June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Using RiboTag and Sun1-sfGFP technologies to map Müller cell-specific epigenetic regulation of stress-induced LIF expression in vivo.
Author Affiliations & Notes
  • Clayton P Santiago
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Casey J Keuthan
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Aisha A Imam
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • John D Ash
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Footnotes
    Commercial Relationships   Clayton Santiago, None; Casey Keuthan, None; Aisha Imam, None; John Ash, None
  • Footnotes
    Support  Funding support to John Ash include NIH R01EY016459-11, Foundation Fighting Blindness, and an unrestricted departmental grant from Research to Prevent Blindness, Inc.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5902. doi:
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      Clayton P Santiago, Casey J Keuthan, Aisha A Imam, John D Ash; Using RiboTag and Sun1-sfGFP technologies to map Müller cell-specific epigenetic regulation of stress-induced LIF expression in vivo.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5902.

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

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Abstract

Purpose : Stressed photoreceptors release signals that stimulate Müller glial cells to induce the expression of trophic factors such as Leukemia Inhibitory Factor (LIF). Understanding the regulation of LIF expression is key to elucidating the mechanisms that control the rate of retinal degeneration. We have previously shown epigenetic regulation of the LIF locus in whole retinal samples. Since Müller glial cells make up approximately only 5% of the retinal cell population, the presence of other retinal cell types may have masked certain regulatory elements. The aim of this study is to confirm the cell type expression of LIF as well as the epigenetic profile of the LIF locus specifically in Müller glial cells.

Methods : All procedures with animals were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Müller glia-specific (GLAST Cre) and neural retina (Chx10 Cre) with either RPL22:HAf/+ or Sun1-sfGFP f/+ mice were injected with TNFα to induce LIF expression. PBS-injected animals were used as control. For RNA analysis, the retinas from RPL22:HAf/+ animals were homogenized, polysome-associated RNA was precipitated by αHA, and RNA was purified for analysis by qRT-PCR. For chromatin analysis, the retinas were fixed and homogenized. Intact nuclei were precipitated by αMyc, fragmented with MNase and prepared for chromatin immunoprecipitation (ChIP). Antibodies against NF-κB, H3K4me3, H3K27me3 or IgG were used. DNA was then purified for analysis by qPCR.

Results : qRT-PCR analysis shows that LIF expression is enriched in Müller cells while other stress markers such as the photoreceptor-specific endothelin-2 is not. ChIP analysis against H3K27me3 and NF-κB denote the presence of poised enhancer-like elements within the first intron of the LIF gene. TNFα-injected mice exhibit increased promoter-associated H3K4me3 levels within the internal transcription start site of the LIF gene in both Müller cells as well as neural retina. Interestingly, this mark is increased at the distal transcription start site in Müller cells, but is masked in samples from the neural retina.

Conclusions : Our data confirms that Müller cells express LIF. We also show that majority of the retinal epigenetic changes taking place at the LIF genomic locus originates from the Müller cells.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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