David L. Spector, Ph.D. - US grants

microscopy; biomedical equipment resource; biomedical equipment purchase;

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop a complete understanding of the spatial and temporal events involved in the regulation of gene expression in living ES cells as they transit from the pluripotent to the differentiated state. An understanding of the nuclear choreography of essential genes regulating the earliest stages of ES cell differentiation is integral to understanding the mechanisms that help shift the ES cell transcriptional program to cell-type specific gene expression and will provide the basis of identifying how alterations in these events alter development. Here, a series of Aims are proposed that will address questions relating to the impact of gene positioning and dynamics on transcriptional regulation using both live cell imaging and molecular approaches. Studies will focus on two transcriptional paradigms: (1) genes that go from the "on" to the "off" state, and (2) genes that switch to monoallelic expression upon commitment to differentiation. The nuclear position of a series of 4 genes encoding the transcription factors Oct4, Nanog, Sox2, and Klf4 will be examined, as transcriptional silencing of these genes serves as a trigger for the initiation of the differentiation process. Changes in the nuclear position/associations of one or more of these genes during the initiation of differentiation will be identified and characterized and the relationship to the transcriptional output of the genes will be examined at the single cell level. Based on these findings a live cell imaging system will be developed to allow the direct study of the spatial and temporal dynamics of the identified gene movements in living cells over time. This system will provide the visualization of the gene and its mRNA transcripts and will allow one to determine if changes in gene positioning/associations is the "cause" or "effect" of changes in the transcriptional output of these genes. Together, these proposed live cell imaging experiments will define the nature of allele dynamics and interactions, and provide insight into the functional consequences of such dynamics/interactions. In the final Aim the same differentiation paradigm will be used to identify genes that become monoallelically expressed upon differentiation. The active and inactive alleles will be characterized and a live cell imaging system will be developed to study the establishment of monoallelic gene expression. Together, the proposed studies will provide important insights into the underlying principles of nuclear organization and gene expression that will be critical to fully understanding the signals that regulate normal developmental progression and how they may be disrupted during disease progression and/or treatments. PUBLIC HEALTH RELEVANCE: This study will identify the nuclear dynamics/associations of a series of genes that are central to the transition of pluripotent embryonic stem cells to the differentiated state. The findings of this study will provide important insights into the underlying principles of nuclear organization and gene expression that will be critical to fully understanding the signals that regulate normal developmental progression and how it may be altered during disease progression and/or treatment.

microscopy; biomedical equipment resource; biomedical facility; transmission electron microscopy; confocal scanning microscopy; protein structure; nucleic acid structure; fluorescence microscopy; scanning electron microscopy;

DESCRIPTION: (Applicant's Description) Advances In-Situ Hybridization and Immunocytochemistry - October 14-27,1998 The Cold Spring Harbor Laboratory proposes to continue a course entitled "Advanced In Situ Hybridization and Immunocytochemistry," to be held in the Fall of 1998-2003. This is a short, two week, intensive course which trains students (ranging from graduate students to Principal Investigators) to enter directly into research that makes use of advanced and/or specialized techniques in microscopy to localize nucleic acids and proteins as well as to provide background on the biochemical concepts underlying these techniques. The techniques will be used to characterize copy number changes of oncogenes and tumor suppressor genes on the DNA-level, and to analyze the cellular distribution and the expression status of mRNAs and proteins relevant to human carcinogenesis (such as proliferation markers and tumor suppressor gene products). The course will enable students to gain first-hand experience in localizing genes, chromosomes, and mRNAs by fluorescence in situ hybridization (FISH), performing spectral karyotyping of human chromosomes, comparing the genomes of normal versus tumor samples by comparative genomic hybridization (CGH), preparing antibody preparations for immunolabeling, performing experiments using different antigen-antibody systems (fluorescence, enzymatic, colloidal gold), as well as examining cells using different state-of-the-art microscopic systems (epifluorescence microscopy, confocal laser scanning microscopy, transmission electron microscopy). The course is taught by internationally recognized leaders in the field who provide hands-on training. Lecturers are invited who give up-to-the-minute reports on current research.

[unreadable] DESCRIPTION (provided by applicant): Our long-term goals are to understand the spatial and temporal events involved in gene expression/silencing in living cells. Understanding the dynamics of these events in vivo will provide the basis to identify changes that occur in cells or tissues associated with various disease states. We propose to utilize a live cell system to allow us to visualize a fundamental cellular process, gene silencing, at the levels of DNA, RNA, and protein, directly in living cells. This system will be used to assess the assembly and dynamics of Polycomb group proteins at a regulatable genetic locus. Using this system we will characterize the temporal pattern of factor arrival and release, and correlate it to changes in chromatin structure. In addition, we will study the dynamics of this specific genetic locus during the transition from the "on" to the "off" transcriptional state, in relation to RNA clearance and nuclear organization. The development and utilization of this live cell system will allow us to gain important insight into the spatial and temporal aspects of gene silencing that cannot be obtained by previously used approaches. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): A group of 5 NIH funded investigators from Cold Spring Harbor Laboratory are requesting funding to purchase a Perkin Elmer UltraVIEW RS-5 live cell imaging system with a Digital Diaphragm to allow for 4-D multi-channel analysis, FRAP and photo-activation studies. This microscope will be used to address immediate questions in the areas of Gene Expression, DNA Replication, Apoptosis, Cellular Senescence and In Vitro Studies of Breast Cancer. These studies cannot currently be performed at Cold Spring Harbor Laboratory due to a lack of the appropriate instrumentation. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop a complete understanding of the spatial and temporal events involved in the regulation of RNA polymerase II transcription and pre-mRNA processing in living cells. An understanding of these parameters in the context of the living cell and organism will provide the basis to identify changes in these events that occur in cells or tissues associated with various disease states. The short-term goals will utilize a previously developed live cell gene expression system to address how a specific genetic locus is modulated at the G2/M and M/G1 transitions in living cells. The spatial and temporal dynamics of factors involved in gene expression will be examined as well as how the transcriptional status of a gene is transmitted to daughter cells during mitosis. In addition, this system will be used to study critical unanswered questions relating to where and when components of the mRNP export machinery associate with nascent mRNPs and how mRNPs are transported through the nuclear pore complex in human cells. Finally, a series of highly innovative experiments will be initiated to bring real-time gene expression studies into the context of a living animal in order to study tissue specific dynamics and changes in gene expression upon skin differentiation. This approach will provide significant insight into gene expression within a tissue and allow one to directly access nuclear dynamics in the context of differentiation and morphogenesis in vivo. Together, these studies will provide significant insight into our understanding of the functional organization of the nucleus with regard to gene expression. Aberrations in nuclear organization have the potential to result in alterations in gene expression, the end product of which may be one of the many pathologies associated with human disease. These studies will bring us a major step closer to studying and evaluating disease models by in vivo molecular imaging and dynamics.

Project 3: D. Spector Long Nuclear-Retained Non-Coding RNAs and Cancer Hannon, Gregory J PROJECT SUMMARY (See instructions): Long nuclear retained non-coding RNAs (IncRNAs) represent a large and relafively unmined class of RNAs that are likely to play critical roles in gene regulafion and disease etiology. A major challenge is to understand the molecular functions of specific IncRNAs both at the cellular level and within the context of an organism. The long-term goal of this project is to identify and characterize IncRNAs that play a critical role in mammary gland development and the inifiation and progression of breast cancer. Here, a series of Aims are presented to dissect out the role of Malati, an abundant IncRNA localized to nuclear speckles and focally amplified in a significant number of metastatic breast cancers. Studies are proposed to develop innovative loss-of-function and gain-of-function mouse models combined with cell biological approaches to assess the function of Malati in normal development and in breast cancer initiation and metastasis. The impact of alterations in the level of Malati on tissue organization will be examined, and its effect on alternative splicing in a tissue-specific manner will be pursued by next-generation RNA-sequencing analyses. Complementary cell biological studies will delve into the role that Malati plays in nuclear organization and its impact on the dynamics of pre-mRNA splicing factors enriched in nuclear speckles. The Malati RNP will be purified using an RNA-tagging strategy and its proteome will be characterized in order to idenfify proteins responsible for its nuclear retention and to provide additional insight into its function. In the final Aim a series of newly identified IncRNAs, that are misregulated in breast cancer, will be prioritized and several will be selected for functional analyses, to identify genes and pathways that they target, and to elucidate the mechanisms by which they contribute to breast cancer tumorigenesis. Together, the proposed studies will provide important insights into the role of several IncRNAs in normal development and cancer and will lead to new opportunities for the identification and characterizafion of a new and excifing class of potential therapeutic targets.

Long nuclear retained non-coding RNAs (IncRNAs) represent a large and relatively unmined class of RNAs that are likely to play critical roles in gene regulation and disease etiology. A major challenge is to understand the molecular functions of specific IncRNAs both at the cellular level and within the context of an organism. The long-term goal of this project is to identify and characterize IncRNAs that play a critical role in mammary gland development and the initiation and progression of breast cancer. Here, a series of Aims are presented to dissect out the role of Malati, an abundant IncRNA localized to nuclear speckles and focally amplified in a significant number of metastatic breast cancers. Studies are proposed to develop innovative loss-of-function and gain-of-function mouse models combined with cell biological approaches to assess the function of Malati in normal development and in breast cancer initiation and metastasis. The impact of alterations in the level of Malati on tissue organization will be examined, and its effect on alternative splicing in a tissue-specific manner will be pursued by next-generation RNA-sequencing analyses. Complementary cell biological studies will delve into the role that Malati plays in nuclear organization and its impact on the dynamics of pre-mRNA splicing factors enriched in nuclear speckles. The Malati RNP will be purified using an RNA-tagging strategy and its proteome will be characterized in order to identify proteins responsible for its nuclear retention and to provide additional insight into its function. In the final Aim a series of newly identified IncRNAs, that are misregulated in breast cancer, will be prioritized and several will be selected for functional analyses, to identify genes and pathways that they target, and to elucidate the mechanisms by which they contribute to breast cancer tumorigenesis. Together, the proposed studies will provide important insights into the role of several IncRNAs in normal development and cancer and will lead to new opportunities for the identification and characterization of a new and exciting class of potential therapeutic targets.

Microscopy Shared Resource - Project Summary/Abstract The Microscopy Shared Resource was developed in concert with the initiation of the Cold Spring Harbor Laboratory (CSHL) Cancer Center in 1986, and has been continuously serving Cancer Center members since that time. The Microscopy Shared Resource is widely used; over the last funding period, it was used by 78% of the Cancer Center membership (29 members). The Shared Resource provides Cancer Center members with access to state-of-the-art microscope instrumentation and sophisticated technical expertise. Among the types of services rendered are wide-field fluorescence microscopy, deconvolution microscopy, confocal microscopy including point-scanning and spinning disk, live cell imaging, super-resolution structured illumination microscopy, standard transmission electron microscopy, immunoelectron microscopy (enzymatic and gold labeling), and evaluation of subcellular fractionation procedures by negative staining or thin sectioning. In addition, training is available for several image analysis programs to allow users to analyze imaging data. All of these services are highly technical and labor intensive. Without the Microscopy Shared Resource, it would be extremely difficult for individual investigators to have access to this sophisticated instrumentation and these types of services, which are critical to their research programs. In addition, the Microscopy Shared Resource serves as a central focal point where users can interact, learn about new imaging technologies, discuss availability of fluorescent proteins/probes or antibodies, and gain insight into imaging approaches that will advance their science. Over the past funding period, the Microscopy Shared Resource moved into a newly renovated building with temperature controlled imaging rooms. In addition, CSHL has acquired support to purchase two new microscope systems: a Zeiss LSM780 point-scanning confocal microscope and a Perkin Elmer UltraVIEW VoX high speed spinning disk laser confocal microscope. In summary, the Microscopy Shared Resource provides the user group with a complete complement of state- of-the-art instrumentation and advanced technical support to help accelerate cancer research and discovery.

Gene Regulation and Cell Proliferation Program Program Leader: David L. Spector Program Co-leader: Christopher R. Vakoc Project Summary Alterations in the regulation of gene expression and promiscuous entry into the cell cycle are defining characteristics of human cancer cells. The Gene Regulation and Cell Proliferation Program (GR) represents an interdisciplinary program with the central aim focused on understanding the regulation of gene expression and cell proliferation in cancer cells. The program has three main focus areas: (1) RNA Biology, (2) Cancer Epigenetics, and (3) Cell Proliferation. Research in this Program over the past five years has produced major advances in our understanding of cancer promoting pathways and has led to the discovery of novel therapeutic strategies now under investigation in, or moving toward, human clinical trials. In the area of RNA biology, researchers in the GR Program have shown that machineries that regulate alternative pre-mRNA splicing reactions include a major class of oncoproteins in human breast and skin cancers. In addition, long non-coding RNAs have been causally linked to the differentiation and metastatic programs in breast cancer, thus motivating the evaluation of anti-sense oligonucleotide (ASO)-based therapeutics in pre-clinical models and in human clinical trials. Researchers investigating small RNA pathways have obtained insight into how cancer cells communicate with their microenvironment using exosomes and evade sensitivity to kinase-targeted therapeutics. An epigenetics focus of this program has led to the discovery that hematological malignancies exploit bromodomain-containing proteins to sustain oncogenic enhancer landscapes. This has led to a rationale to target specific bromodomains in cancer, an approach that is now under investigation in ealy stage clinical trials. Epigenomic profiling of mammary gland cell types is also revealing how a woman's risk for developing breast cancer can be modulated by transient signaling events during pregnancy. Studies in the area of DNA replication are focused on the mechanisms of origin recognition proteins and replicative DNA helicases, including those utilized by tumor-causing papillomaviruses. Technology development continues to be a major focus of the GR program. A novel CRISPR-based functional genomics strategy is revealing core gene regulatory circuitries that sustain the cancer cell state and is also identifying strategies to bolster blood stem cell self-renewal. High throughput screening of ASOs targeting long non-coding RNAs is leading to the identification of targetable vulnerabilites in breast cancers. A continued effort that balances basic research into molecular mechanisms with therapeutic development will lead to continued synergies among members of the Program that is fostered by the Cancer Center. Since 9/1/10, the GR Program published 126 cancer-related research articles, 30 (24%) involved multiple Cancer Center members; 11 (9%) from intra-programmatic collaborations and 25 (20%) from inter-programmatic collaborations. As of 8/1/15, scientists in the GR Program held $3.6 million of direct costs secured from NCI, other peer reviewed and non-peer reviewed, cancer-related research support. Of this, $3.2 million was from NCI and other peer reviewed cancer-related funding sources.

PROJECT SUMMARY - PROJECT 3 Long non-coding RNAs (lncRNAs) represent a large and relatively understudied class of RNAs that have great potential to provide novel opportunities to influence gene regulation and cancer biology. The long-term goal of Project 3 is to identify lncRNAs that are over-expressed in breast cancer, define their role in the disease, and develop approaches to manipulate their expression in vivo to impact breast cancer progression. Here, a series of Aims are presented to determine the roles of Malat1 lncRNA, and several recently identified lncRNAs - Mammary Tumor Associated RNAs (MaTARs) - that are expressed in mouse models of luminal B and Her2/neu mammary cancer. We showed that Malat1 is over-expressed in mammary tumors and its genetic loss or knockdown results in differentiation of the primary tumor and a significant reduction in metastasis. Studies are proposed to identify the steps in metastasis that are dependent upon Malat1 and the critical regions of the RNA contributing to its function. Mouse models containing GFP- or bioluminescently-labeled breast tumor cells will be used to follow metastasis and in vivo antisense oligonucleotide (ASO) knockdown will provide a means to reveal the specific dependencies of Malat1 in the metastatic process. 3D tumor organoid cultures will be used to ascertain functional domains within Malat1 and to identify critical regions that are essential for tumorigenesis. The promoters of mammary tumor genes that are impacted upon Malat1 knockdown are enriched in Sox5 and Tcfcp2l1 binding sites. Studies are proposed to examine the role of these transcription factors, in conjunction with Malat1, to regulate the differentiation of primary tumors and the significant reduction in metastasis upon Malat1 knockdown. A series of studies are proposed to determine the function of 4 recently identified MaTARs that are expressed in a mammary tumor-specific manner. Chromatin Isolation by RNA Purification (ChIRP) coupled to deep sequencing or mass spectrometry will be used to identify interacting DNA sequences and proteins. To evaluate the potential of these MaTARs as therapeutic targets genetic knockout mouse models will be established and crossed with mammary tumor models to assess the impact on tumor initiation, progression and metastasis. RNA-seq will be performed on knockout and wild-type tumors to identify global gene expression changes upon MaTAR loss. The expression level and breast cancer sub-type specificity of human MaTAR orthologs has been examined and will be correlated with overall and relapse-free survival, and metastatic incidence, and appropriate candidates will be studied in patient-derived xenograft mouse models. Together, the proposed studies will define the role of several lncRNAs in breast cancer biology, and determine their potential as therapeutic targets to impact breast cancer progression.

PROJECT SUMMARY The over-arching focus of my research program has been to identify spatial and temporal aspects of regulating gene expression. Our recent emphasis has incorporated mouse embryonic stem cells (ESCs) as a model system to understand gene dynamics and various aspects of gene expression, including the role of long non- coding RNAs (lncRNAs). LncRNAs represent an exciting class of tens of thousands of RNAs among which very few have thus far been functionally characterized. A number of lncRNAs have been shown to exhibit cell- type specific expression, localization to subcellular compartments, and association with human diseases suggesting that they have critical roles in many cellular processes. The long-term goal of this project is to identify the mechanism of action of several lncRNAs identified in an RNA-seq screen that have roles in pluripotency and/or differentiation, and potentially represent different functional classes, Such analysis will provide insight into how these lncRNAs regulate differentiation through their impacts on gene expression and/or nuclear organization. Here, we propose to characterize an exciting nuclear-retained lncRNA, Platr4, that is highly expressed in pluripotent ESCs and is significantly down-regulated upon ESC differentiation. We will examine the impact of Platr4 knockout and over-expression on global gene expression in ESCs. In addition, Platr4 expression will be examined throughout embryonic development and the impact of Platr4 knockout will be examined in developing mouse embryos to identify critical points of function and candidate genes and pathways regulated by Platr4 in early mouse development. Based upon preliminary studies, the role of Platr4 in mesodermal and endodermal lineage commitment will be an immediate focus of investigation. The functional interactions of Platr4 will be identified by an RNA-tagging strategy combined with RNA-seq (ChIRP-seq) to identify genomic interactions of Platr4, and with mass spectrometry (ChIRP-MS) in order to identify proteins interacting with Platr4. Together, the proposed studies will provide important insights into the functional role of Platr4 in lineage commitment and differentiation, and will provide new insights as to how regulatory signals instructed by a long non-coding RNA can impact differentiation and potentially reprogramming.