Kevin C. Wang, Ph.D. - US grants

DESCRIPTION (provided by applicant): Many diseases of the skin have unique anatomic manifestations that have important implications for diagnosis, prognosis, and treatment. It is well known that site- specific development in the skin is shaped by reciprocal epithelial-mesenchymal interactions. Discovering the molecular mechanisms that govern this interaction is vital to our understanding of skin disease. The HOX genes are transcription factors that are regulated in an exquisite temporal and spatial manner to specify proper positional patterning of body structures including the skin. Our laboratory has discovered that a new class of pervasive genes, long noncoding RNAs (lincRNAs), in the HOX loci show striking site-specific expression in skin fibroblasts. One lncRNA located at the distal end of the HOXA locus, termed HOTTIP, is expressed in dermal fibroblasts from distal and posterior body sites. Depletion of this RNA leads to a suppression of expression of contiguous distal HOXA genes, suggesting a role for this ncRNA in distal dermal and epidermal patterning at least in part through transcriptional activation of the HOXA locus. HOTTIP also binds to WDR5, a component of the chromatin modifying complex Mixed- Lineage Leukemia-1 (MLL-1), suggesting it may demarcate domains of chromosomal remodeling via interactions with MLL-1. The goals of this proposal are aimed at: 1) understanding the molecular mechanisms of HOTTIP-mediated gene regulation and transcriptional activation, and 2) defining the functional role for HOTTIP and other WDR5-binding lncRNAs in differentiation and developmental patterning in vivo. The results will expand our knowledge of the role of lncRNAs in skin fibroblast function and positional identity with implications for understanding clinically relevant issues in dermatology such as wound regeneration and site-specific disease manifestations. This award will enable the principle investigator, a dermatology trained physician-scientist, to receive intensive training in skin biology research and develop his own independent research program. Stanford University is providing him full institutional support, extensive resources, and opportunity for collaborations with experts in the field. The department has a strong track-record in mentoring dermatology physician-scientists to independent positions. He will be trained in ethical conduct, experimental design, and laboratory management to transition him to a full-time academic tenure-track position in translational dermatology research where he will spend 90% of his time on research and 10% on clinical activities. PUBLIC HEALTH RELEVANCE: This proposal is focused on understanding at the molecular level how skin from different parts of the body develops unique anatomical features. Many diseases of the skin, such as psoriasis and scleroderma, have site-specific manifestations. Understanding the mechanism of why this occurs has important implications for designing the best treatment options.

Atopic Dermatitis (AD), the most common chronic inflammatory skin disorder worldwide, is driven by both terminal keratinocyte differentiation defects and strong type 2 immune responses. What controls AD disease activity? Why is its response to therapy different from patient to patient? These are poorly understood problems and presents a large unmet need for both effective and safe therapeutics. In this project we seek to provide a molecular handle to explain some of these fundamental questions, by addressing a potentially under-explored source of immune regulation represented by epigenetic mechanisms. Our paradigm-shifting hypothesis centers on the epigenetic regulation of the immune response implicated in the pathogenesis of AD, and on a newly identified class of long noncoding RNAs (lncRNAs), the immune gene priming lncRNAs (IPLs), that exploit pre-formed chromatin topology at specific cytokine nuclear compartments to facilitate their epigenetic priming and activation. We propose to test and expand our hypothesis using technological advances that only now make this possible. I outline here a plan to pursue this opportunity with 3 specific aims. In Aim 1 (the R61 phase), we will establish the upstream molecular events that coordinate the epigenetic state of inflammatory genes. We hypothesize that IPLs are critical mediators that directly interact with the 3-dimensional chromatin architecture to prime chemokine promoters and enhance the pro- inflammatory responses in innate immune cell activation. We will characterize the mechanisms through which IPLs regulate IL-4 and IL-13, keystone interleukins critical to the induction and perpetuation of the Type 2 response in AD. In Aim 2, we will identity novel protein components that directly interact with the IPLs to modulate the chromatin landscape. We will also perform chromosome conformation capture across the IL-4/IL-13 locus to establish whether changing levels of IPL-IL4/13 alters chromosomal looping across the locus. Lastly, in Aim 3 (R33 phase) we will test inhibitors that specifically target IPL-4/13 in established murine AD-like models that recapitulate human AD. The IPL inhibitors will also be evaluated for their effects on the sensory responses known to influence AD behavior, to modulate direct neuronal priming by Type 2 cytokines shown to be a key step in the pathogenesis of AD. Taken together, our data will directly establish how IPLs and 3-dimensional chromatin architecture act in a cooperative manner to reduce gene-intrinsic noise, and allow robust activation of innate immune genes. A more precise fine-tuning of chemokine transcription through direct manipulations of IPL activities represents a highly valuable therapeutic strategy to achieve tailored immunomodulation in AD.

Limitations in current treatment options for many congenital and acquired diseases in humans, including birth defects, cancer, degenerative disorders and diabetes, highlight the need for the development of novel approaches for regenerative medicine to dramatically improve tissue repair. The pluripotent nature of mammalian embryonic stem cells (ESCs) makes them a convenient model for studying aspects of early development and an invaluable starting point for deriving numerous therapeutically relevant cells for regenerative medicine. Despite the remarkable progress made in deciphering mechanisms driving ESC pluripotency, fundamental gaps remain in understanding how human embryonic stem cells (hESCs) regulate the pluripotent state. If we are to use hESCs as a high-fidelity model for embryonic development, and if we wish to improve outcomes of hESC differentiation and the fidelity of cellular reprogramming to pluripotency, then it is imperative that we understand how hESCs fit into the paradigm of mammalian embryonic development. In this project we seek to answer some of these fundamental questions, by characterizing and validating a potential alternative pluripotency state. Our paradigm-shifting hypothesis stems from our unexpected discovery that the honey bee queen-maker protein, Royalactin, and its structural analog in mammals, Regina, have unexpected robust pluripotency maintenance effects in mammalian stem cells. We hypothesize that Regina/Royalactin stabilize and capture a pivotal pluripotent state distinct from the existing pre- (naïve) and post- (primed) implantation associated stem cell states. I outline here a plan to molecularly characterize this novel cellular metastable state with 3 specific aims. In Aim 1, we will Isolate and characterize the composition and activity of the receptor complex(es) in ESCs. We hypothesize that Regina/Royalactin, as secreted molecules, likely directly interact with a receptor partner on the membrane of responsive cells to affect gene expression and subsequent cellular behavior. We will identify the receptor(s) through multiple high throughput forward genetics and proteomic strategies. In Aim 2, we will derive and maintain murine and human ESCs to functionally demonstrate that the Regina/Royalactin-mediated state of pluripotency can be related back to the signaling pathways involved in lineage specification and maintenance in the embryo itself. Establishment of a new distinct stage of mammalian pluripotency will be an important advance in our understanding of early lineage commitment. Lastly, in Aim 3 we will elucidate and characterize the critical mechanisms that interface between Regina/Royalactin and downstream epigenetic and transcriptomic events. The genome-wide analyses will be compared to current established conditions to determine whether genetic and epigenetic instability of the ESCs, associated with impaired developmental potential, exists. Taken together, our data will directly establish how a novel endogenous mammalian pluripotency factor instigates fate decisions in ESCs, and provide a new platform to study the principles governing cell potency, epigenetic regulation, and the mechanisms that regulate developmental processes in naïve pluripotent stem cells.