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Malihe Eskandarpour, Arnulf Hertweck, Catherine Evans, Jonathan Lau, Audrey Kelley, Peter S Adamson, David Cousins, Virginia L Calder, Graham Lord, richard jenner; Targeting P-TEFb recruitment and super-enhancers function suppress the development of Th1-mediated EAU disease. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2079.
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© ARVO (1962-2015); The Authors (2016-present)
We aimed to determine how T-bet acts through the enhancer elements to drive Th1 differentiation and also to provide further insight into the mechanism of super-enhancer function using human and mice models.
To identify the lineage-specific genes and groups of genes repressed by JQ1 and/or Flavopiridol in Th1 cells, wild-type C57BL/6 mouse naive CD4+ T cells were cultured under Th1 and Th2 polarizing conditions and run for gene expression profiling using the Mouse GE 8x60K microarrays (Agilent).The libraries were generated for RNA-seq and RNA-Chip and quantified and then sequenced on an Illumina HiSeq (50bp paired-end) and Illumina GAIIx ro HiSeq 2000 sequencer, respectively. To induce EAU, B10RIII mice aged 5-8 weeks were immunized with 300 mg IRBP161-180 emulsified with Complete Freund’s adjuvant, supplemented with 1.5 mg/ml Mycobacterium tuberculosis complete H37 Ra (1:1 v/v). Each mouse also received 0.4mg Bordetella pertussis toxin intraperitoneally. Flavopiridol and JQ1 were then administered by daily intraperitoneal injection and disease progression scored by retinal funduscopy and histology.
We revealed that T-bet acts through super-enhancers to recruit P-TEFb and activate lineage-specific transcriptional elongation. We showed that recruitment of P-TEFb to Th1 genes depends on T-bet activity and both T-bet and P-TEFb function at super-enhancers to activate transcription of enhancer RNAs. We also demonstrated that P-TEFb activity was required for Ifng eRNA production and treating Th1 cells with JQ1 and Flavopiridol resulted in a marked reduction in eRNA levels. This requirement for P-TEFb function renders lineage-specific genes hyper-sensitive to inhibitors of transcriptional elongation both in vitro and in vivo in a mouse model of uveitis. EAU mice treated with Flavopiridol and JQ1 showed suppression of disease which associated with inhibition of super-enhancer function. Flow cytometry of CD4+ T cells sorted from the retina and lymph node revealed that expression of the super-enhancer-associated gene products Ifng, Tnf, Fasl, Il18r1 and Ctla4 were downregulated by Flavopiridol and JQ1.
Using in vitro and in vivo models, we concluded that P-TEFb binds extensively to the super-enhancers of lineage-specific genes and revealed a molecular mechanism for the selective effect of those drugs on genes involved in mediating inflammation.
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