The primary goal of our laboratory is to decipher the genetic network of regulatory factors controlling ocular surface maturation and maintenance, providing new opportunities for diagnosis, prevention and therapy of ocular surface disorders. We previously established that Krppel-like factors Klf4 and Klf5 have critical non-redundant functions in the ocular surface. As Klf4 and Klf5 regulate essential functions in many tissues, it is imperative that we identify their direct and indirect downstream target genes to facilitate development of new therapies targeting individual processes that they control. In this project, we seek to understand the molecular mechanisms underlying ocular surface functions of Klf4 and Klf5 by testing the central hypothesis that `the choice between corneal epithelial cell proliferatin and differentiation is governed by the ratio of the anti- and pro-proliferative activities of Klf4 nd Klf5, respectively, which work through distinctly configured cis-elements in their target gene promoters', by pursuing three Specific Aims.
Aim 1 is to test the hypothesis that `the choice between the corneal epithelial cell proliferation and differentiation is determined by the ratio of anti- and pro-proliferative activities of Klf4 and Klf5, respectively'.
Aim 2 is to determine if Kl4 and Klf5 bind distinct cis- elements in spite of possessing similar DNA-binding domains, thereby elucidating the factors governing their sequence-specificity and non-redundant functions.
Aim 3 will test if Klf4 and Klf5 contribute to corneal epithelial homeostasis both directly by regulating their target genes in the cornea, and indirectly by regulating the morphogenesis and function of ocular adnexae. We will employ state of the art mouse genetics, and cell and molecular biological techniques including (i) the Klf4-conditional null (Klf4CN), Klf5CN, spatiotemporally regulated corneal epithelium (CE)- or conjunctival epithelium (Conj E)-specific ablation of Klf4 (Klf4?/?CE or Klf4?/?ConjE) and Klf5 (Klf5?/?CE or Klf5?/?ConjE) mouse models to exmine the ocular surface functions of Klf4 and Klf5, and (ii) an innovative integrated functional genomics approach coupling in vivo chromatin immunoprecipitation and Next-Gen sequencing (ChIP-Seq) with Klf4?/?CE and Klf5?/?CE corneal gene expression profiles to detect genome-wide distribution of functionally relevant Klf4- and Klf5-binding sites. The anticipated outcomes of this project will have high impact on concepts and treatment of corneal disorders by (a) determining the role of Klf4 and Klf5 in coordinating the corneal epithelial cell proliferation and differentiation, (b) elucidating the functions of Klf4 and Klf5 in morphogenesis of ocular adnexae and their influence on corneal homeostasis, (c) revealing the genome-wide distribution and functional relevance of Klf4- and Klf5-bound cis-elements and (d) providing us with valuable new mouse models useful for studying dry eye.

Public Health Relevance

Ocular surface disorders represent the most common reason for outpatient visits to an ophthalmologist and account for a significant portion of healthcare costs. Better awareness of the molecular mechanisms governing the ocular surface maturation and maintenance is essential for designing novel therapeutic methods targeting ocular surface disorders. This project seeks to elucidate the molecular mechanisms underlying the diverse functions of powerful transcription factors Klf4 and Klf5 in the ocular surface, providing new opportunities for therapeutic intervention in ocular surface disorders.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY026533-04
Application #
9664631
Study Section
Biology of the Visual System Study Section (BVS)
Program Officer
Mckie, George Ann
Project Start
2016-04-01
Project End
2020-03-31
Budget Start
2019-04-01
Budget End
2020-03-31
Support Year
4
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Loughner, Chelsea L; Tiwari, Anil; Kenchegowda, Doreswamy et al. (2017) Spatiotemporally Controlled Ablation of Klf5 Results in Dysregulated Epithelial Homeostasis in Adult Mouse Corneas. Invest Ophthalmol Vis Sci 58:4683-4693
Tiwari, Anil; Loughner, Chelsea L; Swamynathan, Sudha et al. (2017) KLF4 Plays an Essential Role in Corneal Epithelial Homeostasis by Promoting Epithelial Cell Fate and Suppressing Epithelial-Mesenchymal Transition. Invest Ophthalmol Vis Sci 58:2785-2795