Elaine Fuchs - US grants

The objective is an understanding of the biochemical mechanisms that regulate the expression of human genes during differentiation. The human epidermis has been chosen as a model system, since we are able to serially cultivate these cells in vitro under conditions where many of the differentiative properties, including stratification, are retained. A simple squamous epithelium, the peritoneal mesothelium, has been chosen for purposes of comparison. These cells can also be cultured, using a similar system. The major differentiation-specific proteins of the epidermis are the keratins, a group of related proteins (M.W. 40-70kd) that form 8 nm cytoskeletal filaments in all epithelial cells. We have identified two distinct multigene families encoding the keratins. Pairs of these genes are differentially expressed in different epithelial cells and at different stages of keratinization. Commitment to terminal differentiation results in a shift in the synthesis of mRNAs encoding two pairs of smaller keratins to the synthesis of a new pair of larger keratins (MW 56.5 and 67K). We have found that epidermal cells in culture will terminally differentiate if the vitamin A in the medium is removed. In addition to causing increased stratification and s. corneum formation, removal of the vitamin regulates the synthesis of the mRNAs encoding the large keratins. In contrast, increasing the vitamin A level leads to the expression of two keratins (53kd and 40kd) not normally seen in epidermal cells, but made by some cancerous cell lines and also mesothelial cells. Recently, we constructed a cDNA library to human mesothelial mRNA. We have identified cDNAs encoding the keratins of the 53kd and 40kd keratins that are positively regulated by vitamin A. We now propose to isolate and sequence the genes for these proteins in order to define the regulatory sequences responsible for the tissue-specific and vitamin A-specific expression of these genes. We will utilize mammalian gene transfer systems to study the transcriptional and translational regulation of these genes. Finally, using purified cellular receptors for the retinoids, we will investigate whether retinoic acid and retinol act directly or indirectly to influence the expression of these specific keratin genes. It is our aim to elucidate the significance of the expression of this pair of vitamin A-regulated keratins in human epidermis and to determine the precise mechanism by which vitamin A differentially regulates their expression.

The long range objective of this research is an understanding of the biochemical mechanisms that operate and regulate the expression of human genes during growth and differentiation. The human epidermis has been chosen as a model system, since we are able to serially cultivate these cells in vitro under conditions where many of the differentiative properties, including stratification are retained. The major differentiation-specific proteins in these cells are the keratins, a group of closely related proteins of MW 40-70 K daltons that form 80 Angstrom cytoskeletal filaments. Only a subset of these keratins are ever expressed by an epidermal cell at any one time. During the course of differentiation, this subset changes concomitantly with an increase in keratin synthesis, leaving the fully differentiated epidermal cell with 85% of its total protein as keratins. We already know that early changes in the differentiation involve changes in mRNAs whereas late changes involve proteolytic processing. Recently, we isolated near full-length cDNA clones to the different epidermal keratins mRNAs and we showed that there are two distinct classes of keratins. We would now like to determine the nucleic acid sequence of these cDNAs. Since no amino acid sequence data is available, the amino acid sequence predicted from the cDNA sequence will be essential in determining the structural and functional relationship between these two classes of keratins. We will also isolate and characterize genomic clones for the keratins. This will aid us in determining the complexity and chromosomal organization of the keratin genes. We will then prepare subclones of these sequences to probe posttranscriptional regulation of keratin gene expression. Finally, epidermal cells from patients with genetic skin diseases are currently being cultured and alterations in the regulation and expression of the keratins and other differentiation-specific proteins in these cells will be investigated.

The long range objective of this research is an understanding of the biochemical mechanisms that operate and regulate the expression of keratin genes during differentiation and development in human epithelial tissues. The human epidermis has been chosen as s model system, since we are able to serially cultivate these cells in vitro under conditions where many of their differentiative properties, including stratification are retained. The keratins are the major differentiation-specific proteins in these cells, They are a family of >20 fibrous proteins (MW 40-67K) that form 8 nm filaments in the cytoskeletons of all and only epithelial cells. They appear to be specifically tailored to suit the varied protective and structural needs of each epithelial cell. Keratins can be subdivided into two distinct groups, type 1 and type 11, and a member of each is essential for filament assembly. Type 1 and type 11 proteins are frequently expressed as specific pairs, and typically 1-3 pairs are expressed at any one time. During the course of differentiation in epidermis, this subset changes concomitantly with an increase in keratin synthesis, leaving the fully differentiated cell with 85% of its total protein as keratins. Additional changes in keratin patterns are observed during development, in wound-healing, and in a number of human skin diseases, As such, the pattern of keratins expressed by an epithelial cell may provide a powerful diagnostic tool for genetic diseases and cancers of epithelia, In order to assess the potential value of keratin expression in medicine, it is essential that we understand the functional and structural significance of the multiplicity of keratin sequences and their role in differentiation and development. We have isolated and characterized a number of the human genes encoding these keratins, and we have prepared specific cRNA probes to detect different keratin mRNAs, and monospecific antibody probes to detect different keratin proteins. We are currently using these tools to 1) examine the role of different keratin sequences in filament assembly; 2) examine the mechanism by which specific keratin pairs are selected from the repertoire of keratin genes, and how the two types are regulated in balance; 3) explore the function of keratins using an in vivo as well as an in vitro approach; and 4) examine the molecular basis of altered keratin expression in certain genetic skin diseases.

neoplasm /cancer; molecular biology; cell cycle; cell differentiation; tissue /cell culture;

Our objective is to elucidate the mechanisms underlying growth and differentiation in human epithelial tissues. The human epidermis has been chosen as a model system, since we can cultivate these cells under conditions where differentiative properties, including stratification and keratinization, are retained. The major epidermal proteins are keratins. Basal keratinocytes express K14 (50 kd) and K5 (58 kd). As cells undergo terminal differentiation, they downregulate K5 and K14 mRNAs and induce expression of K1 and K10. This switch may enhance filament bundling, an early indication of differentiation. In squamous cell carcinomas, K1 and K10 is reduced, and K6 and K16 appear in suprabasal cells. These keratins are not normally expressed, but are induced transiently during wound- healing. We have shown that both normal and abnormal differentiation are controlled negatively by vitamin A. When elevated to ~10X higher than physiological, synthesis of all suprabasal keratins are downregulated. SQCC cells are more sensitive to retinoic acid (RA) than normal cells, and differentiation can be completely blocked with RA treatment of SQCC lines. Prerequisite to understanding how retinoids control differentiation and why they are useful therapeutically is to elucidate the biochemical behavior of basal cells, i.e. the precursors of the differentiating cells. Central to this study is investigating the sequences and factors controlling basal keratinocyte genes. We have isolated the functional K5 and K14 genes. Using a reporter or tagged K14 gene, coupled with (1) transfection of cultured keratinocytes, and (2) transgenic mouse technology, we have begun to elucidate mechanisms underlying K14/K5 gene expression. Parallel to these studies, we will explore events leading to Ra-mediated inhibition of differentiation. We will examine the kinetics and level of regulation of each Ra-induced change. Of particular interest will be whether K5 and K14 and their keratinocyte transcription factors are regulated by retinoids. Finally, we will (1) examine the role of the steroid hormone-like retinoic acid receptors (RARs) in controlling the RA response, and (2) investigate whether these receptors are involved in the induction of expression of autocrine regulators, including TGF-alpha and TGF-betas, which may act in conjunction with vitamin A to control growth and differentiation. A knowledge of the factors controlling this balance is a prerequisite to understanding how this equilibrium goes awry in the course of human diseases, e.g. psoriasis and basal- and squamous- cell carcinomas. The global aim of this proposal is to coordinate the molecular biology of human differentiation with the application of this research to medicine.

embryonic stem cell; tissue /cell culture; biomedical facility; genetically modified animals; laboratory mouse;

DESCRIPTION (provided by applicant): Our global objective is to elucidate the mechanisms underlying tissue homeostasis and regeneration in mammalian epidermis and its appendages, and to understand how this process goes awry in human skin cancers. A central issue in achieving this goal is to understand how adult skin maintains and transcriptionally regulates a pool of stem cells in the hair follicle (HF-SCs) that drive new growth during the hair cycle and repair the epidermis upon injury. At the start of each new hair cycle, two key signals--Wnts and Bmp inhibitory factors-- converge to stimulate HF-SCs to stabilize 2-catenin and generate proliferative but transient progeny that will then differentiate to make hair. Increasing evidence points to 2-catenin's near universal role in regulating SC proliferation and/or fate determination, and excessive stabilized 2-catenin is frequently seen in human cancers. 2-Catenin acts as a co-factor for the Lef1/Tcf family of DNA binding proteins. In HF-SCs, two family members-- Tcf3 and Tcf4--are co-expressed, where they function redundantly to repress differentiation in the absence of Wnt signaling and promote proliferation and fate commitment in its presence. Interestingly, in cultured embryonic stem cells (ESCs), Tcf3 is an integral component of the pluripotency regulatory complex (Sox2, Oct4, Nanog), not present in adult SCs. How Wnts/2-catenin regulate Tcfs to affect the transcriptional machinery and whether they function as a universal rheostat in controlling key SC genes remains unknown. The hair follicle offers a particularly attractive model for addressing this important issue, as it provides an especially abundant source of SCs which express Tcf3 and respond in a temporal and spatial manner to stabilized 2-catenin. Recent advances in chromatin immunoprecipitation, deep sequencing and HF-SC isolation and purification now make it possible to define the global transcriptional and epigenetic changes in Tcf/Lef-regulated genes that occur when HF-SCs in vivo receive a Wnt signal, and become activated to exit their native niche and progress along a specified lineage. Moreover, with the recent identification of Lhx2, Nfatc1 and Sox9 as additional essential HF-SC transcription factors, global chromatin mapping should illuminate how this transcriptional machinery governs HF-SC behavior and how external stimuli change the chromatin states of key target genes. We've devised strategies to do this and to decipher which direct target genes are most likely relevant to the critical features of stem cell behavior, activation and lineage commitment. Finally, we've developed innovative methodology to rapidly assay the functional significance of our candidates by lentiviral mediated knockdown in HF-SCs in vitro, and in cycling postnatal HFs and development in vivo. By dissecting how these signaling pathways operate to transcriptionally balance stem cell activation, proliferation and differentiation in hair follicle, new targets are likely to emerge for the development of new pharmaceutical agents that can inhibit Wnt signaling and uncontrolled growth in cancers.

Skin is the most visible indicator of the aging process. The overall focus of this proposal is to understand the mechanisms that underlie cell survival and aging in the epidermis of skin. The mammalian epidermis is in equilibrium, as mitotically, the following questions are addressed: (1) Do epidermal keratinocytes that overexpress cell survival factors in transgenic mice display a prolonged proliferative capacity? Does expression of cell survival factors in skin decline with age? (2) Do transgenic epidermal cells overexpressing cell survival factors express an increased level of beta1 integrin (a protein recently shown to be expressed at higher levels in epidermal stem cells)? (3) How does the level of beta1 expression vary with age in control skin, and is there a correlation between the levels of beta expression and decline in proliferative capacity with aging? (4) In the normal population of mitotically active keratinocytes, do those cells that express higher levels of beta1 integrin also express elevated levels of cell survival factors? (5) Do transgenic mice expressing elevated levels of beta1 integrin in their basal layer have a prolonged proliferative capacity, and are they more resistant to UV damage-induced apoptosis? Do they have elevated levels of cell survival factors? (6) Is expression of cadherin- associated proteins altered in Bcl-x and/or beta1 integrin expressing transgenic mice, and does it change with age in normal skin? Do differences correlate with proliferative capacity? This research is a fundamental prerequisite to our global understanding of the molecular mechanisms that controls aging, a process that inevitably affects us all.

The goal objective of this research is to elucidate the mechanisms underlying the control of keratinocyte growth and gene expression in oral epithelial tissues, and to assess how these processes are altered in oral cancers. To achieve this goal, we are focusing on understanding the regulatory controls that govern the genes that encode keratins, the major structural proteins of oral epithelial cells. This understanding will not only be useful to our understanding of how malignancy leads to aberrations in gene control, but in addition, the keratin promoters will be valuable for developing keratinocytes as a vehicle for gene therapy in the treatment of premalignant and/or postoperative head and neck Cancers. The 5' sequences of K14 and K5 are sufficient to faithfully drive expression in the mitotically active keratinocytes of tongue, buccal and tooth epithelia of transgenic mice. Due to their enormous proliferative capacity in culture, keratinocytes have been used successfully for bum operations and are potentially powerful vehicles for drug delivery and gene therapy of oral cancers and other tissue abnormalities. Elucidating how K5 and K14 promoter activity is controlled will be key in developing keratinocytes for these purposes. To this end, we will identify the critical sequences and factors that are central to efficient expression of foreign genes in oral keratinocytes. We will also explore how these factors are themselves regulated, and whether these processes are altered in oral squamous cell carcinomas (SQCCs). We will also explore how retinoids control differentiation in normal and SQCC keratinocytes, and elucidate the key retinoid receptor mediated target genes involved in this process. Such work is particularly important to developing new and improved retinoids for the treatment of oral leukoplakias caused by heavy smoking. We are also interested in identifying the critical molecular differences that distinguish a mitotically active oral keratinocyte from a leukoplakia keratinocyte and from an SQCC keratinocyte. We have devised a strategy to screen for such differences, and to test their importance by examining premalignant and malignant tissues in vivo. Finally, using transgenic mouse technology, embryonic stem cell technology and conditional knockouts with oral keratinocyte promoters, we will engineer in mice the molecular aberrations that we detect in the keratinocytes of oral cancers. These studies will not only allow us to assess the functional significance of such aberrations and their role in tumor progression, but in addition will provide valuable animal models for developing new and improved tools for the-diagnosis and treatment of these cancers. Our laboratory has recently pioneered research that illustrates how an understanding of keratinocyte biology can lead us to the causes of a variety of human genetic disorders. In the coming years, we will continue our endeavors to apply our molecular genetic research to medicine.

embryonic stem cell; tissue resource /registry; biomedical facility; genetically modified animals; diabetes mellitus; laboratory mouse; animal breeding; cryopreservation; transfection; microinjections;

DESCRIPTION: This grant application requests support for the EPITHELIAL DIFFERENTIATION AND KERATINIZATION Gordon Research Conference to be held July 8-13, 2001 at the Tilton School, N.H. Funds are requested to support registration, lodging, and travel expenses of the invited speakers and chairpersons. Partial support is also requested for poster presenters selected to give short platform talks, and to assist several qualified foreign scientists who otherwise could not attend. Applicants will be accepted based on the quality of their research in areas related to this conference, their ability to contribute to the discussion, and the likelihood that their research productivity and creativity will be enhanced by participation. This will be the 12th Gordon Conference on Epithelial Differentiation and Keratinization, which has been held every other year since 1979. The conference has attracted and helped to engage scientists from many disciplines to focus on basic and clinically-applied research in epidermal biology. Progress has been made in understanding these important tissues not only as models for investigating many questions of general scientific interest in cell and molecular biology, development, and genetics, but also for exploring the relation of epithelial biology to our understanding of genetic disorders of epithelial tissues. The 2001 program includes topics of traditional interest to this conference and new ones that reflect the continuing intersection of new scientific fields with epidermal and epithelial biology. The sessions represented by 36 speakers and chairpersons are as follows: (1) Epithelial stem cells, (2) Epithelial polarity, (3) Signal transduction pathways in epithelia, (4) Adhesion, Wnts, Shh and epithelial development, (5) Pathogens, epithelia and cancer, (6) Epithelial adhesion, proliferation and differentiation, and (7) Epithelial architecture

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Our global objective is to elucidate the mechanisms underlying differentiation and development in mammalian epidermis and hair. To achieve this goal, we focus on the regulatory controls that govern [unreadable] expression of keratins, the major structural proteins of these cells. Understanding how this complex [unreadable] pattern of keratin gene expression is established is key to elucidating how these cells develop and [unreadable] how these processes go awry in human skin disorders. Elucidating how K5 and K14 promoter activity [unreadable] is controlled is prerequisite to developing cultured keratinocytes as therapeutic agents for drug [unreadable] delivery and gene therapy, an extension of their current use in burn operations. Our studies have [unreadable] already unveiled some transcription factors, including the AP2 family, in regulating K5 and K14 gene [unreadable] expression, and the Tcf/Lef family of transcription factors in regulating the hair keratin genes. Given [unreadable] the multiplicity of factors involved in regulating transcription in the skin, a complementary microarray [unreadable] approach is needed to provide a database of the global changes in transcription factor gene [unreadable] expression during skin development. Preliminary analyses have already illuminated which of the AP2 [unreadable] family members and enhancer factor associates are likely to be key in governing transcription during [unreadable] keratinocyte specification. Additionally, a database of developmental patterns of gene expression will [unreadable] greatly facilitate our interpretation of AR31737-approved arrays of skin-specific transcription factor [unreadable] knockout mice, and enhance the identification of downstream target genes. The database will serve [unreadable] as the foundation for integrating and achieving our funded goals of AR31737 research aimed at [unreadable] elucidating the regulatory mechanisms that govern epidermal and hair follicle gene expression. The [unreadable] database will be an excellent resource for future dissection of the downstream pathways involved, [unreadable] and for expediting our progress in understanding how cell fate decisions are made in normal and [unreadable] disease states. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our global objective is to develop a molecular understanding of how epithelial stem cells undergo morphogenesis, homeostasis, and wound repair in mammalian skin and bring this research to a clinical setting. Our focus is on how skin stem cells utilize cytoskeletal connections to intercellular and cell substratum junctions to generate tissues. Knowledge of the proteins involved in coordinating actin filament (AF) cytoskeletal dynamics with adherens junctions (AJs) and elucidating how these connections are inversely coordinated with integrin-AF dynamics are key to understanding how self-renewing skin epithelium maintains and repairs its surface barrier and how a stem cell can give rise to a hair follicle (HF). Elucidating how microtubules (MTs) link to AJs is important for understanding how the stratified epidermis maintains a single layer of dividing cells, and how spindle polarity changes when stem cells are activated to produce HFs. A molecular understanding of these dynamics will be obtained by focusing on four cytoskeletal-junction linking proteins that surfaced as being key from our prior studies: -catenin, which coordinates and polarizes AF-AJ dynamics and suppresses AF-integrin dynamics;ACF7, which coordinates and polarizes MT-AF and Par3- mediated dynamics in a wound response;and Par3 and mInsc, whose polarization relies upon -catenin and 1 integrin, and which appear to be critical for orienting spindles in skin morphogenesis. Through biochemical and molecular approaches, we'll define and characterize the associated proteins that are involved in polarizing and remodeling cytoskeletal dynamics during skin morphogenesis and wound repair. Through mouse genetics and novel ShRNA strategies we've developed to rapidly knockdown genes in adult and embryonic skin, the functional significance of these proteins and their varied associations with other proteins will be ascertained. Finally, as the major players in these processes unfold, functional analyses will be combined with microarray data to define the underlying significance of global changes in AJ-cytoskeletal gene expression that occur concomitantly as stem cells receive external cues to remodel their AJ-cytoskeletal connections and initiate stratification and HF morphogenesis. Understanding how these cytoskeletal linkages are regulated in normal skin is a prerequisite to elucidating how defects in these processes lead to genetic disorders, including skin cancers. Past and present AR27883 research provides an excellent illustration of how molecular skin biology can help to generate new and improved tools for the diagnosis and treatment of human skin disease. Public Health Relevance: Stem cells are natural units of tissue repair and homeostasis, and their versatility holds promise for tissue regeneration. This research focuses on deciphering how different tissue structures are derived from multipotent stem cells in the skin. Specifically, we focus on how stem cells within a single layer use cell-cell and cell substratum junctions to remodel their cytoskeleton to generate a stratified, differentiating epidermis or an invaginating hair follicle bud, and how this changes transiently in a wound response. This study is a fundamental prerequisite to understanding how aberrations in these basic properties go awry in skin cancers, including squamous cell carcinomas.

Project Summary The global objective of this research is to elucidate the mechanisms underlying tissue homeostasis and regeneration in mammalian skin and to understand how this process goes awry in human disorders, including cancers. Central to achieving this goal is the purification and characterization of the differerent stem cell (SC) populations within skin and determination of their relative contributions to tissue homeostasis and wound- repair. Past AR050452 research led to purification of hair follicle (HF) bulge cells, and established them as self- renewing SCs that function in hair cycling and wound-repair. These HF-SCs also displayed more robust self- renewing and broader tissue regenerative potential than interfollicular epidermal (IFE)-SCs. Several differences might account for this. One is that like human IFE-SCs, mouse HF-SCs exist in a quiescent state for prolonged periods. Another is that they uniquely express transcription factors such as Nfatc1, which when absent, causes HF-SCs to lose quiescence and cycle HFs continuously. Past AR050452 research has set the foundations to tackle the following key questions: (1). Is HF-SC quiescence critical to their long-term maintenance? What happens when HFs cycle continuously? Do they expend their SCs over time? Can they heal wounds faster? (2) What are the molecular mechanisms governing the resting phase of the HF-SC niche? How does Nfatc1 and its associated transcriptional regulators function to maintain HF-SC quiescence? What are its direct targets and how do they regulate the balance between quiescence and long-term self-renewal? (3) What molecular changes define the difference between stemness and the transit amplifying state? Do stem cell progeny influence this transition and if so how?(4) What are the relative contributions of IFE and HF SCs to wound-repair? Does this differ in superficial vs deep wounds? In young vs adult mice? (5) How do IFE and HF SCs alter gene expression in response to injury? How fast are these changes and what controls the process? To answer these questions, we'll use FACS, RNA-seq, ChIP-seq, conditional gene knockout and RNAi screens in vivo and employ these methods to explore skin stem cells in their native, mutant and wound-induced environments.

Project Summary Our global objective is to develop a molecular understanding of how epithelial stem cells undergo morphogenesis and maintain homeostasis in mammalian skin, and to bring our research to a clinical setting. Our focus is on how, in response to local developmental cues, skin stem cells remodel their cytoskeletal connections with intercellular and cell-substratum junctions in order to generate and maintain tissues. Knowledge of the proteins involved and their physiological roles in coordinating these cytoskeletal and adhesion dynamics are key to understanding how skin epithelia form and are maintained and how cellular organization goes awry in hyperproliferative skin disorders, including cancers. During skin development, a single layer of unspecified epithelial progenitors can both stratify to form the surface barrier and also invaginate to form hair follicles (HFs). As morphogenesis proceeds, resident stem cells are set aside so that in adult skin, self-renewing progenitors maintain and repair the skin's barrier and fuel cyclical bouts of hair regeneration. In prior AR27883 research, we showed that whether normal or malignant, all skin progenitors reside at epithelial-mesenchymal interfaces. Progenitors utilize their underlying basement membrane to polarize and orient their spindle, balance growth and differentiation and migrate. We have shown that in part, WNT-signaling is at the root of these polarized cytoskeletal and adhesion dynamics. However, the epithelium must also sense and respond to cell crowding and mechanical cues. How these signals intersect remain unclear. In the next 5 years, we'll address these critical issues by: (1). Determining the governance and importance of cell density in tissue morphogenesis within the epidermis and HFs. (2). Dissecting the roles of acto-myosin cytoskeletal regulators in tissue morphogenesis. (3). Elucidating how the developing epidermis copes with poorly performing cells for the sake of tissue fitness and how this changes once the stratified skin barrier is established. (4). Elucidating the role(s) of differential WNT- signaling and mechanotransduction regulators within developing hair buds. We'll assess how signaling is sensed in a polarized fashion and how cell density, tension and basement membrane production impacts the decision to invaginate rather than evaginate. (5). Applying the knowledge gained in Aims 1-4 to dissect how and why tissue morphogenesis and maintenance change in malignancy. To meet these aims, we'll combine a variety of molecular and genetic approaches. During our R37-AR27883 MERIT Award, we developed live imaging to interrogate cellular movements during skin development. We also pioneered a powerful in utero method to efficiently and selectively compromise gene function in embryonic and adult skin to unveil the physiological relevance of our findings. Finally, we adapted this technology to CRISPR/CAS, enabling us to conduct rapid conditional knockouts in mice as well. Our past and present AR27883 research, displaying continued success over nearly 4 decades, exemplifies how molecular skin biology can lead to new and improved tools for diagnosing and treating human skin diseases. We aim to continue to make such inroads in the next grant period.

Project Summary Our global objective is to elucidate the mechanisms underlying tissue homeostasis and regeneration in mammalian skin and to understand how this process goes awry in inflammation and cancers. Central to achieving this goal is to determine how stem cells sense and tailor their programs of gene expression in order to perform their particular tasks during homeostasis and wound repair and survive under stressful situations. Past AR31737 research has revealed that adult epidermal and hair follicle (HF) stem cells originate from a common embryonic skin progenitor, but in adult skin, they reside in distinct microenvironments. While maintaining some commonalities, their different niches endow stem cells with distinct molecular properties and instructions to perform separate tasks. Upon injury, the nearest stem cells must respond and adopt a plasticity that enables them to regenerate either epidermis or hair follicles irrespective of the niche from whence they came. We have learned that this `dual lineage' plasticity, transient in a wound state, is hijacked and becomes constitutive in cancer. We've also learned that at the heart of stem cell identity and responsiveness are niche-specific transcription factors (TFs) that act in concert with DNA effectors of environmental signals (e.g. pSMAD1/4 for BMP signaling and LEF1/TCF1 for WNT/?-catenin signaling) to regulate key genes whose expression must rapidly change with the environment. In yrs 39-44 of AR31737 research, we'll now address: What are the chromatin dynamics that enable skin stem cells to choose between epidermal and HF fates? What are the key TFs involved and how do they drive the epigenetics needed to make these fate decisions? How do HF stem cells maintain their fate during homeostasis? How do these stem cells enter a plastic state following injury and then change their identity to epidermal stem cells when they find themselves in the epidermis after repair is complete? Finally, we have discovered that chromatin remodeling in epidermal stem cells is rapid after injury and other types of inflammatory stimuli, but once tissue homeostasis is restored and inflammation has waned, some of these changes resolve slowly. What are the mechanisms underlying this epigenetic memory, and how does it affect tissue fitness? To answer these questions, we will couple in vivo high throughput technologies with our functional interrogation methods, involving in utero lentiviral delivery of epigenetic reporter genes, inducible genes, RNAis and Crispr/CAS guide RNAs to selectively target the skin's stem cells. At the conclusion of this research, we expect to have advanced our knowledge of tissue homeostasis, wound-repair, inflammatory disorders and malignancies and provided new insights to therapeutic strategies.