The long-term goal of this program is to elucidate those molecular mechanisms of mammalian lens development and homeostasis that are directly controlled by DNA-binding transcription factor Pax6. Previous studies have shown that Pax6 is essential for establishing lens lineage and regulation of crystallin gene expression. However, the complete spectrum and range of Pax6's function and the mechanism by which it affects lens development remain to be determined. Using chromatin immunoprecipitation (ChIP), we have now identified a large number of novel genes including Mab21l1 and DNase II? as putative direct Pax6 targets. Mab21l1 is highly expressed in the lens placode and its promoter region contains multiple binding sites for Six3, another important lens-lineage specific regulatory gene. DNase II? is most highly expresed in lens fiber cells just prior to their denucleation. Evidence exists that lens-preferred expression of this gene is under the control of Pax6, AP-2? and Foxe3. In contrast, Hsf4 plays a direct role in the upregulation of DNase II? in differentiating lens fiber cells. We also found that Pax6 not only promotes lens development but it also simultanously suppresses alternative developmental programs such as the expression of neurogenic genes in lens lineage. These findings suggest that Pax6 controls epigenetic mechanims that control individual cell lineage formation in embryonic development. In order to carry out this long-term goal, the following specific aims are proposed: (1) To elucidate transcriptional regulation of Mab21l1, a gene essential for the survival of lens progenitor cells, by Pax6, Six3 and other factors in transgenic mouse and via cell culture experiments. (2) To elucidate transcriptional regulation of DNase II?, an enzyme required for lens fiber cell denuclation, by Pax6, AP-2?, Foxe3 and Hsf4, in transgenic mouse and through a combination of protein-DNA binding studies and cell culture based reporter assays. (3) To identify those novel direct Pax6-targets that are regulated via distal 5'and 3'enhancers and to generate a Pax6-dependent regulatory network that controls lens and forebrain development using chromatin immunoprecipitations analyzed by massively parallel DNA sequencing (ChIP-seq), RNA expression profiling in normal and Pax6 mutated tissues.

Public Health Relevance

This application is relevant to human health as lens cataract is a major cause of worldwide blindness. Pax6 is an essential gene for mammalian eye development, it controls lens lineage formation and terminal differentiation of lens fiber cells. Mutations in PAX6 and other genes studied here (FOXE3, HSF4 and MAF) are known to cause human congenital eye diseases. Mutations in PAX6 also cause a variety of neurological disorders and PAX6 has also been implicated in type II diabetes.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY012200-14
Application #
8403024
Study Section
Special Emphasis Panel (ZRG1-ETTN-E (92))
Program Officer
Araj, Houmam H
Project Start
2000-01-01
Project End
2013-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
14
Fiscal Year
2013
Total Cost
$489,309
Indirect Cost
$194,544
Name
Albert Einstein College of Medicine
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
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Castellanos, Raquel; Xie, Qing; Zheng, Deyou et al. (2014) Mammalian TBX1 preferentially binds and regulates downstream targets via a tandem T-site repeat. PLoS One 9:e95151
Walcher, Tessa; Xie, Qing; Sun, Jian et al. (2013) Functional dissection of the paired domain of Pax6 reveals molecular mechanisms of coordinating neurogenesis and proliferation. Development 140:1123-36
Xie, Qing; Yang, Ying; Huang, Jie et al. (2013) Pax6 interactions with chromatin and identification of its novel direct target genes in lens and forebrain. PLoS One 8:e54507
Shaham, Ohad; Gueta, Karen; Mor, Eyal et al. (2013) Pax6 regulates gene expression in the vertebrate lens through miR-204. PLoS Genet 9:e1003357
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Wontakal, Sandeep N; Guo, Xingyi; Will, Britta et al. (2011) A large gene network in immature erythroid cells is controlled by the myeloid and B cell transcriptional regulator PU.1. PLoS Genet 7:e1001392
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Wang, Wei-Lin; Li, Qingtian; Xu, Jianming et al. (2010) Lens fiber cell differentiation and denucleation are disrupted through expression of the N-terminal nuclear receptor box of NCOA6 and result in p53-dependent and p53-independent apoptosis. Mol Biol Cell 21:2453-68

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