2008 — 2009 |
Sen, George L |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Role of Polycomb Group Proteins in Epidermal Diffentiation
[unreadable] DESCRIPTION (provided by applicant): Background: Epidermal differentiation is a tightly regulated process that begins with actively dividing cells that eventually differentiates into enucleated corneocytes that form the water impermeable layer of the skin. Perturbations in the balance between growth and differentiation can lead to a variety of human skin disorders. Increased proliferation and diminished differentiation can lead to hyperproliferative disorders such as psoriasis, basal or squamous cell carcinomas. Tremendous effort has been placed on identifying tissue specific transcription factors that control epidermal differentiation. Much less emphasis has been placed on a potential role for epigenetic regulation of differentiation. In particular, the polycomb group (PCG) proteins offer an attractive means of repressing epidermal differentiation genes in the absence of differentiation inducing signals. Objective/Hypothesis: The overall goal of this proposal is to further define the factors involved in regulating epidermal differentiation, with a specific focus on the role of PCG proteins in epidermal differentiation. Our preliminary studies indicate that PCG proteins bind to a majority of epidermal differentiation genes in undifferentiated epithelial cells. We propose a model where PCG proteins actively repress epidermal differentiation genes in the undifferentiated state. Upon receiving differentiation signals, PCG proteins no longer associate with the promoters of differentiation genes allowing the chromatin to unwind to allow transcription of epidermal differentiation specific genes. Specific Aims and research design: (1) Genome wide mapping of PCG binding sites in the epidermis: 1A) To determine PCG binding regions in undifferentiated epidermal cells. 1B) To determine the impact of differentiation on PCG protein binding to previously identified (1A) target sites. (2) Characterization of the functional role of PCG proteins in epidermal differentiation: 2A) To determine the effect of loss of function of PCG proteins on epidermal differentiation. 2B) To determine the effect of gain of function of PCG proteins on epidermal differentiation. Relevance to public health: In almost all cancers, epigenetic changes accompany cancer initiation and progression. At the heart of these changes include altered epigenetic regulation mediated by polycomb group proteins. Components of the PCG protein complex such as EZH2 and Bmil are often overexpressed in cancer. Thus, understanding how PCG proteins regulate their target sites in normal cells is of utmost importance in determining how they are altered to contribute to cancer initiation and progression. [unreadable] [unreadable] [unreadable]
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1 |
2010 — 2014 |
Sen, George L |
K01Activity Code Description: For support of a scientist, committed to research, in need of both advanced research training and additional experience. |
Epigenetic Regulators in Epidermal Homeostasis and Neoplasia
DESCRIPTION (provided by applicant): My long-term goal is to spend a career guiding a productive research laboratory teaching students and fellows while making significant contributions to epithelial biology. In particular, I am interested in how cells differentiate into different developmental lineages and retain that cellular memory. I am interested in the epidermis as a model system since the skin has a constant turnover rate with cycles of proliferation and differentiation. These cycles must be tightly regulated or disorders would rapidly arise. I am also interested in how dysregulation of these fundamental processes can lead to human diseases such as cancer. This training proposal will help me attain these goals because of all the techniques and skills I will learn to address the questions of the proposal. Specifically, the training during this award period will provide me with new expertise in epithelial biology, in vivo mammalian model systems, genomics, and cancer biology. My immediate goal is to accomplish the aims of the proposal as described below. Epidermal homeostasis is a tightly regulated process that begins with actively dividing cells that eventually differentiate into enucleated corneocytes that form the water-impermeable layer of the skin. Perturbations in the balance between growth and differentiation can lead to a variety of human skin disorders. Increased proliferation and diminished differentiation can lead to hyperproliferative disorders such as psoriasis, basal or squamous cell carcinomas. Thus, a comprehensive understanding of the regulators of epidermal growth and differentiation would be key to unlocking the molecular mechanisms underlying skin disorders which could potentially lead to the development of target guided therapies for skin disorders. This effort aims to define the role of epigenetic modifiers in epidermal growth, differentiation, and neoplasia. Our previous findings demonstrated that the histone demethylase, JMJD3, controls epidermal differentiation by removal of methyl marks on H3K27me3 marked epidermal differentiation gene promoters. Because of the central role of JMJD3 in epidermal differentiation, functional alterations in JMJD3 may contribute to neoplasia. Aim I will characterize the role of JMDJ3 in human epidermal tumor progression. In order to identify other epigenetic modifiers with a role in epidermal growth and differentiation, we systematically depleted the function of DNA and histone modifying enzymes and found DNA methyltransferase 1 (DNMT1) and Histone deacetylase 2 (HDAC2) to have effects on epidermal growth and differentiation. Aim II focuses on characterizing the role of DNMT1 and HDAC2 on epidermal growth, differentiation and neoplasia.
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1 |
2013 |
Sen, George L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ras Signaling in Skin Stem Cell Proliferation and Fate @ University of California San Diego
DESCRIPTION (provided by applicant): Adult stem cells are the basic building blocks for wound repair and regeneration. Understanding how to stimulate their growth and development is critical to advancing regenerative medicine. Our laboratory studies adult stem cells in the hair follicle because they are accessible and divide at specific developmental ages. Recently, activating mutations in the RAS signaling pathway have been found in children with Costello and cardiofaciocutaneous (CFC) syndromes. These syndromes are associated with multiple skin defects that appear to affect the proliferation and differentiation of skin stem cells. We have recently developed several mouse models, which develop many of the same skin abnormalities as Costello and CFC patients, including excess skin production and hair loss. We propose to use our mouse models to 1) investigate the basis of RAS- induced hair cycle defects, 2) determine the effect of RAS on adult stem cell proliferation, and 3) identify the downstream pathways that cause these defects. By understanding the function of RAS signaling in adult stem cells of the skin, new therapies could be developed to improve organ regeneration and wound repair.
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0.954 |
2015 — 2018 |
Sen, George L Zhang, Kang [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Limbal Stem Cell Fate and Corneal Specific Enhancers @ University of California San Diego
? DESCRIPTION (provided by applicant): The cornea is a specialized epithelium, which protects the eye and maintains transparency for normal vision. Diseases of the cornea or its stem cells (limbal stem cells) affects millions of people worldwide and result from a spectrum of etiologies, ranging from genetic defects to infection, inflammation, or trauma. Vision loss often occurs as the result of transformation of the transparent cornea into a skin-like epithelium. Recent work from our labs and others now reveal that this critical transition may be precipitated by the loss of PAX6 expression. Downregulation of PAX6 causes the loss of cornea features and gene expression and the ectopic activation of skin-like epithelium. Conversely, introduction of PAX6 into skin cells induces many cornea-like features. These studies indicate a critical role of PAX6 for maintaining cornea epithelium, but the mechanisms, which determine cornea vs. skin identity, are still unknown. Our hypothesis is that PAX6 contributes to cornea identity through tissue-specific DNA regulatory elements called enhancers. Enhancers are responsible for coordinating tissue-specific gene expression and for the development of specific cell types. Enhancers are also thought to be of considerable medical importance as genetic variation in and epigenetic regulation of enhancers is thought to play a major role in susceptibility to disease. Despite their biological and medical importance, little is known about the enhancers of the cornea and disease. Advances in chromatin biology and high-throughput sequencing provide new, powerful tools to identify tissue-specific enhancers of the cornea and have the potential to expand our knowledge of cornea disease. Here we propose to (1) identify enhancers, which distinguish cornea from skin epithelium via ChIP-seq; (2) determine how tissue-specific enhancers are regulated in the cornea by chromatin analysis and reporter studies in cultured cells, animal model transplants; and (3) determine the role of PAX6 in the regulation of tissue-specific enhancers. To accomplish these goals, an interdisciplinary approach is needed. The combined expertise and resources of two labs (eye, skin) provides an ideal path to identifying cornea- vs. skin-specific regulation and will result in a better understanding of how ectopic skin features become activated in the cornea. This major effort will result in the identification of genome-wide enhancers in the cornea, critical to stem cell biology and translational studies, and the generation of new genetic reagents to study cornea disease.
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0.954 |
2015 — 2021 |
Sen, George L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulators of Epidermal Growth and Differentiation @ University of California San Diego
DESCRIPTION (provided by applicant): Epidermal homeostasis requires a proper balance between proliferation and differentiation which when altered can lead to hyperproliferative disorders such as squamous cell carcinomas. Regulators of this process may include RNA degradation enzymes such as the exosome which is composed of 11 subunits. We have recently shown that exosome subunits such as EXOSC9, EXOSC7, and EXOSC10 are essential for maintaining epidermal self-renewal by promoting the mRNA degradation of differentiation specific transcription factors in progenitor cells. It is currently unknown the function of the other 8 subunits of the exosome as well as their associated adaptor proteins. Objective/hypothesis: This proposal seeks to understand the gene regulatory mechanisms involved in maintaining normal epidermal homeostasis. We hypothesize that exosome subunits exist in epidermal cells in subcomplexes with distinct functions. Subcomplexes such as EXOSC9, EXOSC7, and EXOSC10 are necessary to maintain progenitor function while other subcomplexes are necessary for differentiation. We also hypothesize that adaptor proteins recruit distinct exosome subcomplexes to target RNAs to either maintain self-renewal or to promote epidermal differentiation. Specific Aims: (1) To determine the role of the exosome subunits in epidermal homeostasis and (2) to determine the role of exosome associated adaptor proteins in epidermal homeostasis. Study Design: To study epidermal homeostasis in a more clinically relevant setting, we established new methods to introduce specific combinations of genetic elements into 3- dimensionally intact human skin, containing human epidermal cells in the context of human dermal stroma and basement membrane, regenerated on immune deficient mice. By using this model, we can perform loss of function experiments on exosome subunits and adaptor proteins in regenerated human skin to characterize their role in epidermal growth and differentiation. We will also use RNA immunoprecipitations followed by next generation sequencing to determine the RNAs associated with exosome subunits as well as adaptor proteins.
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0.954 |
2017 |
Sen, George L |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Post-Trancriptional Regulation of Epidermal Stem and Progenitor Cell Self-Renewal @ University of California San Diego
Project Summary/Abstract Background: Transcriptional mechanisms that regulate epidermal homeostasis have been well established but recently we have discovered that post-transcriptional mechanisms play prominent roles in maintaining epidermal self-renewal. We have shown that the RNA helicase DDX6 is necessary to maintain epidermal self-renewal through the mRNA degradation and translation pathway. By associating with specific members of the translation pathway DDX6 binds to and mediates the translation of self- renewal and proliferation transcripts to maintain self-renewal. DDX6 also associates with mRNA degradation proteins to bind differentiation-inducing transcripts to promote their degradation to prevent premature differentiation. Objective/hypothesis: This proposal seeks to understand the regulation of epidermal stem and progenitor cell self-renewal and differentiation through post- transcriptional mechanisms. We previously identified proteins associated with DDX6 by mass spectrometry and our objective is to characterize the role of each protein in regulating epidermal growth and differentiation as well as the mechanisms of action. Furthermore we seek to determine the specific transcripts that DDX6 and its associated complexes bind. Specific Aims: (1) To determine the role of DDX6 associated proteins on epidermal homeostasis and (2) to identify and characterize the transcripts associated with DDX6 complexes. Study Design: To study epidermal homeostasis in a more clinically relevant setting, we generate 3-dimensionally intact human skin, containing human epidermal cells (that have been permanently knocked down for either DDX6 or its associated proteins) in the context of human dermal stroma and basement membrane, regenerated on immune compromised mice. By using this model, we can perform loss of function experiments on DDX6 and its associated proteins in regenerated human skin to characterize their role in epidermal growth and differentiation. We will also use RNA immunoprecipitations followed by next generation sequencing to determine the RNAs associated with DDX6 complexes.
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0.954 |
2018 — 2021 |
Sen, George L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Post-Transcriptional Regulators of Epidermal Homeostasis and Neoplasia @ University of California, San Diego
Project Summary/Abstract Background: Transcriptional mechanisms that regulate epidermal homeostasis have been well established but recently we have discovered that post-transcriptional mechanisms play prominent roles in maintaining epidermal self-renewal. We have shown that the RNA helicase DDX6 is necessary to maintain epidermal self-renewal through the mRNA degradation and translation pathway. By associating with specific members of the translation pathway DDX6 binds to and mediates the translation of self- renewal and proliferation transcripts to maintain self-renewal. DDX6 also associates with mRNA degradation proteins to bind differentiation-inducing transcripts to promote their degradation to prevent premature differentiation. Objective/hypothesis: This proposal seeks to understand the regulation of epidermal homeostasis and tumor initiation through post-transcriptional mechanisms. We previously identified proteins associated with DDX6 and our objective is to characterize the role of each protein in regulating epidermal growth, differentiation, and progression to neoplasia as well as the mechanisms of action. Furthermore we seek to determine the specific transcripts that DDX6 and its associated complexes bind during homeostasis and tumor initiation. Specific Aims: (1) To determine the role of DDX6 associated proteins on epidermal homeostasis and tumor initiation and (2) to identify and characterize the transcripts associated with DDX6 complexes. Study Design: To study epidermal homeostasis in a more clinically relevant setting, we generate 3-dimensionally intact human skin, containing human epidermal cells (that have been permanently knocked down for either DDX6 or its associated proteins) in the context of human dermal stroma and basement membrane, regenerated on immune compromised mice. By using this model, we can perform loss of function experiments on DDX6 and its associated proteins in regenerated human skin to characterize their role in epidermal growth, differentiation, and progression to neoplasia. We will also use CLIP- Seq to determine the RNAs associated with DDX6 complexes during the progression from normal to neoplastic epidermis.
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0.954 |
2019 — 2021 |
Sen, George L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Human Epidermal Tumorigenesis by the Mrna Degradation Pathway @ University of California, San Diego
Project Summary/Abstract Background: Transcriptional mechanisms that regulate epidermal homeostasis and neoplasia have been well established but recently we have discovered that post- transcriptional mechanisms play prominent roles in maintaining epidermal self-renewal. We have shown that the 3'-5' mRNA degradation pathway mediated by the exosome complex is necessary to maintain epidermal self-renewal. Specifically, the exosome subunits, EXOSC7, EXOSC9, and EXSCO10 are necessary to prevent premature differentiation of epidermal stem cells by targeting and degrading transcripts that code for potent pro-differentiation transcription factors. Objective/hypothesis: This proposal seeks to understand the molecular mechanisms governing the progression from normal to neoplastic skin using a RAS driven human epidermal tumor model. Our preliminary data suggests that a 5 subunit exosome subcomplex is upregulated during tumor initiation and targets/degrades transcripts coding for factors that would inhibit tumor growth and survival. Our objective is to characterize the role of each tumor induced exosome subunit in the progression of normal to neoplastic skin. Furthermore we seek to determine the specific transcripts that each exosome subunit binds during tumor initiation to promote tumorigenesis. Specific Aims: (1) To determine the role of exosome subunits on the progression from normal to neoplastic skin and (2) to identify and characterize the transcripts associated with exosome subunits. Study Design: To study epidermal homeostasis in a more clinically relevant setting, we generate 3-dimensionally intact human skin, containing human epidermal cells (that have been permanently knocked down for exosome subunits) in the context of human dermal stroma and basement membrane, regenerated on immune compromised mice. By using this model, we can perform loss of function experiments on exosome subunits in regenerated human skin to characterize their role in epidermal growth, differentiation, and progression to neoplasia. We will use CLIP-Seq to determine the RNAs associated with the exosome subunits during the progression from normal to neoplastic epidermis.
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0.954 |