, combined with their strong expression patterns in multiciliated cells, confer a robust functional regulation on motile ciliogenesis. Mice deficient for all miR-200 miRNAs die postnatally, exhibiting strong respiratory dysfunction, excessive mucus accumulation and impaired motile ciliogenesis. Using mouse and frog genetics, cell biology and molecular biology approaches, we proposed to carefully characterize the phenotype in miR-200 deficient MCCs in mouse and in human, with a particular focus on motile ciliagenesis. In addition, we propose to investigate the molecular mechanisms underlying the miR-200 functions during motile ciliogenesis. Taken together, these proposed studies will not only deepen our understanding on the molecular basis of motile ciliogenesis, but also provide important insights into the development of new diagnostic markers and therapeutical agents for treating respiratory conditions.  

Project summary Lung cancer is the leading cause of cancer death worldwide, largely due to its highly metastatic nature. Hence, elucidating the molecular mechanisms for tumor metastasis remains one of the most pressing challenges in lung cancer research. To date, most studies on cancer metastasis have focused on protein-coding genes, yet it has become increasingly clear that non-coding RNAs, particularly, microRNAs (miRNAs), are integral components of the molecular network for cancer metastasis, Using a Kras-driven, p53 deficient lung adenocarcinoma mouse model, we compared the miRNA expression profiles between primary and metastatic lung tumors, and identified miR-200 miRNAs as the most downregulated miRNAs in lung cancer metastases. The miR-200 family consists of five homologous miRNAs located at two genomic loci: mir- 200b/200a/429 and mir-200c/141. To characterize miR-200 functions in lung cancer metastasis, we generated KrasLSL-G12D/+;p53fl/fl; mir-200c/141-/- (KP200cKO) mice, which exhibited a significant increase of tumor metastases within a short latency. Interestingly, all metastatic KP200cKO tumors examined exhibited a complete silencing of all miR-200 miRNAs, suggesting that a complete loss of miR-200 redundancy was essential for developing cancer metastasis in this model. Based on these preliminary findings, we hypothesize that miR-200 miRNAs are key repressors of cancer metastasis in Kras-driven, p53 deficient lung adenocarcinomas. Using mouse genetics, CRISPR genome editing, cell and molecular approaches, we propose to comprehensively characterize the importance of miR-200 miRNAs during lung cancer metastasis, and will elucidate the underlying molecular and cellular mechanisms that govern the biological functions and transcriptional regulation of miR-200 miRNAs. Our proposed studies will provide important insights into a highly robust mechanism to repress lung cancer metastasis.

Project summary Genetically engineered mouse models are an indispensable tool for studying human development and disease, enabling us to characterize gene functions in vivo, to investigate the cellular and molecular mechanisms of physiological and pathological processes, and to establish faithful disease models for drug screen. To date, most mouse models are engineered to recapitulate human disease phenotype, yet more sophisticated mouse models are in demand to study disease gene function in a native cellular background, to perform single cell based analyses and to set up high-throughput reporter-based screens for phenotypes or therapy. Fluorescent tagging of endogenous genes is a particularly powerful strategy, allowing the characterization of subcellular localization of proteins, identification of interaction partners, and isolation of specific cell populations in the context of development and disease. Engineering functional tags in an endogenous gene locus of interest preserves endogenous expression and minimizing genomic disruption, but the technology still remains inefficient, costly and laborious. We recently developed CRISRP-EZ (CRISPR RNP Electroporation of Zygotes), an electroporation-based technology that outperforms microinjection in efficiency, simplicity, cost, and throughput for mouse genome editing. Based on the CRISPR-EZ technology, we aim to develop novel technologies to achieve rapid fluorescent tagging of endogenous proteins with unprecedented efficiency, simplicity, throughput and cost saving. First, we will employ the self- complementing split-GFP system to achieve functional GFP tagging of endogenous genes in vivo using CRISPR-EZ. Second, we will develop CRISPR-READI (CRISPR RNP Electroporation and AAV Donor Infection) to engineer full length fluorescent protein tagging on endogenous loci in mice by AAV-mediated HDR editing. Taken together, our technologies will allow efficient and high throughput fluorescent tagging in mouse disease models for comprehensive mechanistic studies and powerful drug screening.

Cilia are microtubule-based cellular protrusions with diverse biological functions, including fluid movement, cellular locomotion, environmental sensing, and signal transduction. Traditionally, most cilia are classified based on differences in ciliary ultrastructure, biological function, and ciliary motility, with primary cilia and motile cilia as the major categories. The primary cilia functions as solitary sensory hubs to transduce extracellular stimuli into intracellular signaling pathways, and the motile cilia exhibited coordinated beating to generate directional fluid movement. Choroid plexus epithelial cells contain multi-sensory cilia that regulate the production of cerebrospinal fluid (CSF) to support neuronal development and physiology. Using serial transmission electron microscopy (TEM) and focus ion beam scanning electron microcopy (FIB-SEM), our preliminary results suggest that the multi-sensory cilia of choroid plexus represent a distinct type of cilia, exhibiting unique ultrastructural features, while resembling aspects of both primary cilia and motile cilia. Defective ciliogenesis in choroid plexus causes hydrocephalus, at least in part, due to CSF overproduction. Choroid plexus cilia are likely to play an important role in Shh signaling, as FoxJ1 deficient choroid plexus cilia no longer respond to Shh treatment in explant culture. We discovered a functional connection between Shh signaling and Aqp1 expression. Hence, we hypothesize that choroid plexus cilia are a unique type of multi-sensory that mediate Shh signaling to regulate CSF production, at least in part, by regulating the expression of water channels and ion transporters. Here, using a combined approach of advanced imaging techniques, mouse genetics, imaging studies, cell biology and molecular biology, we propose to study the ciliary ultrastructures, ciliogenesis mechanisms and biological functions of the multi-sensory cilia of choroid plexus. First, using electron microscopy, FIB-SEM and super-resolution imaging, we will characterized the ultrastructure of choroid plexus cilia, and define their developmental dynamics at different developmental stages. Second, we will employ mouse genetics and genomics studies to identify and characterize the ciliogenesis machineries of choroid plexus cilia. Finally, we will elucidate the molecular mechanisms through which dynamic choroid plexus cilia mediate the Shh signaling to regulate CSF production. Taken together, the proposed studies will structurally and functionally define a new type of multi-sensory cilia in choroid plexus, and will generate important insights on the molecular basis for the regulation of CSF production.