April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Methylation sensitive regulation of the EphA5 promoter by KLF16, a zinc finger transcription factor expressed in retinal ganglion cells
Author Affiliations & Notes
  • Deborah C Otteson
    Optometry, University of Houston, Houston, TX
  • Jian Bo Wang
    Optometry, University of Houston, Houston, TX
  • Footnotes
    Commercial Relationships Deborah Otteson, None; Jian Bo Wang, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5732a. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Deborah C Otteson, Jian Bo Wang; Methylation sensitive regulation of the EphA5 promoter by KLF16, a zinc finger transcription factor expressed in retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5732a.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: In the mouse visual system, the receptor tyrosine kinase EPHA5 plays an important role in the formation of retinotopic maps. Previously identified regions of differential methylation within the EphA5 promoter contain predicted Krüppel-like factor (KLF) transcription factor binding sites. This study tests the hypothesis that KLF transcription factors are methylation-sensitive regulators of the EphA5 promoter.

Methods: Expression plasmids containing coding regions for Klf2, 4, 5, 6, 7, 13, 15 and 16 (gift of Dr. JL Goldberg, U. Miami) were tested for activation of mEphA5-pGL2 promoter constructs. Luciferase assays used transient transfections of 293FT cells, normalized to co-transfected renilla luciferase or β-galactosidase. Developmental expression of KLF16 and POU4 in mouse retinas used immunofluorescence labeling and imaging with confocal microscopy.

Results: Of the 8 KLF transcription factors tested, only KLF16 altered activity of mEphA5 promoter constructs, increasing luciferase reporter expression 4 to 6-fold vs. control (p <0.05). Deletion analysis localized the KLF16 responsive element to -118 to +120 bp relative to the transcription start site. KLF16 activation the mEphA5-118 promoter construct was blocked by mutation of the predicted KLF16 binding site or by prior promoter methylation using HpaII methyltransferase. By immunofluorescence, KLF16 was expressed in the retina in POU4+ retinal ganglion cells (RGCs) at embryonic days 13.5 and 16.5 and in RGCs, presumptive amacrine and horizontal cells at postnatal day 7.

Conclusions: The KLF16 critical region is located within a CpG island flanking the transcription start site of EphA5. This region was previously shown to have increased methylation in the nasal retina, where EphA5 expression is lowest. These data support our hypothesis that KLF16 is a methylation-sensitive regulator of the EphA5 promoter and the timing of expression is consistent with a role for KLF16 in regulating EphA5 expression in RGCs. Interestingly, overexpression of KLF16 was previously shown to inhibit neurite outgrowth and activation of EPHA5 receptor signaling is known to promoter growth cone collapse in RGCs in vitro. The current findings may provide a possible mechanism for at least some of the inhibitory effects of KLF16 on neurite extension.

Keywords: 531 ganglion cells • 739 transcription factors • 497 development  
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×