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Rebecca McGreal, Louise Wolf, Ales Cvekl; Promoter-enhancer interactions and regulation of αA-crystallin locus during lens fiber cell differentation. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1214.
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© ARVO (1962-2015); The Authors (2016-present)
Gene regulatory networks allow system level control of key cellular processes including differentiation. Lens fiber differentiation is strictly controlled through temporal and spatial organization of gene expression, including crystallin genes. However, 3D-organization of active genes within the nuclei is not known. αA-crystallin (Cryaa) ranks among the most abundantly expressed lens genes and proteins. The 16kb Cryaa locus is characterized by a lens-specific chromatin domain marked by two “outside” enhancers, 5’-DCR1 and 3’-DCR3. We hypothesize that the Cryaa locus is involved in multiple inter- and extra-genic interactions that are temporally regulated and show tissue-specific patterns.
Chromosome conformation capture (3C) was used to map intragenic 3D-organization, i.e. DNA looping, of Cryaa locus of mouse lens, heart, and forebrain at stages E14.5, E15.5, E16.5, E17.5, and P1. Cell cycle synchronized αTN4 and mouse embryonic stem (ES) cells were also examined. ChIP-seq data on histone H3K4me1- and PolII-enriched enhancers in lens and forebrain were used to aid data interpretation. Transgenic mice were generated to determine the function of DCR1 as a “core” enhancer and of 2kb 5’-extended DCR1.
In lens, strong DNA looping was detected between the promoter and DCR1, most abundant at E17.5 and least at E14.5. Interestingly, this loop was also found in non-lens tissues, albeit with much lower abundance. No loops in the Cryaa locus were found in ES cell chromatin. In contrast, no evidence for significant promoter/DCR3 looping was obtained although another loop outside the 3’-boundary of the Cryaa locus was detected in different tissues. The DCR1/promoter loop apparently extends downstream corresponding with H3K4me1 and PolII occupied domains. Transgenic studies suggest the extended DCR1 region may play a role in early expression of Cryaa in lens.
Altered interactions between different portions of the Cryaa locus suggest dynamic changes in chromatin organization throughout development. DCR1 is a “core” enhancer region flanked by a 3’-extension (~1.5kb). Ongoing experiments aim to find whether enhancer transcription (eRNA) occurs at DCR1 and identify novel regulatory factors that bind and regulate the Cryaa locus. These data lay the foundation for understanding molecular basis of lens development and gene regulation through 3D-organization of lens chromatin.
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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