Howard Y. Chang - US grants

DESCRIPTION (provided by applicant): Fibroblasts are the principal cells that reside in the dermis of the skin and stroma of all vertebrate organs. Although fibroblasts were thought by many to be homogenous, we have discovered that human fibroblasts from different anatomic sites of the skin and internal organs constitute many distinct, differentiated cell types that carry out unique genetic programs to specify the site-specific design and functions of epithelial organs. The broad goal of this project is to understand the physiologic specialization and developmental regulation of fibroblasts by following the expression level of a large number of genes in cultured fibroblasts. Firstly, using high density cDNA microarrays and computational bioinformatics, we will create a comprehensive molecular map of fibroblast gene expression patterns from diverse site in the human body. This large compendium of fibroblast gene expression patterns will reveal how many different types of fibroblasts exist, how they are related to one another, and where they are located in the human body. The gene expression programs that characterize specific fibroblasts will define the physiologic specialization of different fibroblast cell types and will help to explain the inductive potentials of fibroblasts in development, their functions during tissue repair, and the anatomic specificity of diseases affecting skin and connective tissues. Secondly, the site-specific gene expression profiles of fibroblasts will be used as tools to understand how different types fibroblasts develop. By assessing the gene expression patterns of fibroblasts from different sites cultured together and of fibroblasts cultured in isolation from one another, we will address whether fibroblasts instruct each other to adopt site-specific fates, or whether the differentiation program is controlled autonomously within each cell. Thirdly, adult fibroblasts have the unique property of maintaining positional memory, preserved in the form of Hox gene expression patterns established during embryogenesis. Hox genes encode a family of transcription factors that activate other genes to specify positional identities in development. We will test whether Hox genes are master regulators of fibroblast differentiation by assessing the gene expression patterns of fibroblasts with ectopic or inactivated Hox functions. From the proposed experiments, we hope to learn a great deal about the gene expression programs in fibroblast differentiation. This knowledge will provide many basic insights into wound healing, organ development, anatomic specificity of genetic and acquired diseases affecting the skin and connective tissues; and shed light on how positional identity is acquired and maintained in the human body.

[unreadable] DESCRIPTION (provided by applicant): The human body has a tremendous capacity for healing, but the ability of cells to grow, regenerate tissues, and recruit new blood vessels may also be misused during cancer progression. In many common cancers, especially breast cancer, the ability of tumors to express genes seen normally in a model of wound response is a powerful and accurate predictor of subsequent metastasis. We discovered that specific genetic mutations in human breast tumors drive expression of the wound response signature. Amplification of two genes, CSN5 and MYC, activate the wound response signature and causes increased cell division and invasion. CSN5 activates the post-translational modification of the MYC oncoprotein by ubiquitin, leading to activation of MYC's activity. However, molecular mechanisms of wound signature activation as well as its pathogenic mechanisms in cancer progression vivo are unclear. Therefore, we propose to study how CSN5 and MYC activate the wound signature and breast cancer metastasis in two aims. Firstly, we will determine which enzymatic activities of CSN5 and its associated COP9 complex are important for CSN5 to activate MYC and enhance tumor progression in vivo. An isopeptidase activity of CSN5 is highly amenable to inhibition by small molecule drugs and therefore could present an attractive therapeutic target in breast cancer. Secondly, we will determine how CSN5 selects between two antagonistic ubiquitin ligase pathways to activate MYC. These studies will elucidate a novel pathway implicated in breast cancer progression and pinpoint specific targets for therapeutic intervention. Breast cancer is the second most common cause of cancer death for women in the United States. These deaths are most often caused by the spread of breast cancer to other sites of the body, a process that involves genes normally reserved for wound healing. Research on the wound response genes in breast cancer will help to improve the risk assessment of breast cancer patients and identify targets for cancer therapy. [unreadable] [unreadable] [unreadable] [unreadable]

Project Summary Proper control of gene expression is essential for life. While substantial advances have been made in the discovery of DNA sequences and transcription factors that act combinatorially to regulate transcription, much less is known about later steps in the gene expression program. Nevertheless, it is now clear that substantial regulation of gene expression occurs post-transcriptionally, in pre-mRNA splicing, RNA localization, translation, and RNA decay, and in the coordination of RNAs by RNA binding proteins (RBPs), microRNAs, and RNA chemical modifications. While many sequence motifs are known to mediate post-transcriptional regulation, RNA secondary structures that govern access to these motifs are much less well understood. The long term goal of this project is to enable structural characterization of RNAs on a genome-wide scale. First, we will apply a recently developed method to map secondary structures of most human RNAs in living cells. Second, we will develop methods to follow the fate of RNA secondary structures through the RNA life cycle. Third, we will develop high throughput methods to perturb and test functions of RNA structures. This integrated pipeline of new technologies will enable investigators to identify and decode regulatory RNA elements in the genome with unprecedented speed and accuracy.

[unreadable] DESCRIPTION (provided by applicant): The human body has a tremendous capacity for healing, but the ability of cells to grow, regenerate tissues, and recruit new blood vessels may also be misused during cancer progression. In many common cancers, especially breast cancer, the ability of tumors to express a set of 512 genes seen normally in a model of wound response is a powerful and accurate predictor of subsequent metastasis and death. The prognostic information of the 512 gene "wound signature" was independent of and more informative than traditional clinical and pathologic risk factors. The wound signature was also independent of previously identified prognostic signatures and molecular markers. However, the large number of genes involved renders the wound signature difficult to implement in daily clinical practice. The central goal of this proposal is to develop technologies that will greatly simplify the diagnosis of the wound signature. We propose to develop two technologies toward this end. First, we will develop a method to diagnose the wound signature based on detecting DNA amplification of its two regulator genes. Our recent discovery that the wound signature is a consequence of amplification of two key growth regulator genes predicts that measuring the regulator genes will be sufficient to diagnose the wound signature and to prognosticate patient outcome. Second, we will develop a novel gene picking algorithm and RNA amplification technology that can reproduce the prognostic power of the wound signature by measuring only a small fraction of gene transcripts from tumor samples. At the end of the funding period, we envision the capacity to diagnose the wound signature from standard formalin-fixed, paraffin embedded tumor sections. [unreadable] [unreadable] Breast cancer is the second most common cause of cancer death for women in the United States. These deaths are most often caused by the spread of breast cancer to other sites of the body, a process that involves genes normally reserved for wound healing. Improved technology to identify the wound response genes in breast cancer will improve the risk assessment of breast cancer patients and better guide patients to appropriate therapies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This project covers 3 thematic areas: Applying Genomics and Other High Throughput Technologies, Translating Basic Science Discoveries into New and Better Treatments and Reinvigorating the Biomedical Research Community. Somatic cells are highly stable in adult animals due to robust gene expression patterns, which are stabilized by epigenetic mechanisms. The seminal invention of induced pluripotent stem (iPS) cells, however, provided the surprising conclusion that the differentiated state can be reversed by simple expression of four transcription factors (TFs). This finding proved that even supposedly stable epigenetic modifications of genes are essentially controlled by TFs. We asked whether this concept can be extended to trans-differentiation of one cell type into another, and recently succeeded in converting mouse fibroblasts directly into functional neurons, referred to as induced neuronal (iN) cells, by overexpression of only three lineage-specific TFs. Our findings indicate that TFs suffice to not only reverse a particular pathway of differentiation, but also to redirect the transcriptional regulatory network in a cell into a completely different pathway. This fundamental result answered one of the key open questions in the field, and is the basis of the current proposal. Apart from documenting the dominance of TFs over epigenetic modifications, iN cells could represent an attractive way to derive patient- specific neurons from skin fibroblasts. This may be used to model various neurological diseases or for cell transplantation therapy. This proposal aims to characterize the process of iN cell generation on the molecular level, with the expectation to gain fundamental insights into the biology of the underlying trans-differentiation process. In addition to identifying the molecular events underlying the fibroblast-to-neuron conversion, this study will in particular assess the epigenetic stability of the iN cell state as well as their safety with respect to their potential tumorigenicity, key prerequisites for clinical application of iN cell technologies. Our multidisciplinary approach entails state-of-the art high-throughput sequencing technologies for genome-level interrogation of epigenetic states and transcription, newly developed microfluidic devices enabling genome- wide analyses of small cell populations as well as multiplex gene expression on the single cell level allowing the determination of cellular heterogeneity, electrophysiology, and neurodevelopmental techniques. PUBLIC HEALTH RELEVANCE: This application will develop methods to generate neurons directly from non-neuronal cells, allowing the production of neurons from skin fibroblasts of human patients. Patient-derived neurons could be used for modeling neurological diseases or as cell grafts to treat neurodegenerative diseases like Parkinson's disease.

DESCRIPTION (provided by applicant): Epigenetic aberrations are a key driver of cancer pathogenesis. Long intergenic noncoding RNAs (lincRNAs) have emerged as a pervasive and important class of regulators that serve as the interface between DNA and chromatin modification machinery. The lincRNA HOTAIR is overexpressed in approximately a third of human breast carcinomas and is a powerful predictor of eventual metastasis and death. HOTAIR reprograms the breast cancer chromatin state to alter the positional identity of the cancer cells, enabling invasion and metastatic spread to distant organs. The long term goal of this project is to understand the mechanistic basis of lincRNA action in human cancers. First, using HOTAIR as a model system, we will address the structure-function relationship of a lincRNA and its oncogenic activities. Second, we will examine how HOTAIR targets specific genomic loci to relocalize Polycomb proteins. Third, we will determine whether HOTAIR may serve as a therapeutic target by testing its ongoing requirement during cancer progression. These experiments will provide key insights into how long noncoding RNAs may instigate cancer progression, and should pave the way for new cancer diagnostics and treatments in the future.

DESCRIPTION (provided by applicant): Long noncoding RNAs (lncRNAs) have emerged as a pervasive and important class of regulators of gene expression and chromatin states. We discovered that numerous lncRNAs are induced by DNA damage, and actively regulate the cell's response to DNA damage, including those elicited by environmental toxicants. The long term goal of this project is to understand the regulation and functions of lncRNAs in response to environmental exposures. First, we will identify lncRNAs and open chromatin sites induced by diverse genotoxic agents. Second, we will determine how the DNA damage-inducible lncRNAs modulate the activity of p53, a key transcription factor in the damage response. Third, using two new genomic technologies, we will identify the chromatin regulatory functions and gene targets of toxicant- induced lncRNAs. These experiments will provide key insights into how incipient environmental stress is transmitted into long term changes in gene expression, chromatin states, and ultimately distinct cell fates.

DESCRIPTION (provided by applicant): Despite the rapidly increasing capacity to sequence human genomes, our incomplete ability to read and interpret the information content in genomes and epigenomes remain a central challenge. A comprehensive set of regulatory events across a genome - the regulome - is needed to make full use of genomic information, but is currently out of reach for practically all clinical applications and many biological systems The proposed Center will develop technologies that greatly increase the sensitivity, speed, and comprehensiveness of understanding genome regulation. We will develop new technologies to interrogate the transactions between the genome and regulatory factors, such as proteins and noncoding RNAs, and integrate variations in DNA sequences and chromatin states over time and across individuals. Novel molecular engineering and biosensor strategies are deployed to encapsulate the desired complex DNA transformations into the probe system, such that the probe system can be directly used on very small human clinical samples and capture genome-wide information in one or two steps. These technologies will be applied to clinical samples and workflows in real time to exercise their robustness and reveal for the first time epigenomic dynamics of human diseases during progression and treatment. These technologies will be broadly applicable to many biomedical investigations, and the Center will disseminate the technologies via training and diverse means.

Funds are requested for the fabrication of computational hardware and software powerful enough to support the Bioinformatics analysis pipelines, which includes, processing, analyzing, storing, backing up, maintaining, and sharing of the data generated from high-throughput sequencing

Altered gene regulation underlies many facets of autoimmunity and its treatments. Pathogenic autoantibodies and immune complexes ultimately exert their effects through cellular signal transduction to impact gene expression. The central roles of specific transcription factors in driving immune cell fates, and anti-inflammatory and immunosuppressive drugs that control gene expression - such as steroids, cyclosporine A (CsA), and inhibitors of JAKs, lnterieukin-1 (IL-1), Tumor Necrosis Factor (TNF), and B Cell Activating Factor (BAFF) - suggest the importance of understanding gene regulation in autoimmunity. Rather than simply observing changes in gene expression, recent epigenomic tools have made it possible to determine the causality of gene expression, revealing the specific transcription factors and regulatory elements driving different gene expression programs. However, existing experimental methods require 10 million cells or more per assay, and are complex and laborious to perform. These limitations have largely kept epigenomic analyses out of the reach of the clinical studies of human diseases, including autoimmunity. Here we propose to develop and apply a revolutionary new method called ATAC-Seq to map open chromatin sites genome-wide, to enable facile and rapid epigenomic studies of patients with autoimmune diseases and their response to treatments in real time. We will also explore the role of epigenetics in known genotypes of SLE patients with single nucleotide polymorphisms in genes such as Tyk2, STAT4, and IRF5. The end result will be a set of robust biomarkers and important biological insights into autoimmune and inflammatory diseases. The long term goal of our studies is to include ATAC-Seq in the ACE Shared Research Agenda, where it can be used by ACE investigators as part of their basic science projects, and as a mechanistic assay in ACE clinical trials.

Project Summary Tens of thousands of human genomes have been sequenced, but the central challenge is their interpretation. A comprehensive set of regulatory events across a genome?the regulome?is needed to make full use of genomic information, but is currently out of reach for most clinical applications and biological systems. The proposed Center will develop technologies that greatly increase the sensitivity, speed, and comprehensiveness of understanding genome regulation. We will develop new technologies to interrogate the transactions between the genome and regulatory factors, such as proteins and noncoding RNAs from single cells, and integrate variations in DNA sequences and chromatin states over time and across individuals. Novel molecular engineering and biosensor strategies are deployed to encapsulate the desired complex DNA transformations into the probe system, such that the probe system can be directly used on very small human clinical samples and capture genome-wide information in one or two steps. These technologies will be applied to clinical samples with genomic aberrations to exercise their robustness, and reveal for the first time epigenomic dynamics of human diseases during progression and treatment. These technologies will be broadly applicable to many biomedical investigations, and the Center will disseminate the technologies via training and diverse means.

DESCRIPTION (provided by applicant): Despite the rapidly increasing capacity to sequence human genomes, our incomplete ability to read and interpret the information content in genomes and epigenomes remain a central challenge. A comprehensive set of regulatory events across a genome - the regulome - is needed to make full use of genomic information, but is currently out of reach for practically all clinical applications and many biological systems The proposed Center will develop technologies that greatly increase the sensitivity, speed, and comprehensiveness of understanding genome regulation. We will develop new technologies to interrogate the transactions between the genome and regulatory factors, such as proteins and noncoding RNAs, and integrate variations in DNA sequences and chromatin states over time and across individuals. Novel molecular engineering and biosensor strategies are deployed to encapsulate the desired complex DNA transformations into the probe system, such that the probe system can be directly used on very small human clinical samples and capture genome-wide information in one or two steps. These technologies will be applied to clinical samples and workflows in real time to exercise their robustness and reveal for the first time epigenomic dynamics of human diseases during progression and treatment. These technologies will be broadly applicable to many biomedical investigations, and the Center will disseminate the technologies via training and diverse means.

Project Summary The Chang lab has pioneered discoveries of long noncoding RNAs (lncRNAs) as a pervasive and important class of regulators in human diseases, notably in cancer. LncRNAs can serve as the interface between DNA and chromatin modification machinery, and thus mediate epigenetic aberrations in cancer. The lncRNA HOTAIR is overexpressed in approximately a third of human breast carcinomas and is a powerful predictor of eventual metastasis and death. The long term goal of this program is to understand the mechanistic basis of lncRNA action in human cancers. Our investigations will address (i) the roles of lncRNAs in driving cell-to-to cell epigenetic heterogeneity, a critical issue in cancer metastasis and therapy resistance, using cutting edge single cell epigenomic technology and in vivo genetic models; (ii) the emerging roles of RNA chemical modifications in cancer development; (iii) noncoding mutations that change RNA structure (?ribosnitches?) and use new epigenome editing technologies to pinpoint driver lncRNA genes in human cancers. These experiments will provide key insights into how long noncoding RNAs may instigate cancer progression, and should pave the way for new cancer diagnostics and treatments in the future.